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25th International Symposium
on Microscale BioSeparations MSB 2010
March 21 – 25, 2010
Clarion Congress Hotel, Prague, Czech Republic
www.msb2010.org
25th International Symposium
on Microscale Bio
MSB2010
Institute of Analytical Chemistry AS CR, v.v.i.
Brno, Czech Republic
ISBN 978-80-254-6631-5
Final Program
& Book of Abstracts
Taking Capillary Electrophoresis
to New Heights
• Protein characterization
• Small molecule analysis
• Genetic analysis
• Capillary electrophoresis-mass spectrometry (CE-MS)
Since 1989 when we introduced the first ground-breaking P/ACE system, we’ve led
the industry in capillary electrophoresis (CE) technologies and tools. Our developments
encompass hardware, software and chemistries; with a long list of advancements that
include liquid capillary cooling, SDS-gel for biologics, multiplex gene expression and nano
CE-MS. Always directed by your needs, we continue to focus on new ways to apply the
core technology to key analytical solutions.
New PA 800 plus Pharmaceutical Analysis System
• Increased workflow efficiency
• Simplified user experience
CE-MS
• Novel High Sensitivity Porous Sprayer
• Mass spectrometer optimization
Advances in cIEF
• Reproducible separations
• Simple optimization
And more!
Come visit our stand at MSB 2010, and don’t miss Tuesday’s workshop, “Advances in CE
Technology,” at 1:00 p.m. in Zenit Hall for the latest developments at Beckman Coulter.
All CE-MS applications discussed in this document are for Laboratory Use Only. Not for use in diagnostic procedures.
© Copyright 2009 Beckman Coulter, Inc. Beckman Coulter and the stylized logo are registered trademarks of Beckman
Coulter, Inc. P/ACE is a trademark of Beckman Coulter, Inc.
B2009-10809
Program Overview
MSB 2010 Program Overview
Sunday, March 21
9:00 – 17:30
13:00 – 16:30
17:15 – 17:45
17:50 – 19:15
Registration
Short courses
Opening remarks – Jaroslav Heyrovsky Medal Award
Plenary lectures
Monday, March 22
8:30 – 9:50
10:00 – 11:05
chair
11:05 – 11:25
11:25 – 12:35
chair
12:35 – 15:00
13:00 – 14:00
15:00 – 16:00
chair
16:00 – 16:20
16:20 – 17:40
chair
17:40 – 21:30
Plenary lectures
ZENIT
NADIR
Proteomics
Pharmaceutical analysis
M. Novotný, H. Zou
W. Thormann, M. Gilar
Coffee break
Proteomics
P. G. Righetti, T. Rejtar
S. Fanali, E. DeLorenzi
Lunch break and poster sessions
Waters sponsored lunch presentation
CE-MS
Fundamentals
A. Cifuentes, P. Jandera
T. Laurell, V. Kašička
Coffee break
Food analysis
Metabolomics
J. Hudson, E. J. Maxwell
P. Oefner, M. Breadmore
Poster sessions - Come chat with the Beckman Coulter team over wine and beer
Tuesday, March 23
8:30 – 9:50
10:00 – 11:05
chair
11:05 – 11:25
11:25 – 12:25
chair
12:20 – 15:00
13:00 – 14:00
15:00 – 16:05
chair
16:05 – 16:25
16:25 – 17:50
chair
19:00 – 22:00
Plenary lectures
ZENIT
NADIR
Microfluidics
Instrumentation
A. Manz, H. Becker
D. Kaniansky, J. Preisler
Coffee break
Emerging technologies
Instrumentation
R. Kennedy, S. Cohen
F. Švec, V. Dolník
Beckman sponsored lunch presentation
Instrumentation
Y. Lyubarskaya, V. KošťálS. Jacobson, Hervé Cottet
Coffee break
Pharmaceutical industry
Nanotechnologies
B. Buszewski, H. Stutz
U. Tallarek, J. Dickerson
Conference dinner
Wednesday, March 24
ZENIT
9:00 – 10:25
chair
10:25 – 10:45
10:45 – 12:30
chair
12:30 – 15:00
13:00 – 14:00
14:00 – 15:00
15:10 – 17:10
17:10 – 17:40
Microfluidics
Glycomics
S. Jovanovich, J. Janča
F. Regnier, Z. El Rassi
Coffee break
Next generation DNA analysis High resolution bioseparations
A. Barron, M. Macka
A. Guttman, C. Balog
Lunch break and poster sessions; Permanent MSB scientific board meeting
Agilent sponsored lunch presentation
Come chat with the Beckman Coulter team over coffee and desert
Plenary lectures
Poster Awards and Closing Remarks
NADIR
1
Conference Floor Plan
conference office
Conference Floor Plan
2
Organization
MSB 2010 Organization
Committees
Scientific Advisory Board
Organizing Committee
Annelise Barron
Stanford University, USA
Franta Foret
(Symposium Chairman)
Institute of Analytical Chemistry, Brno
Jeff Chapman*
Beckman Coulter, Inc. USA
Steve Cohen*
Waters Corporation, USA
Franta Foret*
Institute of Analytical Chemistry, Czech Republic
Andras Guttman*
The Barnett Institute, Northeastern University, USA
Stellan Hjerten*
Uppsala University, Sweden
Hanfa Zou
Dalian, China
Steven Jacobson
Indiana University, USA
Franka Kalman*
Solvias AG, Switzerland
Petr Bocek
Bohuslav Gas
Charles University, Prague
Vaclav Kasicka
Institute of Organic Chemistry and Biochemistry, Prague
Karel Kleparnik
Ludmila Krivankova
Ivan Miksik
Institute of Physiology, Prague
Marek Minarik
Genomac International, Prague
Barry L. Karger*
The Barnett Institute, Northeastern University, USA
Robert Kennedy*
University of Michigan, USA
Takehiko Kitamori*
University of Tokyo, Japan
Andreas Manz*
Freiburg Institute for Advanced Studies, Germany
J. Michael Ramsey*
University of North Carolina, USA
Gerard Rozing*
Agilent Technologies GmBH, Germany
Frantisek Svec*
Lawrence Berkeley National Laboratory, USA
Edward S. Yeung*
Iowa State University, USA
* Permanent Committee Members
Venue
Clarion Congress Hotel Prague
Freyova 33
Prague, Czech Republic
www.cchp.cz
3
Jaroslav Heyrovský Medal Award / MSB Student Grants
Jaroslav Heyrovský Medal Award 2010
Jaroslav Heyrovský Medal Award 2010
The highest chemistry award of the Academy of Sciences of the Czech Republic will be presented to Prof. Barry L. Karger, Northeastern University, Boston (USA) on Sunday, March 21, 2010 at 17:30, by Prof. Vladimír Mareček, vice-president of the Czech Academy
of Sciences
Poster awards
Poster presentations will be reviewed by an international panel to select three award winning presentations. This appreciation of the
scientific contributions has been made possible thanks to a generous endowment of AGILENT TECHNOLOGIES. The Poster Awards
will be presented to the winners at the Closing Ceremony on Wednesday, March 24, 2010, 17:10.
MSB 2010 Student Grants
Maria Bohlin
Karlstad University Sweden
Stephanie M. Cologna
Texas A&M University
USA
Naomi Janson
McMaster University
Canada
Chang Liu
University of British Columbia
Canada
Ronny Ludewig
Friedrich-Schiller-University Jena
Germany
E. Jane Maxwell
Canada
Michelle Meighan
Arizona State University
USA
Stefan Mittermayr
University of Debrecen
Hungary
Nantana Nuchtavorn
Mahidol University
Thailand
Mariko Seno
Kyoto University
Japan
Ákos Szekrényes
University of Debrecen
Hungary
Lilla Turiak
Hungarian Academy of Sciences
Hungary
Students Suported by CASSS
Stephanie Cologna
Benjamin Mann
Indiana University
Jane Dickerson
University of Washington
Denitsa Milanova
Stanford University
Seth Madren
Indiana University
Poster Awards sponsored by Agilent Technologies
4
Symposium Proceedings / The MSB Symposium Series
Symposium Proceedings
The deadline to submit your manuscript to the MSB 2010 symposium issue of Journal of Chromatography A is 3rd May 2010.
When preparing your manuscript please carefully follow the journal’s Guide to Authors, which you can find on the online submission
site: http://ees.elsevier.com/chroma
In your cover letter please mention that your manuscript is intended for the MSB 2010 special issue.
The MSB Symposium Series
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B.L. Karger & S. Fanali
E. Yeung
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Y. Baba & K. Otsuka
G. Rozing
R. Kennedy
A. Manz
J. Sweedler
H. Zhou
F. Foret
A. Barron
Gerard Rozing & Franka Kalman
Rong Zeng
5
Technical Program
Technical Program
SUNDAY Short courses
Short course A - Zenit hall, 12:00 – 13:50
Microfabrication Technologies for Microfluidic Devices
Holger Becker
microfluidic ChipShop GmbH, Jena, Germany
Who should attend?
The course is suitable for scientists, technicians and engineers who would like to acquire a comprehensive overview of the field of
microfabrication technologies for microfluidic devices made out of glass, silicon and polymers. The course will cover a wide range
of existing manufacturing technologies and describes the complete development cycle of a microfluidic device from the design to
the ready-to-use device. The course will also provide an opportunity for researchers who already have experience with silicon based
microsystems to extend their knowledge to non-silicon based systems which find more and more applications in microfluidics.
Learning Objectives
1. Identify the suitable material for a given microfluidic application
2. Understand the basic technologies available for the microfabrication of glass, silicon and polymer materials and follow the device
manufacturing process from design to the finished microfluidic device
3. Being able to understand cost-models for the various fabrication technologies
4. Understand modern trends and developments in the field of microfabrication technologies
Topics and Course Organisation
1. Introduction and Objectives
2. A brief history of microfabrication
3. Challenges in material selection
4. Photolithography
5. Silicon microfabrication
6. Glass microfabrication
7. Polymer microfabrication
8. Back-End processing
9. Future trends and conclusion
Short course B - Zenit hall, 14:00 – 16:30
Get the Most out of Your Mass Spectrometer: Use Capillary Electrophoresis Separations
Andre Deelder1, Jerry Feitelson2
1 Leiden University Medical Centre
2 Beckman Coulter
Capillary electrophoresis (CE) is recognized as being a versatile, fast and highly efficient separation technique. Recent advances in
CE-MS coupling have preserved the advantages of CE, and it now emerges as a key technology for sample introduction into MS. Many
scientists are ready to move beyond the existing capabilities of LC-MS for specific applications, and CE-ESI-MS is positioned to provide these new capabilities. This workshop will highlight several successful applications of CE-MS, proving that the capabilities of mass
spectrometry can be significantly enhanced by the use of novel CE interfaces and applications.
Robust and Automated CE-MS is within Reach for Novice Users
David D. Y. Chen1, E. Jane Maxwell1, Xuefei Zhong1, John Hudson2, Chitra Ratnayake2 and Hong Zhang1
1 Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1 Canada
2 Beckman Coulter, Inc., Discovery Solutions
6
Technical Program
The challenge of efficiently processing the often varied volumetric flow rate of CE effluent can be met by using a beveled electrosprayer tip, and the problem of often incompatible requirement of electrolyte composition for the optimum operations of CE and electrospray ionization can be alleviated by introducing a chemical modifier using the junction-at-the-tip configuration. A simple and robust
stainless steel hollow needle electrode with a tapered inner cavity and a beveled outer tip is used to interface CE with MS, resulting in
a stable yet highly sensitive electrospray ion source that is easily automated in CE-MS operations. The interface and its applications for
different types of analyte, including small molecule pharmaceuticals and biomolecules, will be discussed.
CE-MS of Intact Proteins
Rob Haselberg, Gerhardus J. de Jong, Govert W. Somsen
Utrecht University, Department of Biomedical Analysis, Utrecht, The Netherlands
In the biopharmaceutical field there is a growing demand for separation-detection methodologies that permit quality assessment of
proteins in their native state. Capillary electrophoresis (CE) is an attractive separation technique for purity and stability analysis of
proteins. Combination of CE with electrospray-ionization time-of-flight mass spectrometry (ESI-ToF MS) would provide a powerful tool
for protein characterization. This presentation outlines the design and application of robust CE-MS methodologies for intact proteins.
Reproducible CE performance for proteins was achieved by using capillaries coated with layers of charged polymers, which effectively
reduce protein adsorption. The coatings are easy to produce and fully compatible with ESI-MS detection. Stable CE-MS profiles of
basic protein mixtures could be obtained with migration-time RSDs below 1%. Use of a high sensitivity porous sprayer (HSPS) for
sheathless CE-MS interfacing presented significant improvements in protein signal-to-noise ratios, yielding detection limits in the subnM range. Analyses of degraded biopharmaceuticals and drug-protein conjugates further demonstrated that efficient CE separation in
combination with high-mass-accuracy ToF MS allows distinction and identification of highly related protein products.
Exploring the Metabolome by CE-MS: From Study Design to Data Interpretation
Oleg A. Mayboroda and Andre M. Deelder
Leiden University Medical Centre, Biomolecular Mass Spectrometry Unit, The Netherlands
The potential of Capillary Electrophoresis - Mass Spectrometry (CE-MS) as a method for analysis of complex biological matrices was
immediately recognized after the introduction of modern ESI sources. However until recently, CE-MS has been mentioned in the literature as an analytical method that has yet to prove its utility as a routine tool for metabolomics studies. The goal of our presentation is to demonstrate that CE-MS is indeed a mature technology that had already passed “a proof of principle period.” Moreover, we
demonstrate that CE-MS is an essential part of “the metabolomics toolbox” as it helps us to characterize portions of the metabolome
typically difficult for other methods, such as LC-MS, GC-MS and NMR.
CE-HSPS-MS: Toward the Miniaturization of Proteomics
Jean-Marc Busnel1, Jerald S. Feitelson1, Andre M. Deelder2, Oleg A. Mayboroda2
1 Beckman Coulter, Inc., Discovery Solutions Business Center, Brea, CA, USA
2 Leiden University Medical Centre, Biomolecular Mass Spectrometry Unit, The Netherlands
The success of liquid chromatography-mass spectrometry (LC-MS) in biological analysis and in proteomics, in particular, is not only due
to its relatively high resolution and reproducibility but also to the relative simplicity for coupling it either to a fraction collector or to a
mass spectrometer. As compared to LC, capillary electrophoresis (CE) has different characteristics, such as ultralow flow rates, a background electrolyte (BGE) that can be highly conductive, and most importantly, the need to maintain an electric field across the capillary
during the separation. Because of these features, the popular adoption of CE-MS has taken much longer than LC-MS. Indeed, while the
first reports describing interfacing of both techniques were published in the late 80's, research on these topics is still very active today.
Both liquid sheath and sheathless approaches have been demonstrated to couple CE via electrospray ionization to mass spectrometers.
The sheath liquid interface has been the most popular so far despite its significantly lower sensitivity and resolution. Here we describe
experiments to analyze peptide samples of increasing complexity (cytochrome C tryptic digest, E. coli total protein tryptic digest, and
synthetic peptide libraries) using a prototype High Sensitivity Porous Sprayer (HSPS). To increase the mass loading of CE-HSPS-MS, the
compatibility of the interface with online preconcentration methodologies has also been tested. Benchmarking studies compared the
analysis of E. coli tryptic digests using the CE-HSPS-MS platform to conventional bioanalytical platforms, such as CE-MS with a sheath
liquid interface and nanoLC-MS.
7
Technical Program
As compared to conventional approaches, the CE-HSPS-MS platform represents a real breakthrough in terms of bioanalytical performance. Indeed, while the CE advantages of high efficiency and rapid separations are retained, the platform also makes possible the
use of the nanoflow regime of the HSPS-ESI process to provide exquisite sensitivity.
Short course C - Nadir hall, 13:00 – 14:00
Utility of 2D LC for separation of complex samples
Martin Gilar
Waters Corp., Milford, MA, USA
This 1 hour long course will cover the principles of 2D LC and discuss advantages and disadvantages over unidimensional chromatography. Attendees will learn about concepts of peak capacity, separation orthogonality, and their implications for multidimensional
separations. The course will discuss the basic types of experiments, such as column switching, off-line, and on-line 2-D LC. Traditional
as well as novel/unusual 2-D LC approaches, including requirement for mobile phase compatibility, separation mode options, and column dimensions will be explained. Specific application examples will be given with focus on proteomic and peptidomic applications.
About the speakers
Dr. Martin Gilar is a principal researcher in the Biopharmaceutical Sciences department at Waters Corporation. He has a Ph.D. degree
in analytical chemistry with over fifteen years of experience in the separation sciences, namely chromatography, electrophoresis, and
mass spectrometry. His research interest is sample preparation and analysis of biomolecules, such as peptides, oligonucleotides and
proteins, including 2D LC applications. He has published over 30 peer reviewed papers. Dr. Gilar’s received his Ph.D. in analytical
chemistry from Institute of Chemical Technology in Prague (1996). He spent postdoc years in Hybridon Inc. (1996-1998) and Northeastern University in Boston (1998) developing separation methods for antisense oligonucleotides and fraction collector for DNA
molecules. Since 1998 he works at Waters Corp. in Milford, Massachusetts.
Dr. Holger Becker is co-founder and CSO of microfluidic ChipShop GmbH in Jena/Germany. He obtained a postgraduate Diploma
in Science from the University of Western Australia/Perth in 1990 and a Diploma in physics from the University of Heidelberg in
1991. He started to work on miniaturized systems for chemical analysis during his PhD-thesis at Heidelberg university, where he
obtained his PhD in 1995. Between 1995 and 1997 he was a Research Associate at Imperial college/London working in the group of
Prof. Andreas Manz. In 1998 he joined Jenoptik Mikrotechnik GmbH. Since then, he founded and led several companies in the field
of microsystem technologies in medicine and the life sciences. He has published more than 10 original papers and 50 conference
contributions as well as 9 patent applications. He chaired the Industry Group of the German Physical Society between 2004 and
2009 and is the current Chair of the SPIE “Microfluidics, BioMEMS and Medical Microsystems” conference as well as acting as regular
reviewer of project proposals on national and EU level and for several journals devoted to microsystem technologies.
Company sponsored lunch presentations
Zenit hall 13:00 – 14:00
Waters Corporation – Monday, March 22
Evolution of nano-scale Chemistries for Information-rich UPLC-based MS Analyses
Patricia M. Young, Sr. Product Marketing Manager
Waters Corp, Milford, USA
Frequently, analytical labs have samples with trace amounts of an analyte and are required to perform many different tests. Therefore,
when doing LCMS analyses, it is important to get the most information from a small amount of sample. Proteomics researchers are
concerned small changes in expression and post-translational modifications (PTM) that may be expressed in low concentrations in a
complex mixture of proteins or proteome. For drug metabolism pharmacokinetics (DMPK) or metabolic identification (met i.d.), increasingly researchers are working with small samples of body fluids or tissues during development and in clinical trials.
8
Technical Program
Researchers have turned to nano-and capillary-scale chromatography with sub-2-micron packing materials (nanoUPLC) and high resolution MS to detect and quantify small changes in metabolism or protein expression. Typical nanoUPLC uses reversed phase (RP) columns
with internal diameters of 300 micron to 75 micron to overcome dispersive losses incurred typical analytical-scale LC. A standard approach is to use a short pre-column to trap the analytes and flush unwanted solutes to waste. Then the analytes are displaced from the
trapping column to an analytical column packed with sub-2-micron particles to obtain better chromatographic resolution and higher
peak capacity separations that result in better data quality from the MS.
Another way that researchers can increase the peak capacity and information quality is through two-dimensional LC (2D-LC). The most
practiced approach uses strong cation exchange (SCX) followed by reversed phase (RP). A different and highly resolving approach uses
two RP columns: one operated at pH 10 and the second at pH 2. To enable this RP/RP separation, the system must be capable of on-line
dilution between the two dimensions since the on-line fractions from the first dimension will be added directly to the second dimension
column. The next innovation is to integrate these highly resolving chemistries into a UPLC-capable microfluidic device for routine, highly
resolving, information-rich separations.
Complimentary lunch will be provided to delegates who registered for the Waters Seminar.
Beckman Coulter – Tuesday, March 23
Advances in CE Technology Workshop
Chairs: Hans Dewald, Beckman Coulter, Inc., Brea, CA, USA
This workshop will provide an overview of the latest developments at Beckman Coulter, Inc., including instrumentation, novel sheathless CE-MS separations and data, and applications.
Lunch will be provided for all Tuesday workshop attendees.
Agilent Technologies – Wednesday, March 24
All about a Powerful Electrophoresis Portfolio and Applications and Novel Microfluidic Chip based LC/MS Workflow Solutions
Abstract: The seminar will discuss different electrophoretic separatation techniques and its applications reaching from protein fractionation using an Offgel approach to the introduction of the new Capillary Electrophoresis System. An overview of CE-MS for pharmaceutical applications will be presented. In the last presentation, you will hear about a Novel Microfluidic Chip-based LC/MS Workflow
for N-glycan Analysis for Biologics
Agenda with Abstracts:
Agilent in Electrophoresis: From Sample Preparation to Capillary Electrophoresis to Lab-on-a-Chip
Dr. Martin Greiner - Product manager Capillary Electrophoresis, Agilent Technologies
The talk provides a brief overview on the available Agilent portfolio in electrophoretic separation techniques. First part covering
sample preparation and concentration by using an Offgel approach where compounds are separated by Isoelectric Focusing (IEF) and
the fractions are available in solution for further analysis. Part two will introduce the new Agilent 7100 Capillary Electrophoresis and
it’s capability to combine external detectors (LIF, MS, CCD, etc) easily. Another approach in electrophoresis is the Microfluidic-Chip
and assay based 2100 Bioanalyzer for fast separation of RNA, DNA and Proteins or as well as for cell based assays using predefined
methods and complete reagent kits.
An Overview of CE-MS Analysis in the Pharmaceutical Lab - From Small Molecules to Proteins
Dr Serge Rudaz - Laboratory for Pharmaceutical Analysis, University of Geneva, Switzerland
CE-MS is a powerful technique in pharmaceutical analysis, since it can be used for the determination of small compounds as well as
for large biomolecules. This hyphenated technique presents numerous assets such as high efficiency and sensitivity, small sample
consumption and ability to identify the compounds of interest. In this presentation, a sheath liquid interface was used in order to
couple CE with MS (single quadrupole and TOF analyzers) because of its high robustness and versatility. Several examples will be
9
Technical Program / List of Exhibitors & Sponsors
discussed in aqueous and non aqueous BGEs for the analysis of enantiomers, drug substances and proteins present in pharmaceutical formulations and biological matrices.
A Novel Microfluidic Chip-based LC/MS Workflow for N-glycan Analysis for Biologics
Dr Tom van de Goor - R&D Manager Low-Flow and Workflow Solutions, Agilent Technologies
Glycosylation is of great importance for new biologics, changes in the glycosylation profile can lead to dramatic differences in glycoprotein pharmaceutical efficacy, immunogenicity and stability (3). In this presentation, we will describe in detail a new microfluidic
chip designed for N-glycan analysis cleaved off monoclonal antibodies (MAbs). The aim for this novel fully-integrated microfluidic LC/
MS chip is to improve the efficiency of enzyme cleavage of glycans off glycoproteins and consequent analysis of N-glycans. Preliminary data will show the on-chip workflow that involves enzymatic deglycosylation with immobilized PNGase F enzyme, glycan purification, separation, identification and quantitation of N-linked glycans from MAbs. With this chip, the experimental time from antibody
injection to LC/MS result is reduced to 10 minutes while current routine analysis via other methods demands at least 2-3 days. We
will also show that the GlycoPRO-Chip workflow matches well with current analysis methods, such as CE-LIF or MALDI MS.
List of Exhibitors & Sponsors
The organizing committee gratefully acknowledges exhibitors and sponsors for their generous support.
Academy of Sciences of the Czech Republic, v.v.i.
Ministry
of Education,
Youth
and Sports
10
LC06023
Institute of Analytical Chemistry of the ASCR, v.v.i.
Beckman Coulter, Inc.
250 S. Kraemer Boulevard
P.O. Box 8000
Brea, CA 92822-8000 USA
www.celeader.com
Waters, Inc.
34 Maple St.
Milford, MA 01757
www.waters.com
Agilent Technologies, Inc.
5301 Stevens Creek Blvd
Santa Clara CA 95051
United States
www.agilent.com/chem
Thermo Fischer Scientific, Inc.
81 Wyman Street
Waltham, MA 02454
www.thermo.com/massspectrometry
Genentech, Inc.
1 DNA Way
South San Francisco,
CA 94080-4990
USA
www.gene.com
11
Leco Instrumente Plzeň, s.r.o.
Plaská 66
323 00 Plzeň
Czech Republic
www.leco.cz
General sponsor - Czech Republic
Genomac
Genomac International, s.r.o.
Bavorska 856
CZ - 155 41 Prague 5
Czech Republic
www.genomac.com
Watrex
Watrex Praha, s.r.o.
Hošťálkova 42
169 00 Praha 6
Czech Republic
www.watrex.cz/watrex/en/
Delivering the Right Results
CASSS
5900 Hollis Street, Suite R3
Emeryville, CA 94608
USA.
www.casss.org
ECOM, s r.o.
Americká 3, 120 35
Praha 2
Czech Republic
www.ecomsro.cz
Bruker Daltonics, s.r.o.
Zdráhalova 10
613 00 Brno
Czech Republic
www.bdal.com
Elsevier
3251 Riverport Lane
Maryland Heights, MO 63043,
USA
www.elsevier.com
Biomicrofluidics
American Institute of Physics
Suite 1NO1
2 Huntington Quadrangle
Melville, NY 11747-4502,
USA
www.bmf.aip.org
12
Microfluidic ChipShop, GmbH
Carl-Zeiss-Promenade 10
D - 07745 Jena
Deutschland
www.microfluidic-ChipShop.com
AAA Radiotaxi
Základní spojení: 14 0 14
222 333 222
2211 0 2211
Czech Republic
www.aaataxi.cz
eDAQ Pty, Ltd
6 Doig Ave
Denistone East
NSW 2112
AUSTRALIA www.edaq.com
Villa Labeco, s.r.o.
Chrapčiakova 1.
052 01
Spišská Nová Ves
Slovakia
www.villalabeco.sk
AB SCIEX, s.r.o
Krenova 1
Praha 6
Czech Republic
www.absciex.com
Electrophoresis
Journal of Separation science
John Wiley & Sons Inc
350 Main Street
Malden MA 02148
USA.
www.electrophoresis-journal.com
www.jss-journal.com
Mengel Engineerig
Brovaenget 7
DK-2830 Virum
Denmark
www.mengelengineering.dk
VITRUM Praha, s r.o.
Pražská 442
281 67 Stříbrná Skalice
Czech Republic
www.vitrum.cz
Data Apex
Podohradska 1704/1,
15500 Prague,
Czech Republic
www.dataapex.com
Springer, GmbH
Haberstraße 7
69126 Heidelberg
Germany
www.springer.com
ACS-HU
Hungarian Chapter of the American Chemical Society
www.acshc.hu
13
MSB 2010 Program
MSB 2010 Program
Registration daily 9:00 - 17:00
Sunday, March 21
Short Courses
A 12:00 – 13:50 / Zenit Hall
Microfabrication technologies for microfluidic devices
Holger Becker
Microfluidic ChipShop, Jena, Germany
B 14:00 – 16:30 / Zenit Hall - Beckman Coulter
Get the Most out of Your Mass Spectrometer: Use Capillary Electrophoresis Separations
Andre Deelder1, Jerry Feitelson2
1 Leiden University Medical Centre
Robust and automated CE-MS is within reach for novice users
David D. Y. Chen1, E. Jane Maxwell1, Xuefei Zhong1, John Hudson2, Chitra Ratnayake2, Hong Zhang1
1 Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1 Canada
CE-MS of intact proteins
Rob Haselberg, Gerhardus J. de Jong, Govert W. Somsen
Utrecht University, Department of Biomedical Analysis, Utrecht, The Netherlands
Exploring the metabolome by CE-MS: From study design to data interpretation
Oleg A. Mayboroda, Andre M. Deelder
Leiden University Medical Centre, Biomolecular Mass Spectrometry Unit, The Netherlands
CE-HSPS-MS: Toward the miniaturization of proteomics
Jean-Marc Busnel1, Jerald S. Feitelson1, Andre M. Deelder2, Oleg A. Mayboroda2
2 Leiden University Medical Centre, Biomolecular Mass Spectrometry Unit, The Netherlands
C 13:00 – 14:00 / Nadir Hall
Utility of 2D LC for separation of complex samples
Martin Gilar
Waters Corp., Milford, MA, USA
14
MSB 2010 Program
17:15 – 17:30 Opening remarks – František Foret
17:30 – 17:45 Jaroslav Heyrovský Medal awarded to Prof. Barry L. Karger by Prof. Vladimír Mareček, vice-president of the
Czech Academy of Sciences
Opening plenary lectures:
17:50 – 18:35 PL1 Barry L. Karger, Boston, USA – New approaches to protein structure analysis for the life sciences and
biotechnology
18:35 – 19:15 PL2 Miloš Novotný, Bloomington, USA – Glycomic and glycoproteomic profiling in disease biomarker discovery
19:20 – 21:30
Welcome mixer
Monday, March 22
Plenary lectures:
8:30– 9:10 PL3 Pier Giorgio Righetti, Milan, Italy – Plucking, pillaging and plundering proteomes with combinatorial
peptide ligand libraries
9:10– 9:50 PL4 Peter Oefner, Regensburg, Germany – Conquering the metabolome: from extraction to bioinformatics
Lecture hall:
Zenit
Nadir
Chair
10:00 – 10:25
Proteomics
M. Novotný, H. Zou
W. Thormann, M. Gilar
L1 Rong Zeng, Qing-Run Li, Xiao-Bin Xing, Tao-Tao Chen, Chia-Hui Shieh
Shanghai, China
L4 Salvatore Fanali, Zeineb Aturki
Giovanni D'Orazio, Anna Rocco
Rome, Italy
Proteome and phosphoproteome
revealed unique features of mouse
embryonic stem cells
Capillary electrochromatography-mass
spectrometry for the separation of compounds
of pharmaceutical interest and tryptic digest of proteins: comparison with nano-LC-MS and CE-MS
10:25 – 10:45
L2 Tomáš Rejtar, Dipak Thakur,
Dongdong Wang, Sangwon Cha, Barry Karger
Boston, USA Proteomic analysis of minimal sample amounts
L5 Luca Regazzoni, Raffaella Colombo, Laura Bertoletti, Giulio Vistoli, Giancarlo Aldini,
Ersilia DeLorenzi
Pavia, Italy
10:45 – 11:05
L3 Victor Zgoda, Olga Tikhonova, Leonid Kurbatov,
Artur Kopylov, Natalia Moskalyova, Ivan Cheglakov,
Alexander Archakov
Moscow, Russia
Systems biology of HL-60 cell line induced
by ATRA differentiation.
L6 Svenja-Catharina Bunz, Christian Neusuess
Aalen, Germany
Integrated strategies for drug discovery:
capillary electrophoresis, mass spectrometry
and in silico studies
The selective determination of sulfates, sulfonates and phosphates in urine by capillary electrophoresis- mass spectrometry
15
MSB 2010 Program
Lecture hall:
16
Zenit
Nadir
11:05 – 11:25 Coffee break
Proteomics
Chair
P. G. Righetti, T. Rejtar
S. Fanali, E. DeLorenzi
11:25 – 11:50 L10 Václav Kašička, Dušan Koval, Veronika Šolínová,
Jean-Marc Busnel, Martine Poitevin, Gabriel Peltre
L7 Chun xia Song, Mingliang Ye,
Xinning Jiang, Rui Chen, Hanfa Zou
Dalian, China
RP-RPLC Multidimensional Separation with
High Orthogonality for Large-scale
Phosphoproteome Analysis
Capillary zone electrophoresis in strongly
acidic/basic classical and (quasi)isoelectric
background electrolytes applied to separation
and characterization of biopeptides
11:50 – 12:10 L8 Maria Bohlin, Lars Blomberg, Niels Heegaard
Karlstad, Sweden
Affinity studies of ß2-glycoprotein I using capillary
electrophoresis
L11 Simone Portmann, Hiu Ying Kwan,
Regula Theurillat, Andrea Schmitz,
Meike Mevissen, Wolfgang Thormann
Bern, Switzerland
12:15 – 12:35 L9 Romain Verpillot, Antoine Pallandre,
Pelle Olson, Olga Ordeig, Reza Mohamadi,
Jean-Louis Viovy, Jörg Kutter, Myriam Taverna
Chatenay-Malabry, France
Simultaneous analysis of potential biomarkers of
Alzheimer disease, the amyloid peptide quintet,
on microsystem
L12 Yuliya Shakalisava, Fiona Regan
Dublin, Ireland
Enantioselective CE analysis of human
ketamine metabolism in vitro
CE separation strategies for combinations of
anthracycline and taxanes
12:35 – 15:00
13:00 – 14:00
Waters sponsored lunch presentation
Chair
CE-MS
J. Janča, V. Kašička
Fundamentals
A. Cifuentes, P. Jandera
15:00 – 15:20
L13 Csilla Páger, Anna Takácsi-Nagy,
Wolfgang Thormann, Ferenc Kilár
Pécs, Hungary
L16 David Chen, Xuefei Zhong, Chang Liu,
E. Jane Maxwell
New York, USA
Novel methodology to couple isoelectric focusing
with mass spectrometry – experimental and
theoretical advances
Mass balance equation and chemical separation
in two dimensional geometries
15:20 – 15:40
L14 E. Jane Maxwell, Xuefei Zhong, David Chen
New York, USA
L17 Bohuslav Gaš
Improved flow rate compatibility of electrospray
ionization with capillary electrophoresis though
optimization of the emitter geometry
Theory of Electrophoresis: Still a challenge
MSB 2010 Program
Lecture hall:
Zenit
15:40 – 16:00
L15 John Hudson, Jeff Chapman
Brea, USA
Sheathless capillary electrophoresis Mass spectrometry: The high sensitivity
porous sprayer (HSPS) in forensic drug analysis
Nadir
L18 Michael Breadmore, Joselito Quirino,
Wolfgang Thormann
Hobart, Australia
Computer-aided design of on-line enrichment
strategies for electrophoresis
16:00 – 16:20
Coffee break
Chair
Food analysis
J. Hudson, F. Kilár
Metabolomics
P. Oefner, M. Breadmore
16:20 – 16:45
L19 Carolina Simo, Virginia Garcia-Cañas,
Miguel Herrero, Alejandro Cifuentes
Madrid, Spain
L23 Michal Holčapek, Robert Jirásko, Miroslav Lísa,
Milan Nobilis
Pardubice, Czech Republic
Capillary electrophoresis for foodomics
Potential of HPLC/MS/MS in the identification
of drug metabolites
16:45 – 17:05 L20 Yi Ting Hsieh, Tai Chiu, Huan Chang
Taipei, Taiwan
Detection of monophosphate nucleotides
through surface-assisted laser
desorption/ionization mass spectrometry
using gold nanoparticles
L24 Rawi Ramautar, Oleg Mayboroda, Andre Deelder,
Govert Somsen, Gerhardus De Jong
Leiden, The Netherlands
17:05 – 17:25 L21 Pavel Jandera, Veronika Škeříková,
Jiří Urban, Romana Kubíčková
Pardubice, Czech Republic
Monolithic columns with dual HILIC - reversed phase
mechanism for capillary LC of polar compounds. 17:25 - 17:45 L22 Angela Bachi, Pier Giorgio Righetti,
Egisto Boschetti, Elisa Fasoli
Milan, Italy
L25 Ekaterina Nevedomskaya, Rawi Ramautar,
Rico Derks, Gerben Zondag, Ingrid van der Pluijm,
Andre Deelder, Oleg Mayboroda
L26 Philip Britz-McKibbin, Richard Lee, Daniel West,
Stuart Phillips
Hamilton, Canada
Searching for trace proteins in foodstuff via combinatorial peptide ligand libraries (CPLL)
Time-resolved Metabolomics for Quantitative
Assessment of Oxidative Stress with Strenuous Exercise
17:45 – 21:30 Poster sessions – Come meet the Beckman Coulter team - serving drinks at the Beckman Coulter stand
19:00 – 21:30
Invited Speaker dinner
Lecture hall:
Stable and flexible metabolic profiling
by comprehensive CE-ToF-MS
Metabolic profiling of mouse urine by CE-MS:
dissection of osteoporosis biomarkers from TTD mice
Zenit Nadir
17
MSB 2010 Program
Tuesday, March 23
Plenary lectures:
18
8:30 – 9:10 PL5 Andreas Manz, Saarbrücken, Germany – What has cuneiform writing or landing on the moon to do with electrphoresis on chip?
9:10 – 9:50 PL6 Thomas Laurell, Lund, Sweden – Linking acoustophoretic cell and particle processing to MALDI mass -
spectrometry
Lecture hall:
Zenit
Nadir
Chair
Microfluidics
A. Manz, H. Becker Instrumentation
D. Kaniansky, J. Preisler
10:00 – 10:25
L27 Robert Kennedy, Gwendolyn Anderson, Maojun Gong
Ann Arbor, USA
A Miniaturized Western Blot
L30 František Švec
Berkeley, USA
10:25 – 10:45
L28 Steven Cohen, James Murphy, Joseph Michienzi,
Paul Rainville, Angela Doneanu, Michael Tomany
Milford, USA
Expanding the scope of high performance,
high pressure microfluidic chromatography
L31 Vladislav Dolník, William Gurske
Palo Alto, USA
10:45 – 11:05 L29 Nils Danckwardt, Matthias Franzreb, Andreas Guber, Volker Saile
Karlsruhe, Germany
Pump free transportation device for magnetic
L32 Yi Liu, Xiaofang Fu, Yu Bai, Huwei Liu
Beijing, China
11:05 – 11:25
Coffee break
Chair
Emerging technologies
R. Kennedy, S. Cohen
Advanced porous polymer monoliths with controlled
surface chemistry for separation of biomacromolecules
Recent developments in size-separation of proteins
by capillary sieving electrophoresis
Preparation and applications of the capillary coated
dynamically with Carboxymethyl chitosan
particles in a microfluidic channel
Instrumentation
F. Švec, V. Dolník
11:25 – 11:45
L33 Tadahiro Yamashita, Yo Tanaka, Kae Sato,
Takehiko Kitamori
Tokyo, Japan
Vascular tissue engineering in separable microchip
with vascular cells
L36 Hervé Cottet, Nadia Anik, Marc Airiau,
Marie-Pierre Labeau, Chi-Thanh Vuong, Julien Reboul,
Patrick Lacroix-Desmazes, Corine Gerardin
Montpellier, France
11:45 – 12:05
L34 Josef Janča
Zlín, Czech Republic
Can the cells and subcellular particles be
fractionated according to their shape?
L37 Jan Preisler, Pavla Jungová, Viktor Kanický,
Pavel Krásenský, Jarmila Navrátilová, Ondřej Peš
Jan Šmarda, Tomáš Vaculovič
Brno, Czech Republic
Determination of polymer effective charge by indirect
UV detection in capillary electrophoresis
A Multidetection platform for microcolumn separations
MSB 2010 Program
Lecture hall:
Zenit
12:05 – 12:25
L35 Vratislav Košťál, Gregory G. Wolken,
Edgar A. Arriaga
Minneapolis, USA
Elucidating the effects of cytoskeleton on the
electrophoretic properties of individual mitochondria 12:20 – 15:00
13:00 – 14:00
Beckman sponsored lunch presentation
Chair
Y. Lyubarskaya, V. Košťál
Nadir
L38 Dušan Kaniansky, Róbert Bodor, Marián Masár,
Bratislava, Slovak Republic
Column-switching operations as joined capillary zone
electrophoresis with capillary isotachophoresis
while suppressed for laminar and electroosmotic flows
Instrumentation
S. Jacobson, H. Cottet
15:00 – 15:25
L39 Bogusław Buszewski, Ewelina Dziubakiewicz,
Ewa Kłodzińska, Katarzyna Hrynkiweicz,
Paweł Olszowy, Michał Szumski
Toruń, Poland
Considerations on influence of charge distribution on
determination of pathogenic bacteria by
electromigration techniques
L42 Jane Dickerson, Lauren Ramsay, Norman Dovichi
Seattle, USA
15:25 – 15:45
L43 Dzmitry Hlushkou, Robbyn Perdue, Derek Laws,
Richard Crooks,Ulrich Tallarek
Marburg, Germany
L40 Martin Weinbauer, Tobias Pfaller, Gertie Oostingh, Hanno Stutz
Salzburg, Austria
Making the capillary-based 2D gel separation a reality:
Challenges and future of 2D CE
Characterization of recombinant allergen variants – Various CE modes addressing one aim Bipolar electrode focusing: A fast, efficient method
for simultaneous analyte preconcentration and
separation
15:45 – 16:05
L41 Archakov A.I., Bukharina N.S., Ivanov Yu. D., Munro A.
Moscow, Russia
Afm-Technology for visualization, counting, measuring the elasticity and activity of the cytochrome P450
monooxygenase proteins
L44 Bregje de Kort, Gerhardus de Jong, Govert Somsen
Utrecht, The Netherlands
16:05 – 16:25 Coffee break
Chair
Pharmaceutical industry
B. Buszewski, H. Stutz
16:25 – 16:50
L45 Wassim Nashabeh, Michael Davis, Stacey Ma San Francisco, USA Capillary electrophoresis in the biotechnology industry:
Advances and challenges through the last decade
Wavelength-resolved native protein fluorescence
detection in CE: towards integrated probing
of protein purity and conformation
Nanotechnologies
U. Tallarek, J. Dickerson
L49 John Perry, Kaimeng Zhou, Zachary Harms,
Stephen Jacobson
Bloomington, USA
Transport and sensing in asymmetric nanochannels
16:50 – 17:10
L46 Yelena Lyubarskaya
Cambridge, USA
L50 Ivanov Yu.D., Naumova O.V., Popov V.P, Archakov A.I.
Moscow, Russia
Application of micro separations in biopharmaceutical
development.
AFM and nanowire nanochips for protein and cell
detection.
19
MSB 2010 Program
Lecture hall:
Zenit
Nadir
17:10 – 17:30
L47 Jesper Østergaard, Ulrik Franzen, Lin Yang,
Eva Moeller Copenhagen, Denmark L51 Marc Mueller, Joselito Quirino, Pavel Nesterenko,
Gordon Wallace
Hobart, Australia
Characterization of drug-liposome interactions and
liposomal drug formulations using pre-incubation
affinity CE
Graphene nanosheets in capillary electrophoresis
17:30 – 17:50
L48 Meriem Mokaddem, Marie Lecoeur, Pierre Gareil,
Anne Varenne Paris, France On-line CIEF-ESI-MS in glycerol–water media with
a view to hydrophobic protein applications 17:50 – 18:10
19:00 – 22:00 L52 Jan Petr, Anne Varenne, Bruno Teste,
Stéphanie Descroix, Anne Faure, Jean Siaugue,
Pierre Gareil
Paris, France
Characterization of alpha-lactalbumin grafted
nanoparticles by capillary electrophoresis for the
development of a new immunodiagnostics method
GC x GC-TOF MS: challenging approach to human
metabolomic profiling associated with human disorders
Jitka Zrostlíková, Petr Wojtowič, Tomáš Adam,
Tomáš Kovalczuk, Jakub Schůrek
Prague and Olomouc, Czech Republic
Conference dinner - Restaurant Vikárka - Rudolf’s foundry
Vikářská 39, 119 00 Praha 1. Tel: 233 311 962 or 724 527 368; http://vikarka.cz/en/
Wednesday, March 24
20
Lecture hall:
Zenit
Nadir
Chair
Microfluidics S. Jovanovich, T. Laurell Glycomics
F. Regnier, Z. El Rassi
9:00 – 9:25
L53 Bingcheng Lin, Jianhua Qin
Dalian, China Simple, handheld and low cost: To promote
microfluidcs close to real application L57 Andras Guttman, Zoltan Szabo, Tomáš Rejtar, Barry L. Karger
Boston, USA
9:25 – 9:45
L54 Petr Smejkal, Markéta Ryvolová, František Foret,
Andras Guttman, Michael Breadmore, Rosanne Guijt, Fritz Bek, Mirek Macka Dublin, Ireland
Bioanalytical applications of microfluidic electrophoresis with LIF and LED fluorescence detection
L58 Crina Balog, Oleg Mayboroda, Manfred Wuhrer,
Cornelis Hokke, André Deelder, Paul Hensbergen
New advances in sample preparation methods for
CE analysis of n-linked glycans
Mass spectrometric identification of aberrantly
glycosylated human Apolipoprotein C-III peptides
in urine from Schistosoma mansoni-infected individuals
MSB 2010 Program
Lecture hall:
Zenit
Nadir
9:45 – 10:05
L55 Yoann Ladner, Karine Faure, Gérard Crétier
Paris, France Electrochromatography on COC microchips using
methacrylate monolith L59 Gerhild Zauner, Carolien Koeleman, André Deelder,
Manfred Wuhrer
10:05 – 10:25
L56 Jun Wakabayashi, Yo Tanaka, Kae Sato,
Kazuma Mawatari, Yuki Tanaka, Mats Nilsson,
Takehiko Kitamori
Tokyo, Japan
Development of highly efficient intracellular gene detection system on microchip
L60 Maurice Selman, Liam McDonnell,
Magnus Palmblad, Andre Deelder, Manfred Wuhrer
10:25 – 10:45
Chair
Nano LC-MS/MS methods for O-glycome analysis
High throughput IgG glycosylation profiling
by MALDI-FTICR-MS
Coffee break
Next generation DNA analysis
A. Barron, M. Macka
10:45 – 11:10
L61 Stevan Jovanovich, Iuliu Blaga, Paul Butler, David Eberhardt, Corey Garrigues, Ezra van Gelder,
Seth Stern
Pleasanton, USA
Development of a fully integrated library sample
preparation station for Next Generation Sequencing
11:10 – 11:30
L62 Nan Shi, Victor Ugaz College Station, USA Selecting nanoporous hydrogel morphologies to
harness entropic trapping for enhanced DNA
electrophoresis separations High resolution bioseparations
A. Guttman, C. Balog
L66 Kenji Sueyoshi, Takayuki Kawai, Kota Hashiba,
Ryuta Tanaka, Fumihiko Kitagawa, Koji Otsuka
Joanne Horn, Yuan Li, Bill Nielsen, Mary Trounstine
Kyoto, Japan
High performance separation and detection of
biogenic compounds by microscale electrophoresis
L67 L. Renee Ruhaak, Rene Hennig, Carolin Huhn,
Erdmann Rapp, Radboud Dolhain, Andre Deelder
Manfred Wuhrer
Multiplexed CGE-LIF for high-throughput
N-glycan analysis
11:30 – 11:50
L63 Meike Moschallski, Massimo Kubon,
Noor H Abdul Halim, Moritz Sandritter, Kai Fuchsberger, Britta Hagmeyer, Susanne Muench, Karl-Heinz Boven, Andreas Moeller, Rainer Mohrlok,
Martin Stelzle Reutlingen, Germany L68 Evert-Jan Sneekes, Bjorn Haan, Sebastiaan Eeltink,
Remco Swart
Amsterdam, The Netherlands
Microfluidic chip system integrating sample preparation
from physiological samples to DNA analysis
11:50 – 12:10
L64 Xiayan Wang, Shaorong Liu
Oklahoma, USA L69 Samuel Karenga, Ziad El Rassi
Oklahoma, USA
Nanocapillaries: enabling DNA and protein separations in gel-free solutions Investigation on mixed ligand monolithic stationary
phases for capillary electrochromatography
Ultra-high-performance nanoLC-MS/MS analysis of
complex proteomic samples
21
MSB 2010 Program
Lecture hall:
Zenit
12:10 – 12:30
L65 Marcus Gassman, Lucie Benesova,
Barbora Belsanova, Richard Chudoba, Bohuslav Gas, Marek Minarik
Prague, Czech Republic Development and application of new microchip CE DNA assays for point-of-care testing in routine
cancer diagnosis and therapy.
12:30 – 15:00
13:00 – 14:00
14:00 – 15:00
Nadir
L70 Stellan Hjertén
Uppsala, Sweden
Imagination, intuition and considerations in the form
of analogies - three important tools to solve
a scientific problem
Lunch break and poster sessions; Permanent MSB scientific board meeting
Agilent sponsored lunch presentation
Come chat with the Beckman Coulter team over coffee and desert
Plenary lectures:
15:10 – 15:45 PL7 Fred Regnier, West Lafayette, USA – Screening antibody and immunosorbent selectivity by two-dimensional liquid chromatography-mass spectrometry
15:45 – 16:25 PL8 Annelise Barron, Stanford, USA – Long, highly pure recombinant protein drag-tags for free-solution electro
phoretic DNA sequencing
Announcement of the MSB 2011 in San Diego
16:25 – 17:10 PL9 Alexander Tempels, Bea Reeuwijk, Gerard Rozing, Hitoshi Sagawa, Thomas Hankemeier, Waldbron, Germany – Opportunities for CE-MS in Metabolomics
Announcement of the MSB 2012 in Geneva
17:10 – 17:40 Poster Awards and Closing Remarks
Thursday, March 25
Conference Adjournment
Sightseeing tours
22
Lecture Abstracts
Lecture Abstracts
Sunday, March 21st, 2010 / ZENIT + NADIR 17:50
PL1:
New Approaches to Protein Structure Analysis for the Life Sciences and Biotechnology
Barry L. Karger
Barnett Institute, Northeastern University, USA
In both basic life science research and biotherapeutic drug development (biotechnology), there is a critical need for full protein structure characterization. Structure characterization includes, but is not limited to (i) full sequence analysis, (ii) co- and post-translational
modifications, (iii) protein folding and (iv) sublimit analysis. While a variety of analytical tools are available, the most important today
is separation (LC, CE) coupled to MS. In this talk, we will assess the current capabilities of LC/MS and CE/MS to achieve comprehensive
protein structure elucidation for both target molecules that are immunopurified from biological matrices and for recombinantly produced biopharmaceuticals. We will focus particularly on glycosylation analysis and disulfide linkages and describe the role of various
ion fragmentation processes (CID, ETD) in this structure elucidation. Besides examples of technology advancement, we will also present
specific applications where comprehensive structure analysis is important to solve significant problems. Finally, we will suggest future
directions as both separations and mass spectrometry continue to advance in their analytical power.
Sunday, March 21st, 2010 / ZENIT + NADIR 18:35
PL2:
Glycomic and Glycoproteomic Profiling in Disease Biomarker Discovery
Milos V. Novotny
Indiana University, USA
It has long been appreciated that precise and accurate analysis of glycoproteins can be an extremely difficult task. Starting with the
difficulties to isolate minute quantities of glycoconjugates from complex biological materials, the sheer complexity of carbohydrate
structures and their isomerism, the substitution patterns and microheterogeneities at substitution sites of a large, complex protein can
still challenge even the best analytical methods and instrumentation of the present days. Yet, even in the early HPCE symposia, some
interesting developments in analytical glycobiology were reported, although probably “lost” on the audiences which were more eager
to hear about DNA sequencing, peptide mapping, or the somewhat unexpected success of CE with small molecules. The separations
of some glycoprotein isoforms through CZE and IEF, as well as the first successful high-resolution CE-LIF runs of fluorescently- labeled
oligosaccharides, come to mind. The enormous success of biomolecular MS technologies of the last decade in different studies of the
human proteome has recently contributed to enhanced interest in glycosylation as the most ubiquitous and functionally and biomedically important modification of proteins. Since the most important cellular communication is being mediated through sugar-sugar
or sugar-lectin interactions, most human diseases and metabolic disorders involve glycoproteins, as do numerous host-pathogen
interactions. The modern biomedical science thus necessitates high-sensititivity glycomic and glycoproteomic measurements, which,
in turn, are likely to be supplemented by correspondingly miniaturized and selective sample enrichment steps and fast separations.
While drawing heavily from today’s proteomic methodologies, the field of analytical glycobiology has its distinct paradigms and unique
glycomic and glycoproteomic techniques. Recent advances in glycomic profiling and the follow-up glycoproteomic investigations will be
highlighted in this lecture, concentrating on our research and clinical collaborations on breast and pancreatic cancer.
Monday, March 22nd, 2010 / ZENIT + NADIR 8:30
PL3:
Plucking, Pillaging And Plundering Proteomes With Combinatorial Peptide Ligand Libraries
Pier G. Righetti, Egisto Boschetti
Politecnico di Milano, Italy
23
Lecture Abstracts
In any proteome, a few proteins dominate the landscape and obliterate the signal of the rare ones. A host of pre-fractionation techniques has been described, but all of them are besieged by problems, in that they are based on a "depletion principle", often via immuno-subtraction (e.g., in sera, by using a set of 6 to 20 antibodies against the most abundant species). Parasitic co-depletion removes
thousands of low-abundance proteins, nullifying any attempt at bringing to the limelight the “unseen proteome”. A revolutionary
approach consists in the "ProteoMiner Technology", a method enabling the capture of all species present in a proteome, but at much
reduced protein concentration differences. This consists on a combinatorial library of hexapeptide ligands coupled to spherical porous
beads. Such a vastly heterogeneous population of baits means that an appropriate volume of beads could contain a partner able to
interact with all proteins present in a complex proteome. When these beads are contacted with proteomes of widely differing protein
composition and relative abundances, they are able to “normalize” the protein population, by sharply reducing the level of the most
abundant components while simultaneously enhancing the concentration of the most dilute species. Examples are given of analysis of
human urine, sera and cerebrospinal fluid samples. In a red blood cell (RBC) lysate, where haemoglobin alone constitutes 98% of the
total proteins, more than 1500 unique gene products have been found to constitute the remaining 2% proteome. By using this list of
proteins, we have been able to decode a rare RBC disease, congenital dyserythropoietic anaemia, in which the defective gene has been
identified as the one coding for the SEC23B protein, located in chromosome 20. Additionally, these beads can be used to remove host
cell proteins from purified recombinant proteins or protein purified from natural sources that are intended for human consumption, a
matter of serious concern in the Bio Pharm. industry. These proteins typically reach purities of the order of 98%: higher purities often
being prohibitively expensive. Yet, if incubated with "ProteoMiner beads", these last impurities can be effectively removed at a small
cost and with minute losses of main, valuable product.
Righetti P.G.; Boschetti E.; Lomas L.; Citterio A. Proteomics 6 (2006) 3980-3992.
Boschetti E., Lomas L., Righetti P.G. J. Chromatogr. A 1153 (2007) 277-290.
Antonioli P.; Fortis F.; Guerrier L.; Rinalducci S.; Zolla L.; Righetti P.G.; Boschetti E. Proteomics 7 (2007) 1624-1633.
Roux-Dalvai F., Gonzalez de Peredo A., Simó C., Guerrier L., Bouyssié D., Zanella A., Citterio A., Burlet-Schiltz O., Boschetti E., Righetti P.G., Monsarrat B. Mol. Cell.
Proteomics 7 (2008) 2254-2269.
Righetti PG, Boschetti E, Zanella A, Fasoli E, Citterio A. J Chromatogr. A 1217 (2010) 893-900.
Monday, March 22nd, 2010 / ZENIT + NADIR 9:10
PL4:
Conquering The Metabolome: From Extraction To Bioinformatics
Peter J. Oefner
University of Regensburg, Institute of Functional Genomics, Regensburg, Germany
The heavy emphasis on identifying genes as the underlying cause of disease and targets of personalized medicine in current biomedical research has created the impression that we are a mere product of our genes. However, this is far from true; genes almost never
act independently of environmental influences and, as a matter of fact, most common physiological (e.g., height, metabolism) and
pathological (e.g., cancer, cardiovascular disease) phenotypes are rather determined by non-genetic mechanisms such as nutrition,
physical activity and host-microbe interactions. The impact of environmental influences on molecular body composition is particularly
evident in the levels of metabolites in physiological fluids, with genes contributing at most 30 % to the observed variability. Metabolites
may influence body function in a variety of ways: they may damage DNA directly giving rise to somatic mutations, they may affect
DNA methylation and thus gene expression, or they may affect protein structure and function via formation of adducts, respectively
via activation or inhibition of enzyme activity. However, due to great differences in physicochemical properties of metabolites, there
is not a single technique that lends itself to capturing the metabolome in its entirety in a single analysis. NMR spectroscopy as well
as multidimensional liquid and gas chromatographic methods hyphenated to various mass analyzers have become essential tools in
metabolite profiling and fingerprinting, but tremendous analytical and informatics challenges remain with regard to the identification
and quantitation of metabolites, foremost but not exclusively among them being the development of (i) methods for the synthesis of
stable isotope-labeled internal standards, (ii) software for automated alignment, transformation, extraction and integration of NMR
and mass spectral features, and (iii) statistical algorithms such as support vector machines and cross-validation techniques. Ongoing
research efforts employing the aforementioned analytical and informatics techniques include the discovery of biomarkers for the early
diagnosis and prognostication of autosomal dominant polycystic kidney disease, kidney transplant rejection, and metabolic stress in
lactating dairy cows, as well as the study of the central carbon metabolism and methionine, polyamine, and tryptophan pathways in
cancer and graft-versus-host disease.
24
Lecture Abstracts
Tuesday, March 23rd, 2010 / ZENIT + NADIR 8:30
PL5:
What Has Cuneiform Writing Or Landing On The Moon To Do With Electrophoresis On Chip?
Andreas Manz
KIST Europe, Saarbrucken
For the last 20 years my research was focusing on scaling laws, chemical analysis and clinical diagnostics. Miniaturization was driving electrical
circuits to integrated electronic devices, mainly justified by commercial aspects, by benefits of shorter connections and higher integration
density. A similar motivation drives the "lab on a chip" for miniaturizing chemistry and biology. Molecular diffusion, heat dissipation and time
scale is intimately connected and, in consequence, a down-scale of a well characterized experiment by 10× will return identical information
100× faster.
For this purpose, chip technology was adopted to chemistry and the life sciences. Some of it is soft matter (polymers) and some is traditional
glass or silicon technology. The fabrication needs clean rooms and photolithography. Alternatively, self-assembly of soft matter could get similar manufacturing results. Embryology gives many excellent examples on nature's choice. However, engineering in this field is still in its infancy.
Silicon technology (monocrystalline) has another interesting feature: it preserves its structure for a very long time. That was the starting point
for the Human Document Project: to store one document about mankind for a million years.
Tuesday, March 23rd, 2010 / ZENIT + NADIR 9:10
PL6:
Linking Acoustophoretic Cell And Particle Processing To MALDI Mass Spectrometry
Thomas Laurell
Lund University, Sweden
Acoustic standing wave technology offers a label free and non-contact mode of cell and particle manipulation in microfluidic systems. The
most common mode of acoustic standing wave operation in microfluidic channels is at the l/2 resonance. In this case the excitation frequency
is matched to the channel width to support a half wavelength resonance. Under these conditions particles in aqueous media with higher density than water will experience an acoustic force that drives them to an equilibrium position in the centre of the channel. The low mechanical
strain that is imposed on the cells in this process thus opens the route to a wide range of clinical applications. Recent developments describe
whole blood plasmapheresis based on acoustophoresis, which subsequently has been coupled to protein micro arrays for PSA (prostate specific antigen) analysis in undiluted plasma. Since the acoustic primary radiation force is dependent of the particle size this enables the design
of systems that can separate different particles sizes in a complex mixture, so called free flow acoustophoresis (FFA). By providing the FFA
separation channel with multiple outlet channels, defined particle sizes can be collected at each outlet. Continuous flow fractionation of mixed
particles suspensions or multiplex cellular speciation has been obtained utilizing FFA where a key target was blood component fractionation.
Separation of mixed particle suspensions and erythrocytes, leukocytes and platelets in a buffy-coat will be demonstrated. An optional modality
is to perform FFA in a binary mode of operation where cells are separated based on their acoustic contrast factor and where the composition
of the carrier buffer media is tuned such that two normally non-separable species can be separated. The density media is selected such that
particle type A displays a positive contrast factor and the particle type B a negative contrast factor. Thereby the two particle types will migrate
to the pressure node (in the centre) and the pressure antinode (along the side walls) and can thus be collected from different outlets of the
chip. This mode of operation can be regarded as an analogue to conventional density media centrifugation. As a further extension of FFA, an
acoustophoretically driven buffer exchange modality has been developed, utilizing affinity specific microbeads (affinity acoustophoresis). The
ability to extract targeted species from complex mixtures using affinity acoustophoresis will be demonstrated, where affinity acoustophoresis
has been used in decomplexing proteomic samples, prior to MALDI-MS readout. The acoustic standing wave force can also be utilized to trap
cells in a non contact mode in a channel enabling advanced cell stimulation and readout protocols to be developed. A first proof of concept
will be demonstrated in MS readout of a cell membrane penetrating peptide that is exposed to an acoustically trapped erythrocyte population.
Wednesday, March 24th, 2010 / ZENIT + NADIR 15:10
PL7:
Screening Antibody and Immunosorbent Selectivity by Two-Dimensional Liquid Chromatography-Mass Spectrometry.
Wonryeon Cho, Fred E. Regnier*
25
Lecture Abstracts
Department of Chemistry, Purdue University, West Lafayette, IN 47907 U.S.A
Selectivity of both peptide and glycan targeting antibodies was examined by two dimensional liquid chromatography and mass spectrometry. Proteins selected from biological extracts immunospecifically in a first chromatography dimension using antibodies immobilized
by either covalent coupling or adsorption to protein G were desorbed with a denaturing mobile phase and transferred to a 1.5 m nonporous (NP) particle reversed phase chromatography (NP-RPC) column in a second dimension. Protein peak capacity of the NP-RPC
column was approximately 50. Peaks collected from the RPC column were tryptic digested and the peptide fragments identified by
MALDI-MS. The objective of this analytical strategy was to discriminate between protein antigens and non-antigens through identification of their peptides, leading to an evaluation of the selectivity of antibodies and immunosorbents. Quantification of the relative
amount of antigen and non-antigen species captured by immunosorbents was achieved by absorbance, along with the likely capture
mechanism. A limitation of the approach was in discriminating between isoforms of an antigen in which neither the antibody nor the
LC-MS system targeted the differentiating feature in the isoforms.
PL8:
Long, Highly Pure Recombinant Protein Drag-Tags For Free-Solution Electrophoretic DNA Sequencing
Annelise E. Barron
Stanford University, USA
Advances in next-generation sequencing have reduced the cost and time associated with large-scale genomic studies. But the need
remains for targeted, highly accurate sequencing of limited medical samples, for which rapid electrophoresis-based methods are ideal;
e.g., for donor-recipient matching process for organ transplants. Translating CE separations onto microfluidic devices for these applications offers many advantages (faster reads, smaller sample volumes, ease of integration into lab-on-a-chip devices); however, enthusiasm
is dampened by difficulties associated with loading viscous polymer networks into microdevices. Eliminating the need for a polymer
matrix is challenging; DNA mobility in free-solution electrophoresis is size-independent due to the linear scaling of both charge and
hydrodynamic friction with chain length. Free-Solution Conjugate Electrophoresis (FSCE) enables size-based ssDNA and dsDNA separations for DNA sequencing with no separation matrix, through a specific conjugation of exactly identical mobility modifiers to each DNA
molecule in a Sanger mix. Monodisperse, highly pure polyamide drag-tags are chemically conjugated to the 5’ termini of ssDNA oligos or
sequencing primers. An ideal drag-tag, in addition to being totally monodisperse, can be uniquely attached to DNA end-on and is large
enough to provide relatively long and accurate reads by free-solution capillary or chip electrophoresis. Genetically engineered, recombinant proteins were developed for this use. With a 267-amino acid long protein, we are able to distinguish sequence out to 250 bases
of DNA in four colors by free-solution CE, representing a 40% increase in read length over previously published results*, comparable to
the read lengths of the best next-gen sequencing technologies. A drag-tag with 516 amino acids has recently been produced that is
almost completely monodisperse, and sequencing with this drag-tag is underway. The transition from CE to microchip electrophoresis
is expected to go smoothly; these longer drag-tags hold forth the promise of even longer read lengths.
* R.J. Meagher, J-I. Won, J.A. Coyne, J. Lin, A.E. Barron. “Sequencing of DNA by free-solution capillary electrophoresis using a genetically engineered protein polymer
drag-tag.” Analytical Chemistry, 2008, 80(8): 2842-2848.
PL9:
Opportunities for CE-MS in Metabolomics
Alexander Tempels1, Bea Reeuwijk1, Gerard Rozing*1, Thomas Hankemeier2, Hitoshi Sagawa3
1 Leiden-Amsterdam Center for Drug Research, Division of Analytical Biosciences, NL 2333 Leiden, Netherlands
2 Agilent Technologies, 76337 Waldbronn, Germany
3 Human Metabolome Technologies Inc. 997-0052 Tsuruoka, Yamagata ,Japan
Like many other main separation techniques, capillary electrophoresis (CE) had to go through a long incubation period prior to becoming a standard tool. After initial enthusiasm about its abilities in the early nineties of the previous century, practical reality of
application of CE brought the business and technology almost to a stop. Persistence by few instrument manufacturers, engagement in
a number of academic research centers and standard operation in industrial (mainly pharmaceutical analysis) laboratories has formed
26
Lecture Abstracts
the basis of a slow rejuvenation of and confidence in CE- technology.
New impetus and motivation for the revival of CE as an analytical separation technique are:
- Development of biopharmaceuticals and more recently biosimilars
- Advent of metabolomics in particular in combination with MS
In both fields CE appears to be a preferred tool, demanded by the properties of the molecules under investigation.
In this presentation, the authors present recent results of a joint evaluation and validation of a dedicated CE-qTOFMS method for
metabolomics. In particular a rigorous evaluation of a kit designed for analysis of small, charged metabolites will be presented. The
performance of the method was investigated with academic samples and urine spiked with several (labeled) endogenous metabolites.
Dedicated software for handling of CE data was used for peak alignment and peak integration. The validation of the CE-qTOFMS profiling
method was set up to assess its suitability for clinical studies involving urine samples.
Monday, March 22nd, 2010 / ZENIT 10:00 – 10:25
L1:
Proteome and Phosphoproteome Revealed Unique Features of Mouse Embryonic Stem Cells
Rong Zeng*, Qing-Run Li, Xiao-Bin Xing, Tao-Tao Chen, Chia-Hui Shieh
Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China, 320 Yueyang Road, 200031, China
Embryonic stem (ES) cells are pluripotent and capable of self-renewal and, therefore hold promise for cell-based regenerative medicine.
For a thorough understanding of the molecular mechanisms underlying ES cell pluripotency and differentiation, studies focused on a
more comprehensive omics view of human or mouse ES cells ranging from their transcriptome to proteome. The cells were harvested
and subcellular fractionation was applied to reduce the complexity. The two resulting fractions (cytosol and nuclear) were digested
and separated on online yin-yang multiple dimensional liquid chromatography (MDLC), followed by high-resolution LTQ-Orbitrap mass
spectrometry identification. For antibody-based purification, the cell lysate was enriched for phosphorylated serine/threonine proteins
(anti-pS/T enrichment). Meanwhile, TiO2 was also used to complementary enrichment of phosphopeptides. Extensive bioinformatic
analysis was applied to integrated transcriptome, proteome and phosphoproteome, aiming to reveal the unique feature of ES cells in different levels. Based on multiplexed strategies and high-coverage identification of peptide mixtures, we detected 4581 proteins and high
confident 3970 distinct phosphosites from 1642 phosphoproteins. Notably, some prominent phosphorylated stem cell markers with
novel phosphosites were identified by mass spectrometry for the first time. Function annotation to the phosphoproteins showed that
these proteins are acting in various manners as regulation factors in DNA and RNA related biological pathways. We also investigated the
expression correlation between mRNA and protein phosphorylation events in mES. Furthermore, we constructed the database containing transcriptome, proteome and phosphoproteome of stem cell to facilitate the understanding of the mechanism of its pluripotency.
L2:
Proteomic Analysis of Minimal Sample Amounts
Tomas Rejtar*, Dipak Thakur, Dongdong Wang, Sangwon Cha, Barry L. Karger
Northeastern University, Boston, USA
Comprehensive proteomic analysis is an excellent tool to aid in understanding of various biological processes. So far the majority of proteomic
applications relied on analysis of relatively large amounts of material such as analysis of whole tissue sections. However, due to cellular heterogeneity such analyses are only able to estimate average levels of proteins in various cell types. LCM has become a method of choice for collection of
specific cell populations directly from tissues but only limited number of cells (<20,000) is typically available for analysis. Advances in mass spectrometry and introduction of chromatographic columns operating at low nL/min flow rate have allowed for significant enhancement of proteomic
analysis. We have recently introduced 10 µm i.d. open tubular chromatographic columns operating at 20 nL/min, which are suitable for analysis
of complex proteomic samples. We have implemented both 1D and 2D LC strategy applicable to samples of various complexities. In addition, we
have also developed sample preparation strategies that allow for reproducible sample handling using low micrograms of the total material. Sample
preparation methods based on SDS-PAGE analysis followed by in-gel digestion but also in-solution strategies will be shown. Applications of the
developed platform to various types of human and mouse tissues will be discussed. In addition, we have applied this technology to comprehensive
characterization of immunopurified samples and examples of detailed characterization of glycoproteins will be given. 27
Lecture Abstracts
L3:
Systems Biology Of HL-60 Cell Line Induced By ATRA Differentiation.
Victor Zgoda*, Olga Tikhonova, Leonid Kurbatov, Artur Kopylov, Natalia Moskalyova, Ivan Cheglakov and Alexander Archakov V.N.Orekhovich Institute of Biomedical Chemistry RAMS, Pogodinskaya str. 10, Moscow, 119121, Russia
HL60 promyelocytic leukemia cells have a potential to differentiate into granulocytes under inducible effect of all-trans-retinoid acid
(ATRA). In this study we applied a systems biology (metabolomics, proteomics and transcriptomics) approach to study a molecular
mechanism of HL60 differentiation induced by ATRA. Transcriptome analysis of mRNA isolated from HL60 at different time points after
differentiation initiation was performed using a microarray technology. Totally, at least 11202 genes were expressed under conditions
of the cell line culturing among them 134, 207, 364, 393 и 1197 genes were identified as being differentially expressed after first 30
min, 1h, 3h 24 and 96h samples comparing to control, correspondently. Experiments at proteome level were performed using 3D LCMS/MS. This method includes separation of whole cell lysate by macroporous RP-C18 chromatography onto 5 protein fractions with
following “shotgun” proteomics analysis of trypsin digested mRP fractions. As a result over 3200 proteins were identified in the HL60
cell line. Quantitative, label free analysis of obtained proteomics data allowed to reveal the significant increase of expression levels for
30 proteins. Finally, metabolomic analysis of the same samples was performed by the method of LC-MS profiling using high resolution
Q-TOF mass spectrometer. After computational analysis and normalization of the chromatogram the statistically significant changes
of 16th compounds were detected from approximately 500 analysed ones. Combination of high throughput techniques such as those
reported herein can be useful for understanding of the molecular mechanism and downstream signaling of HL60 cells differentiation.
Monday, March 22nd, 2010 / NADIR 10:00 – 10:25
L4:
Capillary Electrochromatography-Mass Spectrometry For The Separation Of Compounds Of Pharmaceutical Interest And Tryptic
Digest Of Proteins: Comparison With Nano-LC-MS And CE-MS
Salvatore Fanali*, Zeineb Aturki, Giovanni D'Orazio, Anna Rocco
Institute of Chemical Methodologies, CNR, Montelibretti - Italy
Capillary Electrochromatography (CEC) is recognized as a powerful technique, useful for the separation and analysis of both charged
and uncharged compounds. High efficiency, high selectivity, short analysis time and low consumption of reagents are some of the
recognized characteristics of this tool. CEC has been successfully used in different application fields such as agrochemical, biomedical,
forensic, pharmaceutical, environmental etc. Although on-line UV detectors have been widely used, recently hyphenation of CEC with
mass spectrometry has been demonstrated providing improvement of sensitivity and possibility for analytes’ characterization.
Although the above mentioned advantages, CEC presents some drawbacks mainly due to the use of packed capillaries that need frits
to retain the stationary phase that could cause bubble formation. This problem is usually resolved applying external pressure to both
electrode compartments. However this set-up is not congruent with MS. As an alternative solution, the use of pressurized CEC (pCEC)
can be convenient to prevent bubble formation without disturbing the MS coupling. In this communication we demonstrate the possibility to perform CEC-MS with packed column with and without external pressure assistance. The system set-up was realized coupling
the CEC capillary with an ESI-ion trap through a liquid-junction interface. A hydrostatic pressure (about 35 mbar) was applied to the
liquid-junction containing an appropriate liquid mixture allowing optimum analytes ionization. CEC-MS was used for the separation
of some compounds of pharmaceutical interest such as basic drugs and non-steroidal anti-inflammatory drugs. In addition, a tryptic
digest mixture of cytochrome C was analyzed and detected by MS. The method was compared with nano-liquid chromatography
employing both a laboratory made and commercial instrumentation.
L5:
Integrated strategies for drug discovery: capillary electrophoresis, mass spectometry and in silico studies
Luca Regazzoni1, Raffaella Colombo2, Laura Bertoletti2, Giulio Vistoli1, Giancarlo Aldini1, Ersilia De Lorenzi*2
1 Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Via Mangiagalli 25, University of Milan, Italy
28
Lecture Abstracts
2 Dipartimento di Chimica Farmaceutica, Viale Taramelli 12, University of Pavia, Italy
Human ß2-microglobulin (ß2-m) is a small protein responsible for dialysis-related amyloidosis, which represents a severe complication of
long term haemodialysis. The stabilization of the native form of the protein through the binding to a small molecule is one of the considered approaches aimed at preventing or even reversing the protein conformational changes that lead to amyloid deposition. Finding a strong
ligand has always been particularly difficult, as ß2-m lacks a specific binding site. An affinity screening previously carried out by affinity
CE (ACE) on a chemical library composed of 200 sulfonated compounds1 has been cross-checked by a newly developed affinity-based
method, using high resolution LTQ-Orbitrap Hybrid Mass Spectrometry. This gave access to a fast feedback of the ACE results and allowed
to fish out interesting hits previously discarded. Advantages and drawbacks in terms of screening throughput, sensitivity, specificity, reagent consumption and accurate measurement of affinity constant between ACE and MS screening have been highlightened. The results
obtained offered the opportunity of carrying out classic QSAR analyses, to unveil the molecular properties which play a determinant role
in the binding, as well as docking simulations, to reveal the possible binding modes for the examined derivatives and their role in ß2-m
polymerization. QSAR descriptors were combined with docking scores, deriving a statistically significant correlation. The predictive ability
of the equation has been used to retrieve further interesting hits, within a commercial database composed of 800000 compounds. Finally,
a combination of affinity capillary electrophoresis, protein refolding experiments by CE in the presence of the hits discovered and in vitro
Thioflavin T fibrillogenesis tests produced information on the bioactivity of the pool of hits discovered. Interestingly, the results obtained
are consistent with the in silico results, as active compounds best accommodate within the monomer-monomer interface of a modelled
protein dimer, which is considered the initial event of the oligomerization that preceds amyloid fibril deposition.
1 C.Carazzone, R.Colombo, M.Quaglia, P.Mangione, S.Raimondi, S.Giorgetti, G.Caccialanza, V. Bellotti, E.De Lorenzi. Electrophoresis 29 (2008) 1502-1510
L6:
The Selective Determination Of Sulfates, Sulfonates And Phosphates In Urine By Capillary Electrophoresis- Mass Spectrometry
Svenja-Catharina Bunz*, Christian Neusuess
Aalen University, Department of Chemistry, Aalen, Germany
Abstract Metabolite identification and metabolite profiling are of major importance in the pharmaceutical and clinical context. However,
highly polar and ionic substances are rarely included since analytical tools are missing. In this presentation the determination of urinary
sulfates, sulfonates, phosphates and other anions of strong acids will be presented. The method comprises a capillary electrophoresis separation using an acidic background electrolyte (pH ≤ 2) and anodic detection by mass spectrometry via negative electrospray ionization
(ESI). In this way only sulfates and sulfonates are detected in the first part of the electropherogram, followed by phosphates and potentially
highly acidic carboxylates. The selectivity for sulfur-containing species is proved using extracted ion electropherograms based on certain
isotopic ratios. > Ethylsulfate (EtS) was chosen as model compound for method development, thus, the applicability of CE-MS for determination of this alcohol consumption marker is shown. A SPE extraction method of ethylsulfate and other organic anions was used to ensure
good migration time repeatability. Several additional compounds can be identified based on the accurate mass determined by the TOF
MS in conjunction with databases. However, numerous detected compounds have not been reported in urinary metabolite databases so
far. Thus, it is demonstrated that the presented method is complementary to existing methods for metabolite characterization in urine.
L7:
RP-RPLC Multidimensional Separation with High Orthogonality for Large-scale Phosphoproteome Analysis
Chunxia Song, Mingliang Ye, Xinning Jiang, Rui Chen, Hanfa Zou*
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy
of Sciences
Protein phosphorylation regulates a series of important biological processes in eukaryotes. However, the phosphorylation sites found
up to now are far below than that actual existence in proteins due to the extreme complexity of proteome sample. Here a new RP-RPLC
2D separation approach was developed for phosphopeptide separation. By this approach, a large number of fractions were collected
from the first dimensional RPLC separation at high pH. And then these fractions were pooled every two fractions with equal time
29
Lecture Abstracts
interval, one from early eluted section and the other one from the later eluted section. The pooled fractions were finally submitted
to RPLC-MS/MS analysis at low pH. It was found the resulting 2D separation was highly orthogonal and yielded more than 30% phosphopeptide identifications over the conventional RP-RPLC approach. This approach took advantage of the high resolution of RPLC and
new fractionation format to improve the orthogonality of two dimensional separations, and provided an highly efficient separation
of phosphopeptides for large-scale phosphorylation analysis. The developed multidimensional separation approach was integrated to
phosphopeptide identification with high-resolution mass spectrometry (LTQ Orbitrap) for large-scale phosphoproteome analysis of
human liver, which resulted in the identification of about 10, 000 phosphorylation sites in human liver by controlling FDR less than 1%.
Though the experiments are not finished yet, it is already become the largest dataset for human liver phosphoproteome.
L8:
Affinity Studies Of ß2-Glycoprotein I Using Capillary Electrophoresis
Maria E. Bohlin*1, Lars G. Blomberg1, Niels H. H. Heegaard2
1 Department of Chemistry and Biomedical Sciences, Karlstad University, Karlstad, Sweden
2 Department of Autoimmunology, Statens Serum Institut, Copenhagen S, Denmark
ß2-glycoprotein I (ß2gpI), also known as apolipoprotein H, is a plasma protein which is involved in the blood coagulation cascade. It binds
negatively charged substances such as heparin, DNA, and anionic phospholipids. A number of functions of ß2gpI have been proposed,
however, the precise function is still not entirely known. Circulating autoantibodies against ß2gpI are associated with increased risk of
thrombotic events, such as thrombosis and reoccurring fetal loss. It is therefore of interest to functionally characterize ß2gpI including
the influence of anti-ß2gpI autoantibodies on the ligand binding behavior of the protein. The characterization of interactions between
biological molecules may be accomplished by capillary electrophoresis under non-denaturing conditions, without the need for immobilization. To avoid charge dependent analyte adsorption to the negative charges of the capillary wall we found the pH hysteresis effect of
silica very useful. An acidic pretreatment of the capillary made it possible to perform a subsequent analysis at neutral pH. We were able
to perform binding studies between ß2gpI and heparin and monosaccharides at different ionic strengths and temperatures in a simple
way. We could also study the effect of mildly denaturing conditions on the binding to the different ligands simply by adding sodium
dodecyl sulfate (SDS), urea and ACN to the background electrolyte. The approach is simple, fast and automatic. The ionic strength,
temperature and other parameters such as denaturing agents could easily be changed to characterize the binding between ß2gpI and
different ligands. Monday, March 22nd, 2010 / ZENIT 12:15 – 12:35
L9:
Simultaneous Analysis Of Potential Biomarkers Of Alzheimer Disease, The Amyloid Peptide Quintet, On Microsystem
Romain R. Verpillot*1, Esther E. Aparicio1, Antoine A. Pallandre1, Valérie V. Nicolas2, Markus M. Otto3, Myriam M. Taverna1
1 Paris-Sud, UMR8612, LPNSS. Faculté de Pharmacie, 92296 Châtenay-Malabry, FRANCE
2 Univ Paris-Sud, IFR 141, microscopy plateform. Faculté de Pharmacie, 92296 Châtenay-Malabry, FRANCE
3 University of Ulm, Department of Neurology, Steinhövelstrasse 1, 89075 Ulm, Germany
Alzheimer’s disease (AD) affects 24 million people today and the prevalence doubles every 5.1 years. The amyloid pathology starts 20-30
years before the first clinical symptoms appear. The aim of this work is to develop a non invasive strategy for the early diagnostic of AD.
Until now, reduction of Aß peptide 1-42 in CSF is considered as a major biomarker of this disease. In particular, the ratio between the Aß
peptides 1-40 and 1-42 in CSF seems to help in the differential diagnosis to other neurodegenerative diseases. The measurement is typically performed using ELISA tests. However, conflicting results may be obtained and in several cases, it is not possible to discriminate AD
from other dementia diseases using currently available assays on routine basis1. These standard Amyloid ß have been recently separated
by SDS-PAGE but this method cannot be considered as fully quantitative2. We report here a CE method on microchip for the separation
and quantitation of five amyloid peptides (Aß 1-42, 1-40, 1-39, 1-38, and 1-37) from biological fluids. This method intends to improve
not only the diagnostic specificity by a multibiomarkers approach but also its precocity by attaining sensitivities compatible with their
measurement at an early stage of the AD. In a previous work, we reported a separation method of these 5 structurally-close biomarkers
by capillary electrophoresis (CE) and UV detection3. Then to enhance the sensitivity, we focused our efforts on a covalent labelling method
by testeing several fluorophores4. Recently, we have adapted these CE conditions to simple and commercial glass microchips. A polymer
30
Lecture Abstracts
coating was dynamically deposited onto the wall of the microchannel to control EOF mobility and to limit peptide adsorption. From this
methodology, we achieved a high resolution for this critical separation. Moreover, we obtained reproducible analyses, which represent
the first step toward a reliable diagnosis. By adapting the recovery method, often employed to quantify protein adsorption by CZE, we
have been able to demonstrate high recoveries of the microchip method toward these peptides. Finally, the method was applied to measurements of these biomarkers in cerebrospinal fluid from patients suffering from AD. In this presentation, we will point out the way we
achieved a high resolution of homologous and structurally very close amyloid peptides into one simple and commercial microchip. This
CE-LIF method can afford very satisfactory LODs allowing to simultaneously quantify 5 potential biomarkers in biological samples. These
results open promising outlook to set a new sensitive and more discriminating diagnostic tool of AD.
This work was supported by an European grant NeuroTAS (STREP-FP6).
1 Wiltfang J. et al Neurochem. 101, 165-173 (2007)
2 Piotr Lewczuk et al Electrophoresis 25, 3336-3343 (2004)
3 Verpillot et al J. chrom. A 1214, 157–164 (2008)
4 Verpillot et al in preparation (2010)
L10:
Capillary Zone Electrophoresis In Strongly Acidic/Basic Classical And (Quasi)Isoelectric Background Electrolytes Applied To Separation And Characterization Of Biopeptides
Václav Kašička*1, Dušan Koval1, Veronika Šolínová1, Jean-Marc Busnel2, Martine Poitevin2, Gabriel Peltre2
1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo 2, 166 10 Praha 6, Czech Republic
2 Ecole Superieure de Physique et Chimie Industrielles (ESPCI), 10 Rue Vauquelin, 75231 Paris, France
Capillary zone electrophoresis (CZE) in strongly acidic/basic classical buffer-based background electrolytes (BGEs) or in isoelectric BGEs
containing a single amphoteric compound, and carrier ampholytes-based capillary electrophoresis (CABCE)1, utilizing narrow pH fractions
of carrier ampholytes (CAs) as quasi-isoelectric BGEs, will be presented as suitable methods for separation and physicochemical characterization of biologically active peptides, e.g. insect oostatic peptides, human and salmon gonadotropin-releasing hormones (GnRHs) and
phosphinic pseudopeptides, i.e. peptide isosteres with one peptide bond substituted by phosphinic acid group (-PO(OH)-CH2-). The acidity
(ionization) constants (pKa) of ionogenic groups of these peptides cover a broad range; pKa values of phosphinic acid group and C-terminal carboxyl group fall within the range 1.5 3.0, whereas pKa of amino group of lysine or phenol group of tyrosine may be higher than
10. Hence, for controlling the separation selectivity, for determination of ionic mobilities of fully ionized peptides and for measurement of
pH dependence of their effective mobilities at extreme pH values, it is necessary to perform their CZE analyses in strongly acidic and/or
basic BGEs. The problems resulting from application of these strongly acidic/basic BGEs (high ionic strength and conductivity of the BGE,
high Joule heating and temperature increase inside the capillary and very small electroosmotic flow in the strongly acidic BGEs) can be
overcome by careful selection of the separation conditions and employing appropriate corrections. From the measured pH dependence of
effective mobilities of the above peptides, the pKa values of their ionogenic groups and the actual mobilities of their ionic forms could be
determined by non-linear regression analysis2. Unlike CZE in classical high-conductive BGEs, CZE in isoelectric BGEs (iminodiacetic acid,
aspartic acid)3 and CABCE in quasi-isoelectric BGEs (prepared by isoelectric focusing fractionation of Servalyte pH 4–9 CAs to 0.2 pH unit
cuts) utilize the inherent advantage of isoelectric BGEs, low electric conductivity, and provide fast separation of peptides at high electric
field strength. With identical selectivity, the separation efficiency of CABCE proved to be as good as that of classical CZE for the insect
oostatic peptides and better for diastereomers of phosphinic pseudopeptides and GnRHs.
The work was supported by the GACR, grants no. 203/08/1428 and 203/09/0675, and by the Research Project AV0Z40550506 of the
ASCR.
1 Busnel J.M., Kilár F., Kašička V., Descroix S., Hennion M.C., Peltre G.: J. Chromatogr. A 2005, 1087, 183
2 Koval D., Kašička V., Jiráček J., Collinsová M.: Electrophoresis 2006, 27, 4648
3 Šolínová V., Kašička V., Sázelová P., Barth T., Mikšík I.: J. Chromatogr. A 2007, 1155, 146
4 Koval D., Busnel J.M., Hlaváček J., Jiráček J., Kašička V., Peltre G.: Electrophoresis 2008, 29 3759
31
Lecture Abstracts
L11:
Enantioselective CE Analysis Of Human Ketamine Metabolism In Vitro
Simone Portmann1, Hiu Ying Kwan1, Regula Theurillat1, Andrea Schmitz2, Meike Mevissen2, Wolfgang Thormann*1
1 Department of Clinical Pharmacology and Visceral Research, University of Bern, Bern, Switzerland
2 Division of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Bern, Bern, Switzerland
Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary and
human medicine. Nevertheless, metabolism and pharmacokinetics of ketamine have not been characterized sufficiently. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations has been developed and applied to the determination of the
human ketamine metabolism in vitro. It is based upon analyte liquid/liquid extraction at alkaline pH and the use of a CE buffer composed
of tris/phosphate at pH 2.5 containing 10 mg/ml of multiple isomer sulfated beta-cyclodextrin as chiral selector, an approach that permits
the simultaneous anionic analysis of the stereoisomers of ketamine, norketamine, dehydronorketamine and hydroxylated norketamine
metabolites with hydroxylation at the cyclohexanone ring1, 2. The assay was successfully used to determine (i) the single human CYP450
enzymes catalizing the metabolism of ketamine and norketamine in vitro, (ii) the metabolites formed during incubation of ketamine or norketamine in presence of recombinant human CYP450 enzymes (SUPERSOMES) and human liver microsomes, (iii) in vitro pharmacokinetics
of ketamine N-demethylation in presence of single human CYP450 enzymes and human liver microsomes and (iv) the stereoselectivity of
the various metabolic reactions. Six CYP450 enzymes (3A4, 2C19, 2B6, 2A6, 2D6 and 2C9) were identified to catalyze ketamine N-demethylation among which three enzymes (3A4, 2B6 and 2A6) generated norketamine stereoselectively. Two enzymes (2B6 and 2A6) were
found to be associated with the formation of further metabolites (hydroxylated norketamine metabolites and dehydronorketamine). The
results suggest that the metabolism of ketamine in man is stereoselective. The work highlights the value of enantioselective CE in drug
metabolism studies.
This work was supported by the Swiss National Science Foundation.
1 R. Theurillat, M. Knobloch, A. Schmitz, P.-G. Lassahn, M. Mevissen, W. Thormann, Electrophoresis, 28, 2748-2757 (2007).
2 A. Schmitz, R. Theurillat, P.-G. Lassahn, M. Mevissen, W. Thormann, Electrophoresis, 30, 2912-2921 (2009). Monday, March 22nd, 2010 / NADIR 12:15 – 12:35
L12:
CE Separation Strategies For Combinations Of Anthracycline And Taxanes
Yuliya Shakalisava, Fiona Regan
DCU, Dublin, Ireland
The versatility of capillary electrophoresis (CE) was demonstrated for the separation of different types of anticancer drugs – anthracyclines
and taxanes. The current use of these drugs in combination therapy sparked the interest in this application. The possibility of the simultaneous analysis of anthracyclines and taxanes can significantly increase a sample throughput of a clinical laboratory. The study shows the
potential of CE for such a challenge: anthracyclines and taxanes were separated by capillary zone electrophoresis (CZE), micellar eletrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC). MEEKC method was successfully applied to these
compounds for the first time and was characterised by very short separation time, high efficiencies of peaks and was proven to be generic
for the separation of different combinations of anthracyclines and taxanes. This is highly beneficial for clinical analysis in cancer therapy
when the combinations of prescribed drugs can be varied. MEKC and high speed MEEKC methods were proven to show good potential
in their application to plasma samples. The paper reports the investigation of ultrafiltration as a sample pretreatment option in order to
achieve appropriate limits of detection and to examine the bound and unbound drug concentration in plasma.
L13:
Novel Methodology To Couple Isoelectric Focusing With Mass Spectrometry – Experimental And Theoretical Advances
Csilla Páger1, Anna Takácsi-Nagy1, Wolfgang Thormann2, Ferenc Kilár*1
32
Lecture Abstracts
1 Institute of Bioanalysis, Faculty of Medicine, University of Pécs, Szigeti út 12., 7624 Pécs, Hungary
2 Department of Clinical Pharmacology, University of Bern, Bern, Switzerland
The coupling of isoelectric focusing in capillaries to mass spectrometry is a challenge, since the presence of the carrier ampholytes and
other polymeric additives is an obstacle to ionization of proteins and peptides. Although, relatively low concentrations of CAs can be used
without compromising the resolution, upon mobilization of the separated protein zones, ampholytes may flow into the ion source of the
mass spectrometer where they can cause ionization suppression of the protein analytes. The other classical problem in isoelectric focusing
is still persisting, as well, i.e. the precipitation of the focused proteins at their isoelectric points. Much effort has been made to solve the
problem, but a universal remedy remains elusive. Methods to remove the ampholytes prior to MS detection are presently hardly applied as
they often are too complex and/or sacrifice too much of the protein resolution obtained by CIEF. In this novel approach, we offer a solution
to solve both problems. On one side the carrier ampholytes will not interfere with the samples, and at the same time, the focused sample
components will not have zero charge when entering MS.
The method is based on the sequential injection of ampholyte and sample zones in uncoated capillary (J. Chromatogr. 813, 1998, 349360), which provides high flexibility in using different ampholytes, with different pH ranges, and the displacement of the whole setup with
the support of the electroosmotic flow. The main advantage of the technique is the possibility to use ampholytes with pH range(s), which
do not include the pI values of the samples to be investigated. In this method the sample molecules can be completely excluded from the
ampholyte zone, and introduced in the MS without the disturbance of the polymeric substances. Since the molecules after the separation
will migrate outside the ampholyte zones, they will bear charges, which – in the case of proteins – will protect them from precipitation.
For the tracing of the migration and placement of samples in the ampholyte zone(s) the low-molecular-mass pI markers (substituted aminomethylphenol dyes with pIs of 5.3, 6.4, 6.6, 7.9 and 10.4) and proteins were used. The lecture will present the successful separation and
optimization of the method applying ampholytes with narrow and broad pH ranges, different catholyte and anolyte zones, and the effect
of pH of the electrolytes used for the focusing. Results with model calculations confirmed the mechanism of the process in the isoelectric
focusing using the “sandwich” or “half-sandwich” setup.
The work was supported by the grants GVOP-3.2.1-0168 and OTKA-NKTH-NI-68863.
L14:
Improved Flow Rate Compatibility Of Electrospray Ionization With Capillary Electrophoresis Though Optimization Of The Emitter
Geometry
E. Jane Maxwell
Department of Chemistry, University of British Columbia
A major challenge in the design of effective interfaces for capillary electrophoresis – electrospray ionization – mass spectrometry (CE-ESIMS) lies in managing the difference in flow requirements for the separation and ionization processes. While the bulk flow rate of CE separations can range from zero to over a thousand nanolitres per minute, depending on the capillary wall treatment and background electrolyte
(BGE), most electrospray emitters give optimal performance over a much smaller range. In order to address this limitation, an investigation
into the impact of electrospray emitter geometry on flow rate compatibility was carried out using a simple but robust interface, consisting
of a stainless steel needle into which the separation capillary is inserted, forming a junction within the needle tip. A chemical modifier
solution may be combined with the capillary effluent immediately before the solution exits the needle in order to improve compatibility of
the BGE with electrospray. Electrospray performance for blunt, tapered and beveled needles was assessed over a wide range of flow rates
and electrospray potentials. Whereas the Taylor cone for both blunt and tapered emitters is located at the needle orifice, electrospray from
a beveled tip occurs from the sharpest point along the beveled edge, while the size of the Taylor cone varies with the flow rate. Compared
to symmetrical emitters, those with a beveled geometry provide increased stability and signal intensity over a wider range of flow rates. Monday, March 22nd, 2010 / ZENIT 15:40 – 16:00
L15:
Sheathless Capillary Electrophoresis - Mass Spectrometry: The High Sensitivity Porous Sprayer (HSPS) In Forensic Drug Analysis
John C. Hudson*, Jeff D. Chapman
Beckman Coulter Inc., Brea, CA, USA
33
Lecture Abstracts
Separation of charged molecules such as drugs and their metabolites is best accomplished in the Forensic setting by Capillary Electrophoresis-Mass Spectrometry (CE-MS). Capillary Electrophoresis (CE) provides the resolution, reproducibility and precision for identification of these compounds from extracts of blood and urine. The combination of CE with Mass Spectrometry then gives the necessary
unequivocal confirmation necessary for the presentation of such evidence in legal proceedings. Sheath liquid interfacing for CE-MS
offers adequate sensitivity for some applications but the sensitivity is compromised along with resolution by the dilution effect of the
sheath liquid. Sheathless interfacing increases sensitivity by an order of magnitude or more while maintaining the high resolution capability provided by capillary electrophoresis. The High Sensitivity Porous Sprayer (HSPS) interface for CE-MS has shown great promise
in maintaining this resolution and providing the sensitivity necessary for demanding areas of forensic drug analysis. Examples of the
application of the HSPS to routine forensic analysis of drugs and metabolites will be presented. The HSPS interface is for Laboratory
Use Only; not for use in diagnostic procedures.
L16:
Mass Balance Equation And Chemical Separation In Two Dimensional Geometries
David D. Y. Chen*, Xuefei Zhong, Chang Liu, E. Jane Maxwell
Mass balance equation has been used extensively in computer simulation programs that describe the migration and separation of
analytes in various separation techniques, including capillary electrophoresis and microfluidic devices. While these simulation programs
have helped to provide much insight into the analyte migration and distribution behaviour in columns, which restrict the analyte migration in one dimension, some of the programs can be used to expand the traditional techniques into new areas when the electrostatic
force and fluid dynamic velocity fields are considered. The use of COMSOL Multiphysics in various situations to model microscale fluid
flow profile and mass transport phenomena will be discussed. One situation involves the flow of solutions around a capillary terminus
in a CE-MS interface to assess the possibility of extra column band broadening. The simulation program was used to provide guidance
in designing experimental conditions to minimize dead volumes and increase peak symmetry. In another situation the program is used
to compute the analyte migration behaviour in a system where more than one voltage and pressure are applied in different locations
in interconnecting channels. A mixture flowing through a channel can be directed into separate channels each containing a pure compound, thus demonstrating the reverse of the mixing process.
L17:
Theory of Electrophoresis: Still a Challenge
Bohuslav Gaš
Charles University in Prague, Faculty of Science
The conservation law or the continuity equation is a set of partial differential equations that result from the balance law of continuous processes in separation sciences. The partial differential equations are powerful tools of the continuum world and allow studying many important
phenomena in the theory of electromigration. The first solution to the partial differential equations was performed by Kohlrausch, who derived
his regulating function. Instead of solving directly the partial differential equations there was a simpler approach to obtain information on
behavior of constituents in electromigration, which was utilized with a great success. This approach was based on moving boundary model
and overall mass balance equations. Further progress in understanding electromigration could be done when solving a linearized system of
equations of electromigration in a matrix form and calculation eigenvalues of the system. We used such approach for development of PeakMaster, a software tool for basic inspection of the background electrolyte properties. By means of PeakMaster the background electrolytes can be
optimized as regards sensitivity in detection, efficiency or selectivity of separation. However, in spite of significant efforts by mathematicians,
most of the fundamental problems in obtaining an analytical solution of continuity equations remain unsolved. Unexpectedly, recently we
discovered by means of computer experiments, that there exists a new class of electrolyte solutions, which have complex eigenmobilities.
According to the theory of dynamical systems, such electrolyte systems must be unstable and behave chaotically when conducting electric
current. This was really confirmed in reality: it leads to the oscillation of the electrolyte concentration and creation of periodic structures in the
separation channel. The present paper overviews the solution of continuity equations from the historical attempts until these days. We show
that in spite of a substantial progress made towards their solution, these equations may still hide new surprising features. 34
Lecture Abstracts
L18:
Computer-Aided Design Of On-Line Enrichment Strategies For Electrophoresis
Michael C. Breadmore1*, Joselito P. Quirino1, Wolfgang Thormann2
1 Australian Center for Research on Separation Science, University of Tasmania, Hobart, Australia
2 Institute of Clinical Pharmacology, University of Bern, Bern, Switzerland
One of the most cited limitations of electrophoresis is its poor concentration detection limits, especially when compared to HPLC. There are now a number of ways in which this can be addressed through on-line concentration methods such as stacking and sweeping. However, many of these have a strong dependence on the composition of the sample matrix and this can contribute significantly to the
irreproducibility of the method. Over the past few years, we have been using computer simulation software to develop a fundamental
understanding of the behaviour of on-line enrichment in capillary electrophoresis. High resolution simulations under conditions used
in the experimental laboratory have been performed for a number of on-line enrichment strategies including dynamic pH junction,
sweeping, field amplified sample stacking and isotachophoresis. The impact of variations in sample matrix will be discussed and strategies through which these can be minimised through improved design of the on-line enrichment strategy will be presented.
L19:
Capillary Electrophoresis For Foodomics
Carolina Simo, Virginia Garcia-Cañas, Miguel Herrero, Alejandro Cifuentes*
Department of Food Analysis, Institute of Industrial Fermentations, National Research Council (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
Assessment of food quality is fundamental to guarantee the authenticity and safety of foods required by consumers and legislation.
This includes the control of technological processes and their effects on food constituents, determination of food nutritional value,
characterization of food components, or the more recent studies on detection and characterization of new food ingredients with demonstrated beneficial effects on human health. These issues are of key importance for industries, research institutions and regulatory
laboratories working in Food Science and Technology. Nowadays, new analytical approaches are emerging to face the everyday more
complex problems and exigent demands in Food Science and Technology. In this regard, we have recently defined for the first time in
a SCI journal the new field of Foodomics (A. Cifuentes, J. Chromatogr. A, 1216 (2009) 7109; M. Herrero et al., Electrophoresis 2010, 31,
205–228) as a discipline that studies the Food and Nutrition domains through the application of omics technologies. Consequently, Nutrigenomics and Nutrigenetics can be considered as subdisciplines of the more general Foodomics field. Thus, Foodomics would cover
e.g., the development of new transgenic foods using molecular tools, the genomic/transcriptomic/proteomic and/or metabolomic study
of foods for compounds profiling/authenticity and/or biomarkers detection, new investigations on food functions via nutrigenomics or
nutrigenetics approaches, etc. In this context, the use of capillary electromigration methods can show interesting possibilities. In this
work, the potential of CE in Foodomics will be demonstrated through different applications carried out by our group.
L20:
Detection Of Monophosphate Nucleotides Through Surface-Assisted Laser Desorption/Ionization Mass Spectrometry Using Gold
Nanoparticles
Yi Ting Hsieh*1, Tai Chia Chiu2, Huan Tsung Chang1
1 Department of Chemistry, National Taiwan University, Taipei, Taiwan
2 Department of Applied Science, National Taitung University, Taitung, Taiwan
We have demonstrated the determination of monophosphate nucleotides through the surface-assisted laser desorption/ionization
mass spectrometry (SALDI-MS) using hexadecyltrimethylammonium bromide (CTAB)-adsorbed gold nanoparticles (CTAB-Au NPs) as
selective probes and matrices. The binding of four monophosphate nucleotides—adenosine 5’-monophosphate (5’-AMP), guanosine
5’-monophosphate (5’-GMP), uridine 5’-monophosphate (5’-UMP) and cytidine 5’-monophosphate (5’-CMP)—to the surfaces of these
35
Lecture Abstracts
CTAB-Au NPs induced their aggregation, which allowed selective concentration of the four analytes from sample solutions. Our results
revealed that CTAB was effective to suppress the matrix-related ion background and to improve ionization efficiency of the analytes.
By using CTAB-Au NPs that were prepared at a Au NPs/CTAB molar ratio of 1:200, our SALDI-MS approach provided the detection of
limits at a signal-to-noise ratio of 3 of 5, 10, 25, and 25 nM for 5’-AMP, 5’-GMP, 5’-UMP and 5’-CMP, respectively. We have successfully
validated the practicality of this approach through the analysis of 5’-AMP, 5’-GMP, 5’-UMP and 5’-CMP in the mushrooms extracts and
5’-GMP in monosodium glutamate (MSG). Our approach provides advantages of simplicity, sensitivity, and reproducibility (shot-to-shot
variation less than 25%) for the determination of the monophosphate nucleotides from solutions.
L21:
Monolithic Columns With Dual HILIC - Reversed Phase Mechanism For Capillary LC Of Polar Compounds
Pavel Jandera*, Veronika Škeříková, Jiří Urban, Romana Kubíčková
University of Pardubice, Dept. of Analytical Chemistry
Some naturally occurring compounds are strongly polar and can be better separated in hydrophilic interaction (HILIC) than in reversedphase (RP) liquid chromatography systems. We prepared zwitterionic polymethacrylate monolithic columns for micro-HPLC by in-situ
co-polymerization in fused-silica capillaries. The retention and separation selectivity were varied by adjusting the concentrations of
sulfobetaine monomer, ethylene dimethacrylate cross-linker and porogen solvent in the polymerization mixture. Capillary columns prepared under optimized polymerization conditions could be employed for dual HILIC-RP mechanism separations of samples containing
compounds differing in polarity, such as phenolic acids, amino acids, flavone glycosides and aglycones and other polar compounds.
Dual-mechanism separations are possible on a single column in aqueous-organic mobile phases at low concentrations of water in the
HILIC mode, and at higher concentrations of water in the RP mode. For this purpose, either isocratic elution steps, or alternating gradients of increasing and decreasing gradients of acetonitrile concentration could be used. The properties of the monolithic zwitterionic
columns were compared with capillary columns packed with aminopropyl silica particles. The monolithic sulfobetaine column shows
similar separation selectivity as the conventional commercialy available ZIC-HILIC silica-based columns, but higher retention and better
separation under reversed-phase conditions. The performance of the monolithic sulfobetaine dual-mode capillary columns is illustrated
by practical application examples.
L22:
Searching For Trace Proteins In Foodstuff Via Combinatorial Peptide Ligand Libraries (CPLL)
Angela Bachi1, Pier Giorgio Righetti2, Egisto Boschetti3, Elisa Fasoli*2
1 San Raffaele Scientific Institute, 20132 Milan, Italy
2 Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, 20131 Milan, Italy
3 Bio-Rad Laboratories, 92430 Marnes-la-Coquette, France
Searching for trace proteins in foodstuff via combinatorial peptide ligand libraries (CPLL) Angela Bachi (2), Pier Giorgio Righetti (1), Egisto
Boschetti (3), Elisa Fasoli (1) 1. Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, 20131
Milan, Italy. 2. San Raffaele Scientific Institute, 20132 Milan, Italy 3. Bio-Rad Laboratories, 92430 Marnes-la-Coquette, France Background: the
proteomes of natural foodstuffs contain a highly unbalanced interval of protein concentrations, with a dozen or so proteins representing the
vast majority (>90%). For this reason the discovery and the identification of minor components represent a formidable challenge. Method: we
have devised and applied a set of baits, part of a most variegated combinatorial library and composed of hexapeptides, to the discovery of
the low-abundance proteome in three types of foodstuff of animal origin: egg white and yolk and cow’s milk. Results: in the case of egg white
only 78 unique gene products were tabulated and our findings have almost doubled that value to 148 unique protein species, of which 107
proteins discovered by the use of CPLLs. As regards egg yolk, in the plasma 100 unique gene products were tabulated in the literature, while
we have found 255 unique protein species (215 proteins by CPLL technology). In the cow’s milk (whey), less than 80 unique gene products were
known and we identified 149 unique proteins, of which 100 not previously described. In addition, after treatment with CPLL, a polymorphic
alkaline protein, an immunoglobulin (Ig), gave a positive signal after blotting and incubation with sera of allergic patients and it was identified
as a minor allergen. Conclusion: CPLLs offer a unique opportunity for bringing to the limelight “invisible” species, by achievement of proteins
lists important for isolation and functional characterization of proteins, possibly having novel pharmaceutical and biomedical applications.
36
Lecture Abstracts
L23:
Potential of HPLC/MS/MS in the identification of drug metabolites
Michal Holčapek*1, Robert Jirásko1, Miroslav Lísa1, Milan Nobilis2
1 University of Pardubice, Faculty of Chemical Technology, Dpt. of Analytical Chemistry
2 Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS Praha a.s. and AV CR
Applications of HPLC/MS/MS with atmospheric pressure ionization techniques in the identification of phase I and phase II drug metabolites in complex biological matrices are reviewed for both in vitro and in vivo drug biotransformation1. The optimization of chromatographic conditions provides a required separation selectivity, analysis time and also good compatibility with the mass spectrometric
detection. The incorporation of additional detectors (photodiode-array UV, fluorescence and polarimetric) between HPLC and MS can
bring valuable analytical information supplementing MS results. The relation among the structural changes caused by phase I (oxidation, reduction, dealkylation, etc.) and phase II (conjugation with glucuronic acid, sulfate, amino acids, etc.) metabolic reactions and
corresponding shifts in the retention behavior in reversed-phase systems is discussed as the supporting information for the metabolite
identification. The first and basic step in the interpretation of mass spectra is always the MW determination based on the presence of
protonated molecules [M+H]+ and sometimes adducts with ammonium or alkali metal ions, observed in the positive-ion full scan mass
spectra. The MW determination can be confirmed by [M-H]- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS
is worthy tool for the further structural characterization because of the occurrence of characteristic fragment ions, either MSn analysis
for studying the fragmentation patterns on trap-based analyzers or high mass accuracy measurements for elemental composition determination on QqTOF. The correlation between typical functional groups found in phase I and II drug metabolites and corresponding
neutral losses will be illustrated on selected examples. The choice of suitable ionization technique and polarity mode in relation to the
metabolite structure is discussed as well.
1 M. Holčapek, L. Kolářová, M. Nobilis, Anal. Bioanal. Chem. 216 (2008) 1962
This work was supported by project MSM0021627502 sponsored by the Ministry of Education, Youth and Sports of Czech Republic and
project 203/08/1536 sponsored by the Czech Science Foundation.
L24:
Stable And Flexible Metabolic Profiling By Comprehensive CE-Tof-MS
Rawi Ramautar1, Oleg A. Mayboroda*1, Andre M. Deelder1, Govert W. Somsen2, Gerhardus J. De Jong2
1 Leiden University Medical Center
2 Utrecht University
Combined capillary electrophoresis-time-of-flight mass spectrometry (CE-ToF-MS) is a powerful technique for the fast and efficient
analysis of ionogenic compounds. The application of CE-ToF-MS to metabolic profiling of body fluids, however, may be hindered by
reproducibility and coverage problems. In this presentation a novel approach for reproducible and comprehensive metabolic profiling
by CE-ToF-MS will be outlined. Very stable CE performance is accomplished by the use of noncovalent capillary coatings comprised of
double and triple layers of charged polymers. These easy-to-produce coatings provide high migration-time reproducibility and good
tolerance against sample matrix compounds. Moreover, one capillary with different coatings can be used for both cationic and anionic
compounds demonstrating the flexibility of this approach for providing an extended coverage of metabolites in only two CE runs. Incorporation of in-capillary preconcentration by pH-mediated stacking further aids the detection of low-level metabolites. The performance
of this novel CE-ToF-MS platform for metabolic profiling of body fluids has been studied for large groups of urine samples. Using multivariate techniques, it is demonstrated that the CE-ToF-MS platform allows high quality metabolomic data to be obtained. Accordingly,
it can be applied for delineation of the urinary metabolome in a clinical setting. For example, the applicability and usefulness of the
CE-ToF-MS platform will be outlined by the elucidation of metabolites involved in complex regional pain syndrome (CRPS) and urinary
tract infection (UTI). Multivariate statistical analysis of the recorded profiles revealed biomarker candidates, which were subsequently
identified by accurate mass and/or MS-MS using QToF-MS. We also demonstrate the complementary nature of CE-ToF-MS and reversedphase LC-ToF-MS for metabolic profiling of human urine.
37
Lecture Abstracts
L25:
Metabolic Profiling Of Mouse Urine By CE-MS: Dissection Of Osteoporosis Biomarkers From TTD Mice
Ekaterina V. Nevedomskaya1, Rawi Ramautar1, Rico Derks1, Gerben Zondag2, Ingrid van der Pluijm2, Andre M. Deelder1,
Oleg A. Mayboroda*1,
1 LUMC, Leiden, The Netherlands
2 DNage BV, Rotterdam, The Netherlands
Osteoporosis poses serious burden to modern healthcare. However, due to its complexity and multifactorial character, the mechanisms
of its development and progression are not well understood. A novel animal model proposed for age-related osteoporosis is TTD mice.
In this study we performed metabolic profiling of urine from these and wild type mice. We demonstrate that capillary electrophoresis
time-of-flight mass spectrometry (CE-ToF-MS) can be used for profiling of different classes of metabolites within a single run. Multivariate data analysis revealed that the most pronounces metabolic differences between mutant and wild type mice are present among
females, consistent with a more severe osteoporotic phenotype in female TTD mice. The identities of the compounds responsible for
discrimination between samples were obtained and their biological significance is discussed. Thus, CE-ToF-MS-based metabolic profiling
is suitable to uncover metabolites that differentiate wild type from TTD mutant mice and may contribute to novel insights into the
mechanisms underlying age-associated osteoporosis as well as the discovery of early biomarkers. Monday, March 22nd, 2010 / NADIR 17:25 – 17:45
L26:
Time-Resolved Metabolomics For Quantitative Assessment Of Oxidative Stress With Strenuous Exercise
Philip Britz-McKibbin*1, Richard Lee1, Daniel West2, Stuart M. Phillips2
1 Department of Chemistry and Chemical Biology, McMaster University
2 Department of Kinesiology, McMaster University
Despite several decades of active research, the success of large-scale clinical trials involving antioxidants remains confounding given the
beneficial and detrimental effects of reactive oxygen/nitrogen species in human health. Herein, we outline an untargeted metabolomics
strategy with high temporal resolution for quantitative assessment of oxidative stress using erythrocytes as a model reporter cell system by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). A healthy volunteer was recruited to perform
a sub-maximal prolonged ergometer cycling trial until exhaustion with frequent blood collection over a six hour time interval, which
included pre-, during and post-exercise periods while at rest. A follow-up study was subsequently performed by the same subject after a
high-dose oral supplementation with N-acetyl-L-cysteine (NAC) prior to performing the exercise protocol under standardized conditions.
Time-resolved metabolomic analyses of filtered red blood cell (RBC) lysates by CE-ESI-MS in conjunction with univariate/multivariate
data analysis revealed a significant attenuation of cellular oxidation associated with high-dose oral NAC intake, which allowed for the
identification of multiple early- and late-stage biomarkers of oxidative stress. This work demonstrates the proof-on-concept that timely
nutritional intervention can effectively attenuate exercise-induced oxidative stress by unanticipated perturbations in global metabolism
that has promising implications in sports and clinical medicine.
Tuesday, March 23rd, 2010 / ZENIT 10:00 – 10:25
L27:
A Miniaturized Western Blot
Robert T. Kennedy*, Gwendolyn J. Anderson, Maojun Gong
University of Michigan, Ann Arbor, USA
One of the most important electrophoresis methods is the Western blot. In this method, proteins are separated by SDS-PAGE, transferred to a blotting membrane, and then detected on the membrane by interaction with an antibody. Western blotting is popular because
it provides the qualitative information of both approximate molecular weight and immunoreactivity, it can be used for semi-quantitative
analysis, and is highly reliable. Despite the enormous popularity and utility of this method, little work has been reported on generating
38
Lecture Abstracts
a capillary or microfluidic format. We have developed a capillary Western blot in which SDS-protein complexes are separated by sieving CE and captured on a membrane. A key technical achievement was development of an appropriate method of transfer from the
capillary to membrane. Evidence has been obtained that this method will be faster, more mass sensitive, and higher-throughput than
current Western blot methods. Results from this method and a microfluidic analog will be described.
L28:
Expanding the Scope of High Performance, High Pressure Microfluidic Chromatography
Steven A. Cohen*, James P. Murphy, Joseph Michienzi, Paul D. Rainville, Angela Doneanu, Michael J. Tomany
Waters Corporation, Milford, USA
Our development of a high performance ceramic based microfluidic HPLC system has recently focused on optimizing a system with
the equivalent chromatographic scale of a 300 micron ID capillary column operating in the flow rate range of 1 – 12 microliters per
minute. This is motivated by the desire to simplify the interface with electrospray mass spectrometry by employing a gas nebulization system instead of a nanospray interface that is used with 75 micron ID columns, yet provides a significant boost in sensitivity as
compared to 2 mm ID column separations. In addition, the larger diameter columns are compatible with direct injection procedures
and do not require trapping columns for reasonable injection volumes. Ceramic devices are ideally suited for this larger scale as their
superior fracture resistance is compatible with the greater force exerted with pressures up to 15 kpsi when packed with sub-two
micron particles. This is much in contrast to organic polymer or glass/silica devices that are not compatible with UHPLC pressures. Fluid modeling for the larger diameter devices was employed to optimize the fluidic geometry with the resulting optimized path having
larger radius curves than our previous 75 micron ID devices. Connection with the electrospray interface was accomplished with a new
miniaturized spray nebulization source with an easy quick connect design that does not require tools or fittings and yields negligible
band broadening. With larger diameter columns, it is now practical to analyze many small molecule analytes; examples including basic
drugs and carbohydrates will be shown including improvements in sensitivity versus 2 mm columns that approximate those predicted
by theory with little loss in resolving power. Tuesday, March 23rd, 2010 / ZENIT 10:45 – 11:05
L29:
Pump Free Transportation Device for Magnetic Particles in a Microfluidic Channel
Nils Z. Danckwardt*1, Matthias Franzreb2, Andreas E. Guber1, Volker Saile1
1 Institute for Microstructure Technology
2 Institute of Functional Interfaces
In this work, a novel transportation system for magnetic particles which allows the transport of magnetic particles along a channel
without pumping of the fluid is presented. Magnetic particles are used because of their susceptibility for magnetic fields and their capability to couple to specific biomolecules. By this, magnetic particles are an elegant and easy to automate way to handle biomolecules
in analytical assays. In the proposed device, soft magnetic structures are grouped in a pattern on each side of a microfluidic channel.
By overlaying an alternating homogeneous external magnetic field, the magnetic particles can be transported along the channel. The
soft magnetic structures induce gradients to the external field and thus a force is applied on the magnetic particle. The particles move
towards the position with the highest flux density. By switching between different external field vectors different field maxima are
induced and the particle is transported. Two different types of simulation were carried out. The effect of the geometry of one soft
magnetic structure was assessed to observe the magnetization in function of the slenderness and the spatial direction of the magnetic
field. Based on this magnetic simulation the magnetic field data was used for a particle movement simulation written in MatLab-code.
The particle movement simulation takes the hydrodynamic drag into account and indicates the speed as well as the preferred pathway
of the particles. The effect of the pattern of the soft magnetic structures was then analyzed and the geometry was optimized towards
an optimal transport speed. From the simulated data, the particle velocity as well as the effective transport speed along the channel
axis can be deduced. The simulation results were validated with a demonstrator device. A PMMA block was milled to accommodate
the microfluidic channel and to position the soft magnetic structures. The microfluidic channel was formed by insertion of a hose. This
allows the replacement of the hose to vary the do/di ratio and to separate the fluidic and the magnetic part of the device. The soft
magnetic structures are made of permalloy and positioned into cavities along the hose. Experiments were carried out with two pairs of
39
Lecture Abstracts
Helmholtz coils and the movement was observed with a stereomicroscope. Simulation and experimental results cohere. Experimental
results show that the transportation speed for magnetic particles is in the mm/s order. The transport efficiency is near 100% depending
on the particle concentration.
Tuesday, March 23rd, 2010 / NADIR 10:00 – 10:25
L30:
Advanced Porous Polymer Monoliths with Controlled Surface Chemistry for Separation of Biomacromolecules
Frantisek Svec
The Molecular Foundry, E.O. Lawrence Berkeley National Laboratory, Berkeley, CA 94720-8197, USA
Since their birth in the late 1980s and first application in the separations of proteins, porous polymer monoliths were used in numerous
other applications such as solid phase extraction, enzyme immobilization, capillary electrochromatography, gas chromatography, ion
chromatography, and HPLC. Their advantages include ease of the preparation, robustness, high permeability to flow, mass transfer via
convection, and a vast variety of chemistries. Recently, most of our efforts are focused on capillary and chip formats for micro- and
nanoscale applications. These small formats are advantageous for the separations of samples which volumes are small. The pore surface chemistry of polymer monoliths plays an important role in all of their applications. The easiest way to control this chemistry is to
copolymerize in a single step monomers with desired functionalities. However, when a different chemistry and/or different percentage
of the functional monomer are required, the polymerizing system must be re-optimized since any change in the polymerization mixture
also affects porous properties of the monolith. A more convenient approach comprises optimization of copolymerization of a mixture
including a reactive monomer enabling different chemistries to be obtained via chemical modification of reactive groups without any
undesired changes in morphology. This approach can also be used to attach preformed functional polymers to the surface. Similarly,
we developed a universal photografting approach consisting in in situ polymerization of functional monomers from the pore surface
affording monoliths with a variety of functionalities. Yet another option that emerged recently and provides control over the surface
chemistry without affecting the structure is the attachment of functional nanoparticles bearing a specific chemistry to the pore surface. An example we pursued recently includes attachment of gold nanoparticles to the functionalized monolithic capillary and use of this
device for selective fishing-out of thiol containing peptides to simplify complex mixtures originating from digested proteins. This concept was recently extended to a variety of nanosized objects such as carbon nanotubes, fullerenes, hydroxyapatite, and metal oxides.
L31:
Recent Developments In Size-Separation Of Proteins By Capillary Sieving Electrophoresis
Vladislav Dolnik*, William A. Gurske
Alcor BioSeparations LLC, Palo Alto, USA
Capillary sieving electrophoresis (CSE) is a well established method for size-separation and characterization of proteins. SDS capillary
sieving electrophoresis (SDS CSE), which was the very first application of capillary electrophoresis in sieving media, became a standard
method for analysis of proteins and determination of their molecular weight. Initially proteins were believed to bind equal amount
of ionic surfactants, thus enabling proteins to be electrophoreticaly separated according to their molecular weights. Nevertheless,
excessive intrinsic charge and massive glycosylation cause numerous deviations from this basic rule of CSE. As a result, many proteins
depart from molecular-weight based separation during CSE. We developed several protocols to chemically modify proteins suppressing
excessive intrinsic charge and normalizing size-based migration in CSE. Further, we improved CSE of proteins by developing Capillary
Zone Electrophoresis with Cationic Hitchhiking (CZECH) based on electrophoresis in the presence of a cationic surfactant. The method
is characterized by excellent reproducibility of migration times (less than 0.3 %) and unmatched separation efficiency as a consequence
of efficient suppression of electroosmotic flow. Short analysis time is another benefit of the novel method. These competitive advantages make CZECH a strong candidate for advanced method of protein analysis, including 2-dimensional capillary electrophoresis and
capillary electrophoresis with Western blot.
40
Lecture Abstracts
L32:
Preparation And Applications Of The Capillary Coated Dynamically With Carboxymethyl Chitosan
Yi Liu, Xiaofang Fu, Yu Bai, Huwei Liu*
Department of Chemistry, Peking University, Beijing 100871, China
A simple, precise and reproducible dynamic coating method was developed with carboxymethyl chitosan (CMC) as the coating material.
The dynamic coating of the capillary internal surface was accomplished by simply rinsing the capillary with CMC solution for 1 min
in-between runs, with no regeneration procedure or buffer additives. The electroosmotic flow (EOF) in the coated capillary was well
controlled by adjusting the pH value of the running buffer, and the absorption of analytes onto the capillary inner wall was effectively
eliminated. Main parameters of coating condition were optimized, and the reproducibility and stability had been well proved. Extensive
applications of these CMC-dynamically coated capillaries in sample separations were then firmly confirmed, by using proteins, aristolochic acids, organic acids and inorganic anions as model analytes. A high sensitive detection of α-lactalbumin was achieved with a LOD
(S/N=3) of 3.5nM, and the number of plates was as high as 1,200,000/m. In addition, the combination of the CMC coating with none
aqueous capillary electrophoresis (NACE) and capillary electrophoresis mass spectrometry (CE-MS) proved to be practical. All results
showed that the CMC-dynamically coated capillaries had special properties and some superiority over the uncoated ones.
L33:
Vascular Tissue Engineering In Separable Microchip With Vascular Cells
Tadahiro Yamashita*, Yo Tanaka, Kae Sato, Takehiko Kitamori
The University of Tokyo, Japan
Recent progress in tissue engineering has led to clinical successes in regenerative medicine for several functional tissues. However, a
major limitation for tissue engineering remains a lack of methods to produce thick tissues due to the restrictions of passive diffusion
of biomolecules. There is thus a need for in vitro construction of small vascular tissues to overcome this metabolic limitation. To construct small vasculature, we conceived a new method using a microchip focusing on its structure of microchannel. We have previously
reported that the vascular-like tissues with luminal structures can be successfully prepared by culturing human aorta endothelial
cells (HAECs) in microchannels. While this method has potential to reconstruct the vascular for tissue engineering use, it is difficult to
recover the cultured tissues from microchannels, because of enclosed structure of microchannels. In this present paper, we developed
a separable microchip for recovery of cultured HAECs from microchannels, which permits reversible attachment/detachment between
upper and lower substrates. To achieve the concept of the separable microchip, there are two problems. Firstly, when the solution flows
into the microchannel of the separable microchip, the leakage of the solution was induced from slight gap between upper and lower
substrates by capillary forces. To solve this problem, hydrophobic Octadecylsilane (ODS) was modified onto the surface of the substrates, and the Laplace pressure caused by hydrophobic surface prevented the leakage from the microchannel. Secondly, the harvest
of cultured HAECs was typically required for enzymatic proteolysis such as trypsin and collagenase. The enzymatic treatment inflicts
damage to cell-cell junctions by hydrolyzing various membrane-associated proteins, resulting in demolishing tissue-like structures. To
recover the cultured tissue from the culturing surface, we grafted Poly (N-isopropylacrylamide) locally onto the surface of the microchannel. PNIPAAm-grafted surface is well-known to exhibit reversible hydrophilic/hydrophobic property changes, and enable to control
cell adhesion/detachment by temperature alterations. Vascular endothelial cells were cultured for several days in the microchannel.
Then, warmed gelatine solution was introduced into the microchip. By reducing the temperature, gelatine solution turned solid and
cultured tissue detached from the microchannel spontaneously. At last, the microchip was separated to two substrates, and cultured
cells were recovered from microchannel sustained by solidified gelatine string. Now, we are attempting to coculture HAECs with human arterial smooth muscle cells (HASMCs) to enhance the mechanical strength of the tissue, and succeeded in culturing HASMCs in
microchannel. This report is the first demonstration of non-invasive recovery of vascular tissues cultured in microchannels, and will be
useful for the regeneration of various tissues with complex structures.
41
Lecture Abstracts
L34:
Can The Cells And Subcellular Particles Be Fractionated According To Their Shape?
Josef Janča
Department of Physics and Materials Engineering, Tomas Bata University in Zlin, Czech Republic
Analytical fractionation and characterization of living cells and subcellular particles represents a challenging task in contemporary
separation science and technology1. The fractionation is usually performed on the basis of differences either in size or density of the
concerned species. Various microfluidic devices were constructed that can be used for the fractionation of the nanoparticles and micrometer-sized particles, for concentration and manipulation of the individual particulate species, and as a part of the analytical lab-ona-chip (LOC) integrated units. Separation mechanisms in such microfluidic systems are based on the coupling of several forces acting
on the particles like gravitational force, inertial lift forces, Dean drag forces, electrical and magnetic field forces, etc., which generate
the selective migration of the particles. Our recent studies2, 3 of the thermophoretic movement of model synthetic particles exposed to
temperature gradient in microfluidic channel elucidated complex mechanisms by which the spherical, micrometer-sized particles were
retained. The elucidation of various mechanisms governing the separation of model particles opened the way to apply the thermophoresis to the fractionation of the living cells and subcellular particles. The subsequent research of the possibilities to exploit Ludwig-Soret
effect of thermophoresis for the fractionation of living cells by applying the method of Microthermal Field-Flow Fractionation resulted in
a first successful separation of different bacteria4, 5. These preliminary experiments inspired more detailed investigation of the behavior
of living bacteria in temperature gradient6. The conclusions that can be drawn from the most recent experimental study indicate the
possibilities to fractionate the cells and subcellular particles on the basis of differences in their shape. The experimental findings are
supported by the theoretical arguments of extended irreversible thermodynamics7.
1 D. S. Kompala and P. Todd, Editors, Cell Separation Science and Technology, ACS Symposium Series 464, American Chemical Society, Washington, D. C., 1991
2 J. Janča, Microthermal Field-Flow Fractionation: Analysis of Synthetic, Natural, and Biological Macromolecules and Particles, HNB Publishing, New York, 2008
3 J. Janča and J. Stejskal, J. Chromatogr. A, 1216, 9071 (2009)
4 V. Kašpárková, V. Halabalová, L. Šimek, J. Růžička, and J. Janča, J. Biochem. Biophys. Methods, 70, 685 (2007)
5 J. Janča, V. Kašpárková, V. Halabalová, L. Šimek, J. Růžička, and E. Barošová, J. Chromatogr. B, 852, 512 (2007)
6 J. Janča et al., paper in preparation
7 D. Jou, J. Casas-Vázquez, G. Lebon, Extended Irreversible Thermodynamics, Springer-Verlag, Berlin, Heidelberg, New York, 2001
L35:
Elucidating The Effects Of Cytoskeleton On The Electrophoretic Properties Of Individual Mitochondria
Vratislav Kostal*, Gregory G. Wolken, Edgar A. Arriaga
University of Minnesota, Minneapolis, MN, USA
Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) has become an important tool for studying the properties
of isolated organelles. Using CE-LIF we have observed large variations in the electrophoretic mobilities of individual mitochondria
isolated from the same tissue or cell preparation. Understanding what causes such electrophoretic variation is essential in designing
effective electrophoretic separations of mitochondrial subpopulations. We hypothesize that the presence of cytoskeleton attached to
the mitochondrial surface contributes significantly to such variations. This hypothesis is based on the essential role of cytoskeleton as
a scaffold for mitochondria movement within the cell. Here, we demonstrate that individual mitochondria have cytoskeletal remnants
and further propose that the presence of cytoskeleton affects their electrophoretic mobility and isoelectric point (pI). Mitochondria
labeled with a DsRed-2 fusion protein were isolated from L6 rat myoblasts and labeled with Alexa488-tagged phalloidin, which selectively binds the cytoskeletal protein F-actin. CE-LIF analysis reported that about 30% of mitochondria have detectable levels of F-actin
(LOD for Alexa 488 phalloidin was 5.4 zmol). By treating the mitochondrial surface with trypsin prior to labeling with Alexa488-tagged
phalloidin, the levels of this cytoskeletal label decreased dramatically thereby confirming the cytoskeletal presence on the surface of
mitochondria. Distribution of electrophoretic mobilities of treated and untreated mitochondria changed (mean mobilities were 3.3 and
3.9×10–4 cm2V–1s–1, respectively). Further, we determined the pI distributions of mitochondria with capillary isoelectic focusing (cIEF).
Here, the mitochondria labeled with nonylacridine orange were focused in a pH gradient and detected individually by LIF detection.
Using fluorescent internal standards we found the mitochondria pIs ranging from pH 3 to 7 with a mean pI of 6.4. Currently, these
42
Lecture Abstracts
findings are being applied to the separations of mitochondrial subpopulations of normal and age-related giant mitochondria and for
the investigation of mitochondrial aggregation during CE separations.
This work was funded by the National Institutes of Health (R01-AG20866).
L36:
Determination Of Polymer Effective Charge By Indirect UV Detection In Capillary Electrophoresis
Hervé Cottet*1, Nadia Anik1, Marc Airiau2, Marie-Pierre Labeau3, Chi-Thanh Vuong2, Julien Reboul4, Patrick Lacroix-Desmazes4,
Corine Gerardin4
1 Institut des Biomolécules Max Mousseron,2 place Eugène Bataillon, 34095 Montpellier, France
2 Rhodia Recherches et Technologies, 52 rue de la haie Coq, 93308 Aubervilliers
3 Rhodia Inc., 350 G. Patterson Bvd, Bristol, PA 19007, USA
4 Institut Charles Gerhardt 8, rue de l'Ecole Normale - 34296 Montpellier - France
Recently, we demonstrated that the sensitivity of detection in capillary electrophoresis using indirect UV detection can be used to
determine polymer effective charge1. In this detection mode, the polyelectrolyte (solute) displaces a certain quantity of probe contained
in the background electrolyte and having the same charge as the solute. This quantity of displaced probe is directly correlated to the
effective charge of the solute. Contrary to other electrophoretic methods generally used for monitoring changes in effective charge,
this methodology is not based on the effective mobility of the polyelectrolyte (i.e. migration times) but on the sensitivity of detection
(i.e. peak areas). Experimental values of effective charge obtained for statistical copolymers of poly(acrylamide-co-sodium 2-acrylamido2-methylpropanesulfonate), for homopolymers of poly(diallyldimethyl ammonium chloride), poly(acrylic acid), and poly(metacrylic acid),
are compared to the Manning theory of charge condensation. Interestingly, this methodology can be used for the characterization of
macromolecular architecture since the effective charge of copolymers highly depends on the repartition of the charged monomers
along the polymer chain. As an example, this methodology can easily distinguish statistical copolymers from diblock copolymers of
similar chemical charge densities. The variation of the sensitivity of detection of statistically charged copolymers in the indirect UV
mode with the chemical charge density will also be discussed in details.
1 N. Anik, M. Airiau, M.-P. Labeau, C.-T. Vuong, J. Reboul, P. Lacroix-Desmazes, C. Gérardin, H. Cottet. Macromolecules, 2009, 42, 2767-2774
L37:
A Multidetection Platform for Microcolumn Separations
Jan Preisler*1, Pavla Jungová1, Viktor Kanický1, Pavel Krásenský1, Jarmila Navrátilová2, Ondřej Peš1, Jan Šmarda2, Tomáš Vaculovič1
1 Department of Chemistry, Masaryk University
2 Department of Experimental Biology, Masaryk University
An instrumental platform based on a universal off-line interface for deposition of capillary electrophoresis (CE) or liquid chromatography (LC) effluent on a suitable target will be described. The deposited fractions of analytes, such as peptides, proteins, metalloproteins
or inorganic compounds may be analyzed in off-line mode using matrix-assisted laser desorption/ionization mass spectrometry (MALDI
MS). Laser-induced fluorescence (LIF) detection may be used for visualization of analytes on target. Analytes may be subjected to
reactions on target, such as digestion for protein identification. Additionally, inductively-coupled plasma mass spectrometry (ICP MS)
detection mode, nature of which is complementary to soft MALDI MS, may be exploited.
The detection ability is demonstrated on metallothioneins. Information about protein mass, identity and content of elements, such
as metals or phosphorus are available from a single sample. LIF detection of proteins is found to be sensitive, fast and generally useful
for tryptophan-containing proteins. Although not as sensitive as the on-column LIF detection, on-target LIF detection may be used to
select fractions containing proteins. Principle of substrate-assisted laser desorption (SALD) for sample introduction from the target
to ICP MS is explained in detail; influence of laser power density, presence of additives and role of substrate is discussed. The limits
of detection for common metals are in the range of 50 – 500 fg, which allows using SALD for off-line coupling of CE to ICP MS. The
coupling is demonstrated on separation of Cr(III) and Cr(VI) species; subpicomolar detection limits and high separation resolution have
43
Lecture Abstracts
been achieved1. SALD has been also applied to investigate the role of copper ions and disulfiram in cytotoxicity regulation in myeloid
leukemia cells2. Detection limit of ~26 fg for copper was sufficient for determination of copper in U937 cells at physiological levels;
processes in cells were elucidated based on the obtained data. Furthermore, a new sample introduction for ICP MS, which is related
to SALD, will be presented.
We gratefully acknowledge financial support by the Czech Science Foundation (Grant No. 203/09/1025) and the Ministry of Education,
Youth and Sports of the Czech Republic (MSM0021622415).
1 Peš, O.; Jungová, P.; Vyhnánek, R.; Vaculovič, T.; Kanický, V.; Preisler, J. Anal. Chem., 80, 8725–8732, 2008
2 Navrátilová, J.; Jungová, P.; Vaňhara, P.; Preisler, J.; Kanický, V.; Šmarda, J. Int. J. Mol. Med., 94, 1923-1930, 2009
L38:
Column-Switching Operations As Joined Capillary Zone Electrophoresis With Capillary Isotachophoresis While Suppressed For
Laminar And Electroosmotic Flows
Dušan Kaniansky*, Róbert Bodor, Marián Masár
Department of Analytical Chemistry, Faculty of Science, Comenius University, Mlynska Dolina CH-2, SK-84215 Bratislava, Slovakia
Using capillary electrophoresis (CE) is favored for the opened separation system and, due to this, the single-column capillaries with
50-75 μm I.D.s or so is not currently applied for higher I.D.s. For CE is dominated capillary zone electrophoresis (CZE). In general, CZE is analytically restrictive and, for example, consider: (a) concentration limits of detection for the analytes, (b) concentration ratios for the analytes
and (c) resolutions for larger numbers of matrix constituents. CZE with the opened system and for EOF is transferable to ITP. Unfortunately,
such a concept might be restrictive for a standstill operation in ITP. CE with the closed separation system and suppressed EOF is solving
to enhance for limits as mentioned in the previous paragraph (reads the electrophoresis chapters by Vacík as included for the book by Z.
Deyl, F.M. Everaerts, Z. Prusík and P.J. Svendsen (Eds.), Electrophoresis, Part A, Elsevier, Amsterdam, 1979) and, apparently, as applicable
either CZE or ITP. The single-column technology, considered both the opened and closed systems, is analytically restrictive, especially, for
multicomponent sample constituents. Such limits are solvable for using the column-coupling technology (CC-CE). Unfortunately, the CC-CE
opened system (electrophoresis transport along with the laminar and EOF transports) is very complex to join the all transport processes as
these might have significant velocity fluctuations. CC-CE with the closed separation system (eliminating the laminar and EOF transports) is,
for example, broadly applicable as joining ITP with CZE. Currently, we were focused to enhance as regarding ITP-CZE. For this combination
was employed to mix a given sample solution with appropriately chosen discrete spacers (DS). In fact, such a mixed sample is loaded to
the ITP(DS) stage and, subsequently, the sample constituents as separated isotachophoretically. ITP(DS), reached for the steady state, is
transferable to the CZE stage. In the CZE stage is significantly enhancing for the CZE resolution of the sample constituents. In addition, it
was very important to understand our experiments with the CE simulation (SIMUL by Bob Gaš with co-workers). Further, the ITP(DS)-CZE
combination was developed to reach two-dimensional ITP-CZE separations for the sample constituents (especially, benefiting for a fully
automated CE instrumentation). These were documented analytical benefits as based for the 2D ITP(DS)-CZE combination.
This work was supported by the Slovak Grant Agency for Science (VEGA 1/0672/09 and 1/0882/09), the Slovak Research and Development
Agency (VVCE-0070-07).
L39:
Considerations On Influence Of Charge Distribution On Determination Of Pathogenic Bacteria By Electromigration Techniques
Bogusław Buszewski*1, Ewelina Dziubakiewicz1, Ewa Kłodzińska1, Katarzyna Hrynkiweicz2, Paweł Olszowy1, Michał Szumski1
1 Nicolaus Copernicus University, Faculty of Chemistry, Chair of Environmental Chemistry and Bioanalytics
2 Nicolaus Copernicus University, Faculty of Biology and Earth Sciences, Department of Microbiology
Microorganisms are the new “smart” particles that can change their surface properties based on interactions with each other and the surrounding environment. This change in surface characteristics dictate how bacterial cells may interact when forming biofilms or aggregates.
On the other hand the fused silica capillary surface, similarly as a packing material particles in HPLC, shows numerous types of interactions
44
Lecture Abstracts
with the solvent and the solutes. In the case of biomolecules strong adhesion and aggregation effects are observed. Bacterial adhesion and
surface colonization are highly correlated with the physicochemical properties of pathogens surface (zeta potential). Interactions between molecules vary from weak association to strong electrostatic or covalent bonds. These interactions are unstable and this analytes can
easily be removed from adsorbed surfaces by different solvents or by physicochemical and mechanical factors. Nevertheless, when biomolecules are in close proximity to a fused silica surface or coated capillary wall they can form short-range specific interactions, which are
very stable. In this work physicochemical surface characteristics of bacteria were measured to establish their role in bacterial adhesion
and aggregation on the basis on electrophoretic behavior of three selected bacteria species, namely H. pylori, S. aureus, E. coli. The number
and the shape of peaks obtained on the electropherograms were connected with the zeta potential measurements and in-line microscope
observation using specially designed CE – fluorescence stereomicroscope setup. These results suggest that the lower the zeta potential
the higher number of smaller peaks were detected. The direct microscopic observation of electrophoretic movement proved the presence
of many small aggregates originating from individual or clustered bacterial cells. Although the exact mechanisms are not completely understood, it is believed that extracellular proteins (e.g., outer membrane proteins, flagella, etc.), polysaccharides and lipopolysaccharides play
also important role in strengthening bonds between a biomolecules and a substrate (stationary phase) or between other active molecules.
This work was financially supported by Polish Ministry of Science and High Education Grant no: N204 165 31/3730, Foundation for Polish
Science (FNP) project Mistrz and Start and Alexander von Humboldt Foundation. Tuesday, March 23rd, 2010 / ZENIT 15:25 – 15:45
L40:
Characterization of Recombinant Allergen Variants – Various CE Modes Addressing One Aim
Martin Weinbauer1, Tobias Pfaller2, Gertie J. Oostingh2, Hanno Stutz*1
1 Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg, Austria
2 Division of Immunology and Allergy, Department of Molecular Biology, University of Salzburg, Austria
The pronounced prevalence of allergic disorders induced by inhalative allergens causes an increasing burden on public health systems.
Therefore improvements in diagnosis and curative therapy are required. Recombinant allergen products are indispensable tools to ensure
component resolved diagnosis and provide patient tailored specific immunotherapy (SIT). Although recombinant allergens are manufactured in high and consistent purity they may contain impurities introduced during the manufacturing procedure (process related-) but also
modified variants of the target allergen (product related impurities), the latter e.g. derived from chemical modifications during purification.
Consequently, separation methods have to become disposable possessing the capability to reveal these impurities in the course of quality
assessment and ensure batch-to-batch consistency as well as conformity of the target allergens to their natural counterparts. This also
includes the integrity of epitopes. Furthermore recombinant hypoallergens are designed with (partial) disruption of epitopes by deliberately
induced amino acid exchanges in order to diminish detrimental side effects in SIT. In addition, the effect of environmental pollutants on
allergens can be mimicked by their chemical modification thus producing recombinant model compounds for a subsequent investigation
of their altered immunogenicity. Accordingly, physicochemical and immunochemical characterization of allergen products is mandatory.
For this purpose, a portfolio of orthogonal techniques is recommended by the ICH, mostly addressing individual properties. At contrary,
CE has the potential to cover several aspects of characterization simultaneously by the application of different CE modes. Thus, CE can
provide an integrated approach. Protein adsorption is one of the major obstacles in designing highly stable and facile to control CE separations. Beside dynamically coated capillaries, a recent generation of semi-permanent successive multiple ionic polymer layer (SMIL) coatings
applying alternating layers of polyanionic and polycationic polymers has been optimized, accelerating the EOF and substantially improving
the stability of allergen separations. CIEF separations using complex combinations of various carrier ampholytes were applied for profiling
allergen products and pI calculations of individual constituents. For the immunochemical characterization monoclonal antibodies (mAbs)
have been produced in-house and characterized by ESI-TOF-MS and surface plasmon resonance prior to their application in affinity CIEF.
Incubation of allergen products with mAbs was either prior to their injection or in-capillary. The outlined integrated strategy has been applied to recombinant products representing wild-type like allergens, carbamylated or in-house nitrated allergen variants and hypoallergens.
L41:
Afm-Technology for Visualisation, Counting, Measuring the Elastisity and Activity of the Cytochrome P450 Monooxygenase Proteins
Archakov A. I.*1, Bukharina N. S.1, Ivanov Yu. D.1, Munro A.2
45
Lecture Abstracts
1 IBMC RAMS, Moscow, Pogodinskaya str. 10.
2 Manchester University, Manchester , UK
AFM was used for the analysis of aggregation state of single proteins and their complexes of from the cytochrome P450cam (CYP 101),
P450scc (CYP11A1) and P4502B4 (CYP2B4)-containing monooxygenase systems. The proteins and the protein binary monomers and ternary protein complexes, forming within these systems, were visualized. Counting of the number of these proteins and protein complexes
allowed to define the ratio between them. By optical biosensor usage, it was shown that the binary complexes in CYP101 and CYP11A1systems containing intermediate protein Pd or Ad - were inactive, and ternary complexes only were active. In CYP2B4-system both the
binary and ternary complexes are active. The AFM was used for measuring of single enzymes activity by the example of P450BM3 (CYP102)
and horseradish peroxidase (HPR). It was demonstrated that oscillation amplitude of single enzymes in activated state compared with the
ground state increased for the both types of enzymes. Elasticity of single proteins was determined by the example of putidaredoxin ( Pd)
from the protein deformation as affected by sharp and supersharp probe AFM. Integral elasticity 35,71 ÷ 139,43 MPa and local elasticity
was measured 0,3÷5,7 MPa.
L42:
Making The Capillary-Based 2D Gel Separation A Reality: Challenges And Future Of 2D CE
Jane A. Dickerson*, Lauren M. Ramsay, Norman J. Dovichi
University of Washington
Two-dimensional capillary electrophoresis (2D-CE) with laser-induced fluorescence (LIF) detection is used for separation of complex biological samples. We have been developing a 2D CE system that is analogous to the separation in a traditional 2D gel, by first separating
the proteins by their isoelectric point and then by size. There are several challenges involved in coupling cIEF with CSE. Mobilizing and
transfer of the focused pI zones to the second dimension is one issue being investigated. 2D cIEF/CSE has many advantages over traditional
2D-gel electrophoresis, including higher sensitivity, much faster analysis time, less labor intensive and potential for automation. Biogenic
amines are labeled by chromeo-P503, a pyrilium compound that reacts with primary amines to produce a positively charged pyridinium
ion. Fluorescently labeled biomolecules are separated by 2D-CE and detected inside a sheath-flow cuvette with a 473 nm solid state laser
and fiber-coupled avalanche photo diode (APD). 2D-CE-LIF is capable of detecting zeptomole (10-21) quantities of P503-labeled proteins. In
2D-CE, two capillaries, with i.d.s of 50 µm, are aligned at a buffer filled interface. The proteins are separated by reverse capillary isoelectric
focusing (cIEF) in the first dimension. Fractions are electrokinetically transferred to the second capillary, and separated by capillary sieving
electrophoresis (CSE). This technique is used to separate proteins in cellular homogenates prepared from biopsy tissue from patients with
Barrett’s esophagus (BE). Barrett’s esophagus is the only known precursor to esophageal adenocarcinoma; investigation of the differences
in protein expression associated with BE may be used as a prognostic indicator of disease progression.
L43:
Bipolar Electrode Focusing: A Fast, Efficient Method for Simultaneous Analyte Preconcentration and Separation
Dzmitry Hlushkou1, Robbyn K. Perdue2, Derek R. Laws2, Richard M. Crooks2, Ulrich Tallarek*1
1 Department of Chemistry, Philipps-Universität Marburg, Marburg, Germany
2 Department of Chemistry and Biochemistry, Center for Electrochemistry, and the Center for Nano- and Molecular Science and Technology, The University of Texas
at Austin, TX, USA
A method for simultaneously concentrating and separating charged analytes in a buffer-filled microfluidic channel is reported. Analytes
can be simultaneously concentrated and separated, with localized enrichment factors up to 600 within 400 s. The electric field gradient is provided by a complex interplay of electrophoretic, electroosmotic, bulk convective, and diffusive mass and charge transport in
a hybrid PDMS/glass microchannel with embedded floating electrode1–3.
The thin floating electrode attached locally to the wall of the straight microchannel results in a redistribution of the local field strength
after application of an external electric field. Together with faradaic reactions taking place at the bipolar electrode and buffer reactions,
as well as bulk convection based on cathodic electroosmotic flow, an extended field gradient is formed in the anodic microchannel
segment. It imparts a spatially dependent electrophoretic force on the charged analytes, and in combination with bulk convection re46
Lecture Abstracts
sults in electric field gradient focusing at analyte-specific positions. Analyte concentration in the enriched zone approaches a maximum
value which is independent of its concentration in the supplying reservoirs. Numerical simulations which couple the electrokinetics,
hydrodynamics, electrochemistry and buffer reactions provide insights into the underlying fundamental principles for the experimental
findings. A simple approach is shown to unify the temporal behavior of concentration enrichment factors under general conditions.
1 Dhopeshwarkar, R.; Hlushkou, D.; Nguyen, M.; Tallarek, U.; Crooks, R. M. J. Am. Chem. Soc. 2008, 130, 10480-10481
2 Hlushkou, D.; Perdue, R. K.; Dhopeshwarkar, R.; Crooks, R. M.; Tallarek, U. Lab Chip 2009, 9, 1903-1913
3 Laws, D. R.; Hlushkou, D.; Perdue, R. K.; Tallarek, U.; Crooks, R. M. Anal. Chem. 2009, 81, 8923-8929
L44:
Wavelength-Resolved Native Protein Fluorescence Detection In CE: Towards Integrated Probing Of Protein Purity And Conformation
Bregje J. de Kort, Gerhardus J. de Jong, Govert W. Somsen*
Biomedical Analysis, Utrecht University, The Netherlands
Protein therapeutics take an increasingly important position in contemporary drug development and production. As characterization
of biopharmaceuticals is essential, but also very challenging, there is a growing demand for separation-detection methodologies that
permit quality assessment of intact proteins. Capillary electrophoresis (CE) is an attractive technique for purity and stability analysis of
proteins offering efficient and fast separations. Protein emission spectroscopy, which is based on the native fluorescence of aromatic
amino acid residues, is a selective and sensitive detection technique, which can also provide information on the conformational state
of a protein. This presentation outlines the on-line combination of CE with wavelength-resolved fluorescence detection (WRFlu) for protein analysis.
WRFlu was accomplished using a dedicated fluorescence cell that employs wave-guiding principles to lead protein emission light effectively to a spectrograph with CCD detector. The new CE-WRFlu system yields fast separation and detection of proteins down to 10 nM,
permitting detection of minor protein impurities and degradation products. Full protein emission spectra are obtained during the CE
run (“on-the-fly”) with an optical resolution that allows distinction of spectral differences and shifts as small as 1-2 nm. It will be shown
that protein unfolding and refolding is reflected in the measured protein electrophoretic mobilities as well as in the on-line recorded
protein emission characteristics. Overall, the presented results will demonstrate that CE-WRFlu provides a highly promising tool for the
simultaneous monitoring of (changes in) protein purity and protein conformation.
Tuesday, March 23rd, 2010 / ZENIT 16:25 -16:50
L45:
Capillary Electrophoresis in the Biotechnology Industry: Advances and Challenges through the Last Decade
Wassim Nashabeh, Micheal Davis, Stacey Ma
Genentech, A member of the Roche Group, South San Francisco, USA
Since the introduction of first CE application in the routine control system for Rituxan a decade ago, capillary electrophoresis (CE)
has been increasingly utilized as a key analytical tool to support various areas of product development in the biotechnology industry.
The applications include formulation studies, process development, product characterization and validated lot release and stability
testing of both commercial and clinical products. In this talk, an overview of the major applications implemented at Genentech will
be presented. The applications are divided into the following areas: 1) CE as a replacement for slab gel electrophoresis, particularly,
capillary electrophoresis-sodium dodecylsulfate and capillary isoelectric focusing; 2) CE to monitor protein charge heterogeneity as
an orthogonal technique to the traditional ion-exchange chromatographic methods, 3) CE for carbohydrate analysis, including both
oligosaccharide and monosaccharide analysis. Overall, the advantages of these CE-based methodologies include automation, ease in
quantification, rapid analysis time, enhanced resolution, and robustness when compared to the traditional methods. In recent years, a
strategy based on platform assay and high throughput formats have been implemented in order to meet the demands for the increased
demand. In this talk, various practical applications of those methodologies will be discussed along with future directions and needs
for CE in the biopharmaceutical industry.
47
Lecture Abstracts
L46:
Application Of Micro Separations In Biopharmaceutical Development
Yelena Lyubarskaya
BiogenIdec, Inc., Boston, USA
Analytical separations, such as electrophoresis and chromatography with different modes of detection, have been widely used in
biopharmaceutical development for analysis and characterization of protein therapeutics, as well as for biopharmaceutical process
characterization. Although the amount of analyte often is not an issue in the biopharmaceutical development setting, microscale separation techniques are widely applicable, as they provide benefits of high resolution, speed and high throughput, as well as amenability
to automation. Two applications of capillary electrophoresis for analysis and characterization of purity and stability of therapeutic
glycoproteins and recombinant monoclonal antibodies will be demonstrated. CE-SDS analysis on a chip for determination of protein
degradation, aggregation and posttranslational modifications will be demonstrated, and compared to SDS-PAGE and traditional CE-SDS.
Application of imaging capillary isoelectric focusing (icIEF) for determination of protein charge heterogeneity will be described and its
performance discussed in comparison with IEF gel, CE-IEF and ion exchange chromatography (IEC). In addition, an example of using
microscale separations for characterization of biopharmaceutical manufacturing process will be presented. Chinese hamster ovary
(CHO) cells are widely used in biotechnology to produce protein therapeutics. Proteomics analysis has been recently utilized for qualitative and quantitative characterization of CHO cells and cell culture processes. The proteomics analysis is based on two dimensional
nanoscale HPLC with mass spectrometric detection. This approach allows for identification of protein biomarkers related to cellular
productivity in CHO cells. Such biomarkers could potentially be useful for cell line selection and engineering of high producing cells to
enhance the upstream process for biopharmaceutical production.
L47:
Characterization Of Drug-Liposome Interactions And Liposomal Drug Formulations Using Pre-Incubation Affinity CE
Jesper Østergaard*1, Ulrik Franzen1, Lin Yang2, Eva H. Moeller1
1 Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark
2 School of Pharmacy, University of Otago, Dunedin, New Zealand
Liposomes are subject to huge interest in the pharmaceutical sciences due their structural similarities with biological membranes and
the use of liposomes as drug delivery vehicles. Like biological membranes, liposomes are composed of a phospholipid double layer
making them attractive models in studies of adsorption, distribution and permeation of drug substances. CE, an efficient tool for
investigating molecular interactions, has been applied to drug-liposome interactions mainly using the EKC and CEC modes and only
in a few cases utilized for characterization of drug delivery systems. In this contribution, our experiences with pre-incubation affinity
CE for quantification of drug-liposome interactions and characterization of liposomal drug delivery systems are presented. CE in the
frontal analysis mode was suitable for investigating the interactions between positively and negatively charged drug substances and net
negatively charged liposomes1, 2. Apparent liposome–aqueous phase distribution coefficients for 14 drug substances (log Dmem range
1.35 to 3.63) were obtained. Excellent agreement between results obtained by equilibrium dialysis and CE was achieved2. In another
line of study, distribution of propranolol into DPPC liposomes in the absence and presence of 4 bile salts were quantified using frontal
analysis CE. The addition of bile salts, used for increasing drug membrane permeation, led to increases in propranolol distribution.
Further, liposomal formulations of the acylated 28-aa appetite stimulating peptide hormone ghrelin were administered subcutaneously
to rats and CE was applied in order to rationalize the pharmacokinetics3, 4. Pre-incubation CZE and frontal analysis CE showed that
the cationic ghrelin exhibited little affinity for pure DPPC and phosphatidylcholine:cholesterol (70:30 mol%) liposomes and significantly
higher affinity for negatively charged dipalmitoylphosphatidylcholine:dipalmitoylphosphatidylserine (70:30 mol%) liposomes4. The effect
of removing the n-octanoyl side chain of ghrelin on liposome affinity and the actions taken to minimize capillary wall adsorption will be
discussed. A PEGylated liposomal formulation of the anti-cancer agent oxaliplatin was studied by CE. Electrophoresis was performed in
the solution of the formulation (10% (w/v) sucrose and 1 mM calcium gluconate). The zeta potential of the formulation was calculated
from electrophoretic mobility. CE was successfully applied to quantify drug interactions with the empty PEGylated liposomes and monitor the progress of the liposome preparation. Summarizing these studies, the advantages of pre-incubation affinity CE as compared
to EKC and CEC in relation to formulation development will be discussed.
48
Lecture Abstracts
1 Østergaard et al., Electrophoresis 2008, 29, 3320-3324
2 Franzen et al., Electrophoresis 2009, 30, 2711-2719
3 Moeller et al., Int. J. Pharm. DOI 10.1016/j.ijpharm.2009.05.067
4 Østergaard & Moeller, Electrophoresis, DOI 10.1002/elps.200900394
L48:
On-Line CIEF-ESI-MS In Glycerol–Water Media With A View To Hydrophobic Protein Applications
Meriem Mokaddem*1, Marie Lecoeur2, Pierre Gareil1, Anne Varenne1
1 Chimie-ParisTech, Université Pierre et Marie Curie-Paris 6, Laboratory of Physicochemistry of Electrolytes, Colloids and Analytical Sciences (PECSA), 11, rue Pierre et
Marie Curie 75231 Paris cedex 05, FRANCE
2 University of Lille 2, Department of Pharmaceutical and Biological Sciences
Electrospray ionization mass spectrometry (ESI-MS) offers many advantages over traditional UV detection for CIEF separations. The
coupling of both techniques combines resolving power with high precision mass determination. Although it does not provide physical
separation according to molecular mass, CIEF-MS appears to be a valuable alternative to 2-D PAGE separations. In effect, it also provides
information on pI and molecular mass, but with added potential advantages including better ease of automation, greater speed and
direct mass measurements, and likely enhanced sensitivity. In this context, a new original protocol intended for on-line CIEF-MS1 of all
types of proteins is presented herein. To our knowledge, this automated on-line coupling allies for the first time (i) an effective electric
continuity during the whole analysis by a partial filling of the capillary with 60:40 (length/length) catholyte/proteins–ampholyte mixture; (ii) a 30:70 (v/v) glycerol/water anticonvective medium2 that is compatible with MS detection and helps for hydrophobic protein solubilization and (iii) consistency with unmodified bare fused-silica capillaries, as the glycerol/water medium strongly reduces EOF, which
precludes the use of expensive and short-lifetime coated fused-silica capillaries. The anolyte and catholyte compositions were selected
to be compatible with MS detection. They also allow the localization of the pH gradient zone in the electropherogram by adding MS
markers. Further optimization with respect to peak intensity and efficiency, total analysis time and precision on pI determination, was
performed by investigating ampholyte concentration, focusing time, glycerol content, drying gas temperature, nebulizing gas pressure
and sheath liquid flow rates. This new protocol was tested for the separation and the characterization of milk proteins in samples of
intermediate complexity, thus establishing this methodology.
1 Mokaddem, M., Gareil, P., Varenne, A., Electrophoresis 2009, in press
2 Busnel, J-M., Varenne, A., Descroix, S., Peltre, G., Gohon, Y., Gareil, P., Electrophoresis 2005, 26, 3369-3379
L49:
Transport and Sensing in Asymmetric Nanochannels
John M. Perry, Kaimeng Zhou, Zachary D. Harms, Stephen C. Jacobson*
Department of Chemistry, Indiana University, Bloomington, IN 47405, USA
There is considerable interest in shrinking the lateral dimensions of micromachined channels to nanometer length scales for sensing
and separation applications. Some aspects of microchannel transport transfer directly to operation of smaller nanoscale channels, but
nanofluidic systems can be significantly influenced by phenomena such as double layer overlap, surface charge, diffusion, and entropic forces, which are either insignificant or absent in larger microchannels. We present a method of fabricating in-plane nanofluidic
channels in high modulus poly(dimethylsiloxane) (h-PDMS), which can easily replicate nanoscale geometries below 100 nm. Ion transport properties in these nanochannels are measured to determine how the channel geometry influences electrokinetic transport. For
example, funnel-shaped nanochannels rectify ion current, and nanofunnels with smaller taper angles exhibit higher rectification. These
devices are also used to sense transiting particles, e.g., viruses, to characterize particle properties. Nanofunnel fabrication, ion transport
properties, and virus characterization will be discussed.
49
Lecture Abstracts
L50:
AFM And Nanowire Nanochips For Protein And Cell Detection
Ivanov Yu. D.*1, Naumova O. V.2, Popov V. P.2, Archakov A. I.1
1 Institute of Biomedical Chemistry RAMS, Mosvow Russia, 119121
2 Institute of Semiconductor Physics SB RAS, Novosibirsk, Russia
AFM-based nanothechnological methods were used for HCVcore and HBsAg protein detection in AFM-fishing mode. For this purpose,
AFM-biochips with immobilized anti-HCVcore anti-HBsA were created. Then biospecific fishing of corresponding protein partners on these
biochips from solution at the first stage and AFM counting of immunocomplexes at the second stage were carried out. The replacement
of reversible fishing to the irreversible one at the first stage enabled to increase up to 10-16 M the sensitivity of protein AFM detection due
to elimination of thermodynamic barrier of complex formation reaction (for example, HCVcore Ag). AFM biospecific fishing was used for
detection of HBV- and HCVcore–containing virus particalsin serum. Also, we created the Si-nanowire biosensor in cooperation with ISP
RAS (Prof. A .L. Aseev’s group). To demonstrate the possibility of nanowire biosensor for the cells’ registration, the nanowire biochip with
immobilized antibodies against DH 5α cell was created. This biochip was used for biospecific fishing and registration of DH 5α cells. Tuesday, March 23rd, 2010 / NADIR 17:10 – 17:30
L51:
Graphene Nanosheets in Capillary Electrophoresis
Marc B. Mueller1, Joselito P. Quirino*2, Pavel N. Nesterenko2, Gordon G. Wallace1
1 Intelligent Polymer Research Institute, ARC Centre of Excellence for Electromaterials Science, University of Wollongong
2 Australian Centre for Research on Separation Science, University of Tasmania
Graphene is produced through chemical synthesis: graphite becomes oxidised and forms graphene oxide (GO) that can then be reduced
back to graphene, which we call chemically converted graphene (CCG). GO is a one atom thick aromatic carbon sheet with a molecular
weight of approximately 7 million. The aromatic carbon lattice of GO contains defects in the form of hydroxy, ethoxy and carboxyl groups.
Some of these groups are left after reduction of CCG to form a stable colloidal dispersion. While GO is an electrical insulator, self-assembled
CCG films show substantial conductivity (approximately 3 to 300 Scm-1, depending on way of deposition and further treatment). Here, we
present capillary zone electrophoresis (CZE) of graphene and open tubular capillary electrochromatography (OT-CEC) using graphene as
chromatographic material.
Current methods used to characterise graphene, are atomic force microscopy (AFM) and zetapotential measurements. While AFM can
show the sheet size distribution of a graphene dispersion, the zetapotential shows only an average charge of the whole measured dispersion, and none of the methods can show or separate the size or charge carried by the nanosheets.
Nanoengineering, such as self-assembly, relies on controlling the sheet size to increase performance of coatings and other applications that
rely on conductivity, surface roughness, strength and geometrical form of nanoparticles. Methods such as centrifugation showed hardly
any sheet size separation. CZE in tetrapropylammonium hydroxide using fused silica capillaries, on the other hand, allowed us to separated
graphene according to its sheet size and the charge carried (mainly due to the attached carboxylic groups during the chemical production
of GO and CCG). Both influences the conductivity and quality of self-assembled GO and CCG layers.
Due to its huge size compared to other aromatic molecules, CCG and GO shows strong pi-pi interaction with any aromatic molecule nearby. OT-CEC separation of neutral aromatic molecules (naphtalene, phenanthrene, fluorene, biphenyl, and anthracene) was successfully
performed in mixtures of acetonitrile and sodium borate in water using capillaries coated with polydiallyldimethylammonium chloride and
one layer of GO sheets. The CZE results are important to understand CCG and GO dispersion properties and allow for quality control of
graphene dispersions which is important in nanoengineering, e.g. for self-assembling graphene layers that can be used to design flexible,
transparent graphene electrodes, graphene-drug composites for electrically stimulated drug release or bionic surfaces for stimulation of
nerve and muscle cells. Additionally, graphene can be used as chromatographic material in OT-CEC of aromatic molecules. The OT-CEC
platform will allow us to screen chemical candidates that will be used together with graphene.
50
Lecture Abstracts
L52:
Characterization Of Alpha-Lactalbumin Grafted Nanoparticles By Capillary Electrophoresis For The Development Of A New Immunodiagnostics Method
Jan Petr*1, Anne Varenne1, Bruno Teste2, Stéphanie Descroix2, Anne Charlotte Faure3, Jean Michel Siaugue3, Pierre Gareil1
1 Laboratoire Physicochimie des Electrolytes, Colloïdes et Sciences Analytiques (PECSA); UMR CNRS 7195; ChimieParisTech, Paris, France
2 PECSA; ESPCI-ParisTech, Paris, France
3 PECSA;Université Pierre et Marie Curie-Paris 6, Paris, France
The use of nanoparticles (NPs) in immunodiagnostics is a challenging task for many reasons, among which the need for miniaturization.
In view of the development of a microsystem dedicated to an original, miniaturized and fully automated immunodiagnostics which
aims to mimic in vivo interactions, magnetic zwitterionic bifunctional aminated / polyethyleneoxide maghemite core / silica shell NPs
functionalized with allergenic alpha-lactalbumin were synthesized. Capillary electrophoresis (CE), which has been recognized as a valuable technique for the physicochemical characterization of colloidal particles, was used for their characterization. Proper analytical
performances were obtained through semi-permanent capillary coating with didodecyldimethyl-ammonium bromide (DDAB), polybrene
(PB) or permanent capillary wall modification by hydroxypropylcellulose (HPC). First, NPs (both grafted by alpha-lactalbumin and nongrafted) were characterized by CE. The influence of experimental conditions (e.g. buffer component nature, pH, ionic strength, and
electric field strength) on sample stability, electrophoretic mobility and dispersion, was investigated using either DDAB- or HPC-coated
capillaries. The proper choice of experimental conditions allowed the separation of the grafted and the non-grafted NPs. Later, the
aggregation phenomena of NPs were evaluated by Taylor dispersion analysis (TDA) using a CE instrument. A novel three step TDA approach was developed and employed for the study of electric field effects on the colloidal structure of the NPs. The effect of capillary
coating, ionic strength and NPs dilution was described. Finally, NPs grafted by alpha-lactalbumin were characterized in the presence of
anti-alpha-lactalbumin antibody.
The financial support for this project by the Agence Nationale pour la Recherche (ANR, France) is gratefully acknowledged.
Wednesday, March 24th, 2010 / ZENIT 9:00 – 9:25
L53:
Simple, Handheld And Low Cost: To Promote Microfluidcs Close To Real Application
Bingcheng Lin*, Jianhua Qin
Dalian Institute of Chemical Physics, Chinese Academy of Sciences
A series of fabrication methods and devices characteristic of simple, handheld and low cost were developed in our laboratory, which
will promote microfluidics close to its final goal: real application. A straightforward method for fabricating microfluidic devices by
using sunlight as the UV source was established. This method is based on photolithography, but precludes the need for specialized UV
exposure facility. Substrates coated with photoresist were placed directly under sun in a perpendicular direction to the sunlight for
exposure. Masters produced under optimum exposure conditions were used for fabrication of several microfluidic devices with different
materials, structures or functions. Resultant devices were shown eminently suitable for microfluidic applications such as electrophoretic separation, multiple gradient generating and pneumatic valve-based cell culture. This photolithographic method is simple, low
cost, easy to operate and environmental friendly. The fabrication of paper based microfluidic devices in nitrocellulose membrane by
wax printing for protein immobilization related applications was reported. The fabrication process which can be finished within 10
minutes includes mainly printing and baking steps. Wax patterning will form hydrophobic regions in the membrane which can be used
to direct flow path or separate reaction zones, nitrocellulose membrane has better properties than those of normal paper used in this
technology. We believe the wax patterned nitrocellulose membrane will enhance the capabilities of paper microfluidic devices and bring
new applications in this field. A point-of-care system comprising a lateral flow chip and a hand-held quantification device was set up
for quantifying the concentration of biological analytes, The hand-held device measures gold nanoparticles (GNP) based on the unique
optothermal effect of metal nanoparticles to realize quantification of analytes. A reference spot and a blank spot were designed on the
chip to avoid the disturbance from laser attenuation and GNP variance. This system would allow for the whole assay on site and less
than 4 minutes taken. The detection limit is 8 pM for Au nanoparticles and 12 ng for IgG which was tested as a protein sample by our
system. Measuring GNPs by optothermal effect represents a new method of quantitative detection that could upgrade the common
qualitative dry-reagent strips to quantitative systems.
51
Lecture Abstracts
L54:
Bioanalytical Applications Of Microfluidic Electrophoresis With LIF And LED Fluorescence Detection
Petr Smejkal1, Markéta Ryvolová2, František Foret3, Andras Guttman4, Michael C. Breadmore5, Rosanne M. Guijt5, Fritz Bek6,
Mirek Macka*2
1 National Centre for Sensor Research and School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
Institute of Analytical Chemistry of the ASCR, v.v.i., Veveří 97, 60200 Brno, Czech Republic; Faculty of Chemical Technology, University
3 Institute of Analytical Chemistry of the ASCR, v.v.i., Veveří 97, 60200 Brno, Czech Republic
4 Horváth Laboratory of Bioseparation Sciences, Institute of Analytical Chemistry, University of Innsbruck, A-6020 Innsbruck, Innrain 66, Austria
5 Australian Centre for Research on Separation Science, University of Tasmania, Australia
6 Agilent Technologies, P.O. Box 1280, 76337 Waldbronn
Within commercially available CE-on-a-chip platforms Agilent Bioanalyzer 2100 is well established for applications tailored methods for
DNA, RNA and protein analysis. In this work, its applicability as a CE-on-a-chip platform to a broad range of analytes was investigated.
The necessary technical issues investigated included the use of the DNA chips for generic CE and the choice of injection and detection
parameters. To allow a broader application range including non-aqueous CE (NACE), the compatibility of the existing polymer encased
glass chips with solvents such as dimethylsulfoxide was tested and the suitability of this non-volatile, non-hazardous, non-aquous solvent for NACE was verified and demonstrated with NACE separations of basic fluorescent dyes. While fluorescent labelling prior to the
analysis with direct fluorescent detection or the use of indirect fluorescent detection is needed, the applicability of the instrument is
enhanced by the presence of two excitation light sources: red laser (635 nm) and blue LED (470 nm). Several highly fluorescent dyes
including nile blue (NB) and fluorescein isothiocyanate (FITC) that can be used as labelling tags were evaluated for sensitivity and LOD
values. The suitability of the chip-based CE for indirect LIF detection using fluorescent dye probes and direct LIF determination of NBtagged carboxylic acids, FITC-tagged amino acids as well as derivatised carbohydrates, and other biomolecules was investigated and
will be illustrated by examples. Wednesday, March 24th, 2010 / ZENIT 9:45 – 10:05
L55:
Electrochromatography On COC Microchips Using Methacrylate Monolith
Yoann Ladner, Karine Faure*, Gérard Crétier
Laboratoire des Sciences Analytiques, CNRS - Université Lyon 1, Villeurbanne, France
The analysis of biological or environmental samples can sometimes lead to cross-contamination and carry-over, especially due to the
presence of stainless steel needles in conventional HPLC and CZE autosamplers. To avoid irreproducible results, suppliers recommend
extensive rinse, which may not be sufficient. A simpler answer would be the use of disposable analysers integrating injection, separation
and detection within a single miniaturised device (Lab-on-Chip). For this purpose, the miniaturisation of separation technique has to
be carried out on plastic devices. Cyclic olefin copolymer (COC) substrate is a transparent, chemically inert thermoplastic that can be
easily injection-molded, reducing production costs. It has emerged as one of the most relevant material for the fabrication of separation microsystems. Electrochromatography separation technique, which can deal a wide range of solutes (charged and neutral), can be
implemented in plastic microsystems to answer this issue. The introduction of a stationary phase into microchannels to perform chromatographic interactions represents a technical challenge that find an answer through the use of monoliths. Methacrylate monoliths
were selected to provide an efficient, photopolymerizable stationary phase. Since COC is a highly hydrophobic material, it cannot bear
conventional monolithic formulations and the synthesis of acrylate monolith has to be adjusted so that no water is introduced in COC
microchannels. Therefore in this work we have developed a synthesis of methacrylate monolith using non-aqueous porogenic system.
This formulation has been studied in capillary format, in terms of reproducibility, robustness and chromatographic behavior (retention,
efficiency). The issue of wettability has been addressed. Then the transfer of this synthesis has been carried out in COC microsystems,
where successful electrochromatographic separations have been performed. Localisation of the monolithic column within the microsystem has also been investigated, using capillary forces. 52
Lecture Abstracts
L56:
Development Of Highly Efficient Intracellular Gene Detection System On Microchip
Jun Wakabayashi1, Yo Tanaka1, Kae Sato2, Kazuma Mawatari1, Yuki Tanaka3, Mats Nilsson3, Takehiko Kitamori1
1 The University of Tokyo, Japan
2 Japan Women’s University, Tokyo, Japan
3 Uppsala University, Sweden
Gene analysis in single cell level is quite efficient for personalized medicine or diagnosis of cancer and infection disease. In addition,
immediate identification and early treatment are required for clinical medicine. However, there are no rapid diagnosis methods that
meet the clinical requirement. For example, a test kit of sepsis by in-situ hybridization (ISH) is relatively good at present1. The kit can
detect bacterial DNA in leukocytes which phagocyte the bacteria, and shows higher sensitivity (42%) than that of the most popular
blood culture method (11%). However, the sensitivity of the kit is insufficient and it requires 8-hour complex procedure. In addition, skills
are necessary for reproducible detection. In our group, integration of various chemical processes into a microchip has been achieved
with reduction of the process time, reagents, wastes, and troublesome manual operations. On the other hand, in-situ padlock probe/
rolling circle amplification (RCA)2, a better DNA detection method than ISH, has been developed. This method can distinguish a single
nucleotide difference and get clear signal by DNA amplification and fluorescent detection. It can also provide the local distribution of
DNA molecule in each cell and individual signal is countable as a bright dot. We already achieved microchip-based RCA with primer
solid phased beads in vitro3. By applying the technique to cells, an intracellular DNA analysis method with simple procedure, short
time, and high detection rate will be developed. On this research, we utilized suspended leukemia cell line HL60 cells as a model and
developed a detection system of bacterial DNA for diagnosis of infection disease using a microchip. Optimizing reaction condition of
in-situ padlock probe/RCA on microchip, we targeted mitochondrial DNA since the mitochondrion is an organelle derived from bacteria
and a good model for bacteria in a cell. We designed a microchip, which has a chamber in a glass substrate (30 × 70 mm2). All solutions
were injected with a micro syringe pump through a capillary. Initial reaction conditions were based on those of the previous bulk scale
research4. Before detection of intracellular DNA, we firstly detected DNA bound on agarose beads in order to check the experimental
process. Target sequence and procedure were followed by a cell research. As a result, we identified bright dot signals on beads. Then,
intracellular DNA was detected. Mitochondrial DNA was fluorescently detected after in-situ padlock probe/RCA. Bright fluorescent spots
from RCA products were observed. In this study, we developed a detection system of bacterial DNA for diagnosis of infection disease
using a microchip. By applying this technology, complex procedures in conventional methods would be reduced and easier detection
would be achieved.
1 J. Shimada et al., J. Infect. Chemother., 5, 1999
2 C. Larsson et al., Nat. Methods, 1, 2004
3 A. Tachihara et al., Proc. microTAS 2008
4 H.L. Wamsley et al., J. Clin. Microbiol., 46, 2008
Wednesday, March 24th, 2010 / NADIR 9:00 – 9:25
L57:
New Advances In Sample Preparation Methods For Capillary Electrophoresis Analysis Of N-Linked Glycans
Andras Guttman*, Zoltan Szabo, Tomas Rejtar, Barry L Karger
Barnett Institute, Boston, USA
Capillary electrophoresis (CE) is a high resolution separation technique broadly used in the biotechnology industry for carbohydrate
analysis. The standard, well-established sample preparation protocol for CE analysis of N-linked glycans requires long deglycosylation
times (several hours to overnight), and an additional extra cleanup step after fluorophore labeling with ³100 fold excess reagent. In
this presentation, we report on significant improvements in sample preparation for CE based N-linked glycan analysis. Pressure cycling
technology (PCT) was used to increase the speed of PNGase F - mediated release of N-linked glycans from denatured model glycoproteins. With the application of pressure cycling from atmospheric to as high as 30 kPsi, rapid and complete release of the asparagine
linked glycans was achieved for bovine ribonuclease B, human transferrin and polyclonal human immunoglobulin in minutes. Electric
field mediated analysis of the released carbohydrates utilized fluorophore labeling by 8-aminopyrene-1,3,6-trisulfonic-acid, (APTS) in
the reaction mixture. Importantly, by replacing the conventionally used acetic acid catalyst with citric acid, as low as 1 to 10 glycan to
53
Lecture Abstracts
fluorophore molar ratio (vs the typical 1 to ³100 ratio) maintained the > 95% derivatization. This reduced level of APTS simplified the
removal of excess reagent, particularly important in CE-LIF (injection bias) and CE-MS (ion suppression). Importantly, desialylation was
neither observed during the high pressure cycle-assisted glycan release, nor with the use of the stronger acid catalyst in the fluorophore
derivatization reaction. This new sample preparation protocol readily supports large scale and high sensitivity glycan analysis of biopharmaceuticals by CE with decreased deglycosylation time and reduced APTS requirement for derivatization.
L58:
Mass Spectrometric Identification Of Aberrantly Glycosylated Human Apolipoprotein C-III Peptides In Urine From Schistosoma
Mansoni-Infected Individuals
Crina I. A. Balog*, Oleg M. Mayboroda, Manfred Wuhrer, Cornelis H. Hokke, André M. Deelder, Paul J. Hensbergen
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands
Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multi-cellular parasites that live in the mammalian
host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis
and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of S. mansoni-infected individuals, using a combination of glycopeptide separation techniques and in-depth mass spectrometric
(MS) analysis. The resulting fractions were then analyzed by MALDI-ToF MS and reversed phase nano-LC Ion Trap MS. Using this The urinary
peptides were first trapped on a C18 cartridge and subsequently fractionated using cation exchange chromatography followed by normal
phase chromatography. approach, we have characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived
from the C-terminus of human Apolipoprotein C-III (Apo C-III) in urine of S. mansoni-infected individuals which were not detected in urine of
non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for Apo
C-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of in depth mass
spectrometry analysis in combination with extensive chromatographic fractionation for the identification and characterization of O-glycopeptides, without prior knowledge of either the glycan or the peptide sequence. Furthermore, our data indicate for the first time that as a result
of S. mansoni infection, the glycosylation of a host protein is altered.
L59:
Nano LC-MS/MS Methods For O-Glycome Analysis
Gerhild Zauner*, Carolien Koeleman, André M. Deelder, Manfred Wuhrer
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands
Analysis of protein glycosylation is an important step in establishing the function which these glycans play in health and disease. In general
two strategies can be employed for oligosaccharide analysis: 1.) The glycoprotein of interest can be digested and the resulting glycopeptides
further analyzed for glycan identification. 2.) Releasing methods for oligosaccharides can be applied prior to glycan analysis. Our aim is to
develop suitable strategies for O-glycan detection which should be integrated in a combined N- and O-glycosylation analysis. Here we describe
two different approaches for O-glycan analysis: 1.) A protocol for glycopeptide dissection was applied in which asialofetuin and fetuin were
subjected to unspecific proteolysis by Proteinase K. The resulting (glyco)-peptide samples were resolved by nanoscale hydrophilic interaction
liquid chromatography (HILIC)-online mass spectrometry (MS). O-glycopeptides and N-glycopeptides eluted in different regions of the chromatogram, which facilitated characterization. Glycopeptides were analyzed by ion trap multistage MS. MS/MS and MS3 spectra served for
the oligosaccharide and the peptide identification, respectively. We identified the previously reported N- and O-glycan attachment sites for
asialofetuin/fetuin; in addition, one new O-glycosylation site was found. 2.) While techniques for N-glycan analysis are becoming routine, the
O-glycan analysis remains still a challenging task; we developed a combined O-glycan release and derivatization protocol followed by a simple
sample work-up step and mass spectrometric analysis. O-glycans from glycoproteins were released using dimethylamine (DMA). In order to
prevent degradation of the O-glycans by a reaction called “peeling”, their release was performed in the presence of 1-phenyl-3-methyl-5-pyrazolon (PMP), which promptly reacts with the reducing end of the freshly released O-glycan structures via a Michael type addition resulting in a
2:1 stoichiometry of PMP per glycan. Samples were worked up by vacuum centrifugation and solid phase extraction, followed by nano-LC-ESIIT-MS/MS analysis using a conventional reverse-phase (RP) nanocolumn. In conclusion, we here present two methods that allow 1.) the analysis
of Proteinase K-generated glycopeptides for O- as well as N-glycan analysis and 2.) a combined approach of O-glycan release and labeling for
54
Lecture Abstracts
a fast, comprehensive, and sensitive analysis of O-linked oligosaccharides. The latter method is currently evaluated for biological samples with
more complex O-glycan structures.
L60:
High Throughput Igg Glycosylation Profiling By MALDI-FTICR-MS
Maurice H. J. Selman*, Liam A. McDonnell, Magnus Palmblad, Andre M. Deelder, Manfred Wuhrer
Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Center, Leiden, the Netherlands
The biological activity of polyclonal immunoglobulin G (IgG) is modulated by the N-glycans attached to the Fragment cristallizable (Fc)
part. For example, lack of core-fucoses on these N-glycans may lead to a drastic enhancement of antibody-mediated cellular cytotoxicity (ADCC)1 . Moreover, anti-inflammatory properties of IgGs are dependent on sialylation of the Fc N-glycans2 . Potential strategies
for determination of IgG N-glycosylation involve glycopeptide analysis and released N-glycan analysis by mass spectrometry. Although
both strategies provide information regarding N-glycan heterogeneity, only IgG glycopeptide analysis allows discrimination of different
IgG subclasses and provides N-glycan profiles that are Fc specific. Here, we investigated the potential of different sample preparation
strategies for high throughput glycopeptide clean-up. Human polyclonal IgGs were purified from plasma by protein A affinity chromatography. Glycopeptides were generated by overnight cleavage with trypsin. Glycopeptides were then desalted and purified with
miniaturized 96 well reverse phase or hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction (SPE), spotted onto
polished steel MALDI plates (Bruker Daltonics, Bremen, Germany), overlaid with α-cyano-4-hydroxycinnamic acid (CHCA) and analyzed
on an APEX-ultra 9.4 T FTICR-MS equipped with a dual Apollo II source (Bruker Daltonics). Collisional cooling within the intermediate
pressure MALDI source allowed high mass resolution profiling of all IgG glycopeptides, providing information on IgG Fc fucosylation,
galactosylation, sialylation, and the incidence of bisecting N-acetylglucosamine. Both preparations provided clean and reproducible IgG
glycopeptide profiles. Elution of IgG glycopeptides from RP-SPE is achieved with ACN/water in the presence of TFA. Because sialylated
glycopeptides are unstable under acidic conditions the RP preparations required an additional lengthy drying step, reducing the sample
throughput. Elution of IgG glycopeptides from HILIC-SPE, in contrast, was achieved with water, thus no sample drying step was necessary. We applied the method incorporating 96 well HILIC sample preparation and MALDI-FTICR-MS to analyze a large cohort of healthy
volunteers showing age dependent glycosylation changes. Moreover, this method was applied to study IgG glycosylation changes in the
neurological disorder Myasthenia gravis revealing disease related IgG glycosylation changes. 1 Shields, RL, Lai, J, Keck, R, O'Connell, LY, Hong, K, Meng, YG, Weikert, SH, & Presta, LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves
binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem., 277, 26733-26740
2 Kaneko, Y, Nimmerjahn, F, & Ravetch, JV (2006) Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation. Science, 313, 670-673
L61:
Development Of A Fully Integrated Library Sample Preparation Station For Next Generation Sequencing
Stevan B Jovanovich*, Iuliu Blaga, Paul Butler, David Eberhardt, Corey Garrigues, Ezra van Gelder, Joanne Horn, Yuan Li, Bill Nielsen,
Mary Trounstine, Seth Stern
MBI, Pleasanton, USA
The massive throughput of Next-Generation Sequencing (NGS) has already revolutionized our understanding of biology by revealing unimagined information on the genome, epigenome, transcriptome, and the protein-nucleic acid interactome. Although the science is moving rapidly,
the current NGS workflows have created a severe bottleneck in library sample preparation that is limiting the rate of discovery. With the
introduction of 12-fold multiplexing, 96 samples can now be analyzed at a time—completely overwhelming existing infrastructure at most
facilities. In most sequencing centers, libraries are prepared manually with a skilled technician or scientist taking about two days to prepare
two to four libraries. To help researchers address the NGS sample preparation bottleneck, Microchip Biotechnologies Inc (MBI) is developing
a 24-channel fully automated and integrated library sample preparation system--the Apollo 300. The system will process input fragmented
DNA or total RNA using MBI Certified Reagents and optimized protocols to perform all the molecular biology workflows to produce libraries
ready for QC and amplification. This system uses 24-channel fluidic cartridges incorporating MBI’s patented Microscale On-Chip Valves (MO55
Lecture Abstracts
Veä valves) with a small footprint robot that can accept microtiter plates and other formats. The system will reduce the variation inherent in
manual library preparation, shorten the time to prepare libraries, decrease the reagent usage per sample significantly, allow for unattended
operation, and greatly increase throughput. The status of the Apollo 300 NGS Library Preparation System will be described. Data detailing the
implementation and validation of each workflow step for RNA and DNA library sample preparation will be shown and the integration of the
individual steps into completely automated workflows. A PhiX library created on a MOVe device using the fully integrated process has been
analyzed on the Illumina GA platform with performance equivalent to bench controls. The prospects for processing extremely small input
samples will be discussed as will medical sequencing and the needs of next next-gen sequencing.
L62:
Selecting Nanoporous Hydrogel Morphologies to Harness Entropic Trapping for Enhanced DNA Electrophoresis Separations
Nan Shi, Victor M. Ugaz*
Texas A&M University, College Station, USA
Electrophoretic DNA transport through nanoporous hydrogels is characterized by an entropic trapping (ET) mechanism when the DNA size is
close to the gel pore size. This mode of transport can yield highly desirable transport properties (e.g., enhanced size dependences of mobility
and diffusion for improved separations). But hydrogels have largely been ignored in efforts to understand and exploit ET effects, where the
focus has instead centered on simpler idealized “nanofilter” architectures based on arrays of alternating deep wells and narrow slits (i.e., describable by only two length scales). Here we describe a new model that captures the inherently heterogeneous pore morphology comprising
realistic hydrogel matrices, enabling key features of macromolecular transport in the ET regime to be successfully predicted. We then evaluate
our model in the context of double-stranded DNA electrophoresis using mobility and diffusion data simultaneously acquired in microchipbased experiments, where a photocurable hydrogel matrix allows the pore architecture to be tailored by adjusting the UV light intensity. In
this way, we are able to select polymerization conditions that produce pore morphologies favorable for ET, and harness these effects to achieve
improved separation performance (e.g., resolving power that increases with DNA size, the opposite of what is conventionally observed). In
addition to offering a new avenue to overcome existing resolution and read length limitations in DNA electrophoresis, these insights can help
direct efforts to design nanostructured materials with pore architectures specifically tailored to exploit the benefits of ET-dominated transport.
L63:
Microfluidic Chip System Integrating Sample Preparation From Physiological Samples To DNA Analysis
Meike Moschallski*1, Massimo Kubon1, Noor H. Abdul Halim1, Moritz Sandritter1, Kai Fuchsberger1, Britta Hagmeyer1, Susanne
Muench2, Karl-Heinz Boven3, Andreas Moeller3, Rainer Mohrlok3, 1 Martin Stelzl1
1 NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany
2 Institute for Technical Biochemistry at the University of Stuttgart, Stuttgart, Germany
3 Multi Channel Systems, Reutlingen, Germany
We present a microfluidic chip system that integrates comprehensive steps with sample preparation starting from “real-world” biological
samples until the DNA-analysis on-chip. The system will accept biological sample containing bacteria and transfer them into a low conductivity
medium by on-chip dialysis. From several mL of a sample bacteria are accumulated into a chamber of 38 µL by positive dielectrophoresis. For
this purpose the preconditioned sample is pumped through a microchannel comprising an array of interdigitated electrodes. An AC-voltage
is applied to the electrodes resulting in an inhomogeneous electric field that extends into the channel and induces dielectrophoretic forces
acting on the bacteria. High throughput is achieved by integration of a micromixer which enables trapping of up to 80% of bacteria even at
high flow rates. The concentration of bacteria in the starting sample can be determined in-situ by impedance spectrometry. This also allows
for an on-line monitoring of the accumulation process. In the third step the flow is stopped and the frequency of the electric field is lowered.
This induces lysis of the bacteria without the need for any lysis agents. This is due to a high voltage drop over the bacterial cell membrane at
low frequency in combination with a high osmotic pressure. The lysis of E. coli is confirmed by a live-dead staining assay. The bacteria release
DNA which is amplified and fluorescently labelled during PCR. Finally, the target DNA is successfully hybridized on a DNA microarray developed
for the detection of three single nucleotide polymorphisms (SNP) on the gyr A gene that confers fluorochinolone resistance upon E. coli.
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Lecture Abstracts
L64:
Nanocapillaries: enabling DNA and protein Separations in gel-free solutions
Xiayan Wang, Shaorong Liu*
Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, USA
In this work, we demonstrate use of nanocapillaries for DNA and protein separations in gel-free solutions. The nanocapillaries have radii
ranging from 500 nm to 2.5 mm, and separation efficiencies exceeding 106 theoretical plates/m are achieved. The operation procedure
is simple and straightforward. We fill a nanocapillary with an elute (often a borax solution), inject a DNA or protein sample into the
capillary hydrodynamically, elute the analytes via pressure, and detect the separate analytes on-column using a laser-induced confocal
fluorescence detector. For DNA, we have demonstrated separations of DNA from 76 to 106,000 base pairs in a single run. DNA separations are size-dependent, with longer molecules having shorter retention times. We have applied this technique for separations of realworld genotyping samples to demonstrate its feasibility in biological applications. PCR products (without any purification) amplified
from Arabidopsis plant genomic DNA crude preparations are directly injected into the nanocapillary, and PCR-amplified fragments are
well resolved, allowing for unambiguous identification of samples from heterozygous and homozygous individuals. For proteins, we have
achieved baseline resolution for transferrin, a-lactalbumin, insulin, and a -2-macroglobulin. However, their elution order is not size-dependent owing to the variations of ionic valence and molecular conformation from protein to protein. Since we use nanocapillaries, the
sample consumed is in the fL range and the reagent consumed is in the nL regime, and hence the consumable costs are extremely low.
L65:
Development And Application Of New Microchip CE DNA Assays For Point-Of-Care Testing In Routine Cancer Diagnosis And Therapy
Marcuss Gassmann1, Lucie Benesova2, Barbora Belsanova2, Richard Chudoba3, Bohuslav Gas3, Milos Pesek4, Marek Minarik2
1 Agilent Technologies, Waldbronn, Germany
2 Genomac International, Prague, Czech Republic
3 Faculty of Sciences, Charles University, Prague, Czech Republic
4 Faculty hospital Pilsen, Pilsen, Czech Republic
Over the past several years, the flagship application for DNA separation by capillary electrophoresis, the DNA sequencing, has slowly
been conquered by next-generation approaches (next-gen). These technologies, capable of analyzing events at a single-molecule level, are now being used for a wide variety of applications from the discovery of de novo sequences, massive mutation detection and
screening as well as gene expression by sequencing counting. There is no doubt that the next-gen techniques have already greatly
accelerated many areas of genomic research. However, the adorable ability to produce a draft sequence of an entire genome within a
relatively short period is still of a limited use in routine practice. In a typical clinical diagnostic setup of today only a handful of genetic
markers are being evaluated in each patient, rendering massive parallel approaches still too expensive, hence impractical. Therefore,
in spite of the widespread marginalization of the Sanger capillary-sequencing, capillary DNA separations are, for now, here to stay,
and, furthermore, new applications are being developed. The most prominent are DNA fingerprinting approaches based on fragment
analysis, sizing and quantification. Their most important use is for genetic identity typing in forensic casework, chromosomal typing in
prenatal diagnosis and, finally, DNA/RNA profiling in cancerous tumors or other diseased tissues.
The presentation will demonstrate a newly developed chipCE assay based on a modified BioAnalyzer platform and its clinical use in a
variety of application including detection of somatic mutations and gene amplification for non-invasive cancer monitoring, prediction
of therapy outcome and prognosis.
Supported by EU project OPPC Center for applied genomics of solid tumors (CZ.2.16/3.1.00/22213).
L66:
High Performance Separation And Detection Of Biogenic Compounds By Microscale Electrophoresis
Kenji Sueyoshi, Takayuki Kawai, Kota Hashiba, Ryuta Tanaka, Fumihiko Kitagawa, Koji Otsuka*
57
Lecture Abstracts
Department of Material Chemistry, Graduate School of Engineering, Kyoto University
Towards high performance separation and detection of biogenic compounds, several microscale techniques based on capillary electrophoresis
and microchip electrophoresis (MCE) were investigated. To enhance the detectability in microchip-based micellar electrokinetic chromatography (MEKC), an on-line sample preconcentration and separation technique named “transient-trapping (tr-trapping)” was developed1, in which
improvements of both the detectability and resolution can be achieved by the trap-and-release mechanism. The applicability of tr-trapping
to capillary-based MEKC was examined employing several hydrophilic amino acids (AAs) as model analytes as well as under the condition
of the suppressed electroosmotic flow (EOF). In the former case, the sample amino acids were labeled with the BODIPY dye to increase the
hydrophobicity of each amino acid. A mixture of the labeled AAs (BODIPY-AAs) was successfully resolved by tr-trapping–MEKC with an increased detectability. In the latter, hydrophobic steroids were used as model samples. Consequently, tr-trapping could be carried out under the
suppressed EOF condition and the sufficient preconcentration efficiency, e.g., a 490-fold increase for androsterone, was attained. As another
topic, high performance analysis of sugar chains by MCE using a unique on-line sample concentration technique, i.e., large volume sample
stacking with electroosmotic flow pump (LVSEP) [2], was investigated. In the LVSEP–MCE system, a straight microchannel was filled with a
sample solution followed by applying a constant voltage for the preconcentration and separation. The analysis of real sugar chains was carried
out by LVSEP–MCE on a poly(dimethyl siloxane) microchip. As a result, sugar chain analytes obtained from bovine ribonuclease B were well
concentrated and separated with a 2200-fold improvement of the detectability compared to the conventional microchip zone electrophoresis
employing the pinched-injection technique.
1 Sueyoshi, K.; Kitagawa, F.; Otsuka, K. Anal. Chem. 2008, 80, 1255–1262
2 He, Y.; Lee, H. K. Anal. Chem. 1999, 71, 995–1001
L67:
Multiplexed CGE-LIF For High-Throughput N-Glycan Analysis
L.Renee Ruhaak*1, Rene Hennig2, Carolin Huhn1, Erdmann Rapp2, Radboud Dolhain3, Andre M. Deelder1, Manfred Wuhrer1
1 Leiden University Medical Center, Leiden, The Netherlands
2 Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany
3 Erasmus Medical Center, Rotterdam, The Netherlands
The search for glycan-based biomarkers is still hampered by a lack of high-throughput methods for N-glycans analysis. Even though MS-based
methods which allow fast and repeatable analysis of such compounds have been developed, more quantitative techniques, as well as techniques based on fast separation of glycans are lacking. Recently, the use of multiplexed CGE-LIF using a DNA sequencer has been reported for the
rapid analysis of APTS-labeled glycans1. However, the derivatization method was not optimized, and size-exclusion chromatography was used
for sample preparation, resulting in either rather tedious procedures or largely impure samples, still containing substantial amounts of the
derivatization agent. We here present a high-throughput sample preparation method on the 96 well plate level for the analysis of N-glycans
derivatized with APTS. First, glycans are released enzymatically2. Second, glycans are labeled with APTS under conditions that are optimized
for a high labeling efficiency, short handling times and reduced risk of loosing sialic acids.Noticeably, the commonly used, but toxic reducing
agent NaCNBH3 is replaced by 2-picoline borane, which is non-toxic and yields superior results with respect to the robustness of the labeling.
Third, samples are subjected to HILIC purification on the 96 well plate level. Subsequently, purified APTS-labeled N-glycans are analyzed using
standard DNA-sequencing equipment. The method is robust and shows low relative standard deviations (12%) for the 13 major peaks for the
overall analytical method. Using this sample preparation strategy, which is suitable for robotization, we can analyze 96 samples with a handling
time of 2 hours. This method has been applied to a cohort of serum samples from pregnant women, which were sampled during first, second
and third trimester of pregnancy, as well as 6 weeks, 3 months and 6 months post partum. In addition to the previously reported increased
galactosylation of IgG Fc N-glycans during pregnancy3, which we corroborated, we will present novel findings on alterations in the total serum
N-glycome which are associated with the different stages of pregnancy. Overall, we present an optimized sample preparation technique for
high-throughput N-glycan analysis using multiplexed CGE-LIF. It represents a quantitative, robust and fast alternative to MS-based glycoanalytical strategies applicable to both clinical as well as pharmaceutical glycoanalytical questions.
1 Laroy et al. Nature protocols, 2006, 1 p.397
2 Ruhaak et al. Analytical Chemistry, 2008, 80 p.6119
3 Van de Geijn et al. Arthritis Research & Therapy, in press
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Lecture Abstracts
L68:
Ultra-High-Performance NanoLC-MS/MS Analysis Of Complex Proteomic Samples
Evert-Jan Sneekes, Bjorn* de Haan, Sebastiaan Eeltink, Remco Swart
Dionex Corp., Amsterdam, The Netherlands
Introduction Determination of the proteome and identification of biomarkers is required to monitor dynamic changes in living organisms
and predict the onset of an illness. One popular method to tackle contemporary proteomic samples is called shotgun proteomics, in which
proteins are digested, the resulting peptides are separated by high-performance liquid chromatography (HPLC), and identification is performed with tandem mass-spectrometry. Digestion of proteins typically leads to a very large number of peptides. For example digestion
of a cell lysate easily generates 500,000 peptides. The separation of these highly complex peptide samples is one of the major challenges
in analytical chemistry. Method The main strategy to improve the efficiency of packed columns is either to increase column length or by
decreasing the size of the stationary phase particles. However, to operate these columns effectively the LC conditions need to be adjusted
accordingly. Naturally, the on-line coupling to MS systems has to be taken into account in the optimization process. Preliminary data Here,
we report on the performance of nanoLC columns operating at ultra-high pressure. The effects of column parameters (particle size and
column length) and LC conditions (gradient time, flow rate, column temperature) were investigated with reversed-phase (RP) gradient nanoLC. High-resolution LC-MS separations of complex proteomic peptide samples are demonstrated by combining long columns with 2 µm
particles and long gradients. The effects of LC parameters on performance and the influence on peptide identification are discussed. Novel
aspect Ultra-high performance nanoLC by combined optimization of stationary phase, column length and LC conditions.
L69:
Investigation on Mixed Ligand Monolithic Stationary Phases for Capillary Electrochromatograophy
Samuel Karenga, Ziad El Rassi*
Oklahoma State University, Stillwater, USA
In this oral presentation, we wish to report on novel mixed ligand monoliths (MLM) for capillary electrochromatography of a wide range
of solutes differing in both polarity and size. The MLM capillary columns (100 µm I.D.) were based on different compositions of octadecyl
acrylate (ODA) and naphthyl methacrylate (NAPM) monomers in the presence of trimethylolpropanetrimethacrylate crosslinker and a ternary porogenic solvent composed of cyclohexanol, ethylene glycol and water. The monoliths are neutral having no fixed charges on their
surface; yet they exhibited cathodal electroosmotic flow (EOF). The EOF is attributed to the adsorption of ions from the hydro-organic
mobile phase onto the surface of the monoliths, thus providing a negative zeta potential, which is responsible for the generation of a
moderate EOF. As expected the magnitude of the EOF changed with the composition of the MLM. As the percent of the monomer ODA
in the polymerization mixture was increased the EOF increased to a maximum at 50 % ODA and then decreased at 75 % and 100 % ODA.
This indicates that the ODA ligand in general displays a higher binding for the mobile phase ions than the NAPM monomer. While ODA
provides solely nonpolar interactions, NAPM exhibits both nonpolar and π-interactions with certain solutes. Thus, columns with a given
composition of both ligands would in principle yield a unique selectivity for a given set of solutes that is not matched by columns made up
by either ODA or NAPM alone. In fact, experiments have shown that for solutes that display both nonpolar and π-interactions, there was
an optimum composition of NAPM and ODA that yielded the best selectivity and in turn retention and resolution. Several test mixtures
were used in the evaluation of these MLM columns including polycyclic aromatic hydrocarbons, alkyl ketones, nitroalkanes, alkylbenzenes,
toluene and benzene derivatives, and peptides. Peptide mapping of the tryptic digests of some standard proteins will be presented. Wednesday, March 24th, 2010 / NADIR 12:10 – 12:30
L70:
Imagination, Intuition And Considerations In The Form Of Analogies-Three Important Tools To Solve A Scientific Problem
Stellan Hjertén
Dept. of Biochemistry and Organic Chemistry, Biomedical Center, Uppsala University, P.O. Box 576, SE-751 23 Uppsala, Sweden
59
Lecture Abstracts
There seems to be a decline in basic knowledge in separation science among to-day students, an opinion many of my colleagues in several
countries shares with me. The fundamental reason might be that the requirements to pass an examination in mathematics, physics and
chemistry have continuously been lowered at high schools (and consequently at universities) in many countries. There are several articles
in newspapers, scientific journals etc. on this issue. Therefore, many Ph D students do not have the mathematical, physical, and chemical
knowledge to formulate a correct, precise theory, but still they may be interested in theoretical discussions. This lecture may be of some
interest to them and hopefully also to others. However, I will stress that a correct mathematical treatment is always to be preferred, but not
always accomplishable.
The parameters imagination, intuition and analogous considerations are likely used, more or less consciously, in all research activities,
particularly in the first phase of the exploration of a novel research field. I will illustrate by examples the importance of these parameters to encourage young people to take advantage of this approach already when they enter their research carrier. The reason why I
have chosen to derive known, mathematical expressions by this imagination/intuition/analogy approach is that then anyone can easily
judge the strength and the weakness of the approach, i.e. its accuracy. To derive these expressions in a mathematically correct way
requires good theoretical knowledge. The fact that in all the four examples below we will arrive at the correct expression is, of course,
no guarantee that the method always is applicable, rather that it may have the potential.
1) The derivation by imagination of the Einstein equation u/D=Ze/kT for the relation between the charge Z and the diffusion coefficient
D of a substance; u (the electrophoretic mobility) and D are parameter values at infinite dilution. I will also show how to calculate
u and D values at any buffer concentration when they are known at another concentration, starting with a very general equation
which seems to have been forgotten.
2) The derivation by intuition of the equation for the thermal zone broadening in electrophoresis, superimposed by diffusion.
The conventional method to derive this equation is mathematically relatively complicated.
3) The derivation by intuition/analogy of the equation for the broadening of a zone caused by sedimentation in the vertical segment
of the capillary with superimposed diffusion (in a CE experiment). I have published the equation in J. Chromatogr. A 1053 (2004)
181-199 without deriving it.
4) Considerations in terms of analogies is very often (unconsciously) used to solve a research problem. This approach paved the way
for my formulation of a completely novel method for the design of antibiotica with very little risk of resistance (S. Hjertén, to be
published). Probably there will be no time to refer to this very comprehensive study.
5) All research is diversified and all scientists have their own way to solve a research problem. Therefore I will not formulate any distinct
rules, only mention the above, few parameters which perhaps are of some general character.
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Poster Abstracts
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61
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Poster Abstracts
Poster Abstracts
Tuesday, March 23rd, 2010
POO1:
Rapid Identification and Quantification of Estrogens and their Conjugates in Urine by Capillary Electrophoresis-Electrospray
Ionization-Mass Spectrometry
Naomi L Janson, Philip Britz-McKibbin
Department of Chemistry and Chemical Biology, McMaster University, Hamilton ON, Canada
The objective of this research is to develop and validate a novel strategy for the identification and quantification of major urinary estrogens and their sulfate and glucuronide conjugates by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS).
Current methods of analysis for steroid conjugates in urine include immunoassays, gas chromatography-mass spectrometry (GC-MS),
and liquid chromatography-mass spectrometry (LC-MS). These methods are limited by time-consuming and labour-intensive sample
pretreatment, such as solid-phase extraction, chemical derivatization, and enzyme deconjugation. CE-ESI-MS represents a promising
alternative platform for the rapid analysis of urinary steroid conjugates without ion suppression that significantly reduces complicated
off-line sample handling compared to conventional assays. An experimental design was used to optimize ESI-MS parameters to allow
detection of nanomolar levels of major estrogen conjugates in urine after only a simple centrifugation and dilution step. Ten targeted
estrogens and their conjugates have been resolved by CE-ESI-MS, including isomeric and/or isobaric compounds, to allow reliable
quantification that is free of interferences from endogenous urinary metabolites as verified by spike and recovery experiments. In
addition, prediction of analyte migration behaviour and relative migration times is performed based on the intrinsic physicochemical
properties of the analytes, which represents a unique strategy in CE for the identification of unknown estrogen metabolites in cases
where authentic standards are unavailable.
Wednesday, March 24th, 2010
P002:
Glycoprotein Enrichment from Human serum substractions by Boronic Acid – Lecitin affinity Chromatography (blac) and their
Characterization by Multicapillary Electrophoresis and Mass Spectometry
Ákos Szekrényes1, Stefan Mittermayr2, Csaba Váradi1, Márta Kerékgyártó1, András Guttman1
1 Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Hungary
2 Horvath Laboratory of Bioseparation Sciences, University of Debrecen, Hungary / University of Innsbruck, Austria
Analysis of the human serum glycoproteome is a rapidly developing field holding the promise to reveal disease related alterations.
Boronic acid - lectin affinity chromatography (BLAC) has been reported to be an excellent tool to specifically partition and enrich various classes of glycoproteins providing enough material for downstream characterization by multicapillary electrophoresis and mass
spectrometry. In this work, we have evaluated the effect of mixing various ratios of boronic acid and wheat germ agglutinin lectin (1:1,
1:10, and 1:100) to check their efficiency and specificity in human serum glycoprotein partitioning. The enrichment capability of the
differently mixed BLAC columns was checked at the serum protein (intact proteins) and its subfractions, including reduced / denatured
preparations as well as various proteolitic digests (Trypsin, Pronase and Lys C). In addition to the protein level characterization, similar
evaluation was implemented at the glycan level after PNGase F release of the N-linked carbohydrates of the serum glycoprotein subfractions. The glycoprotein fraction specific BLAC enrichment method in conjunction with multicapillary gel electrophoresis was then used
to reveal N-linked glycosylation profile differences between normal and various stage lung cancer serum samples.
Monday, March 22nd, 2010
P003:
Study of the CE Profile of Alpha-1 Acid Glycoprotein (AGP) Isoforms as Biomarker of Cardiovascular Diseases
Angel Puerta1, Jose Carlos Díez-Masa1, Jose Luis Martin-Ventura3, Pedro Jesus Martin-Alvarez4, Coral Barbas5, Jose Tuñon3,
Jesus Egido3, Mercedes de Frutos1
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Poster Abstracts
1 Instrumental Analysis and Environmental Chemistry Department, Institute of Organic Chemistry (C.S.I.C.), Madrid, Spain
3 Fundación Jiménez Díaz – Universidad Autónoma, Madrid, Spain
4 Institute of Industrial Fermentations (C.S.I.C.), Madrid, Spain
5 Universidad CEU San Pablo, Madrid, Spain
Alpha-1 acid glycoprotein (AGP) is a serum glycoprotein with a pI in the range of 2.8 to 3.8 and a molecular weight around 41-43 kDa1,
2
. AGP presents several isoforms (molecules of the protein differing in its peptidic and/or glycosidic moieties). Changes in the isoforms
of AGP have been related to different pathological states such as cancer and liver or cardiovascular diseases (CVD), among others2-5.
Beside that, AGP is involved in the mechanism of the body response against CVD such as acute myocardial infarction, releasing AGP with
different glycosylation in comparison to normal AGP in plasma5. However, to our knowledge, the role of changes in the isoforms of AGP
as biomarker of CVD has not been addressed. As differences in the glycosylation or in the amino acid composition of the glycoprotein
can lead to differences in its total charge and/or mass, capillary zone electrophoresis (CZE) is a suitable technique for the analysis of
the isoforms of the intact AGP6. In this work, a preliminary study about differences in the CZE profile of intact AGP isoforms between
healthy individuals and patients with CVD has been performed. Two CVD, abdominal aortic aneurism (AAA) and carotid atherosclerosis
(CTA), were taken into consideration. Biological samples (plasmas, sera and supernatants from atherosclerotic plaques) were analyzed
by CZE after immunoaffinity chromatography (IAC) purification and concentration using methods previously developed in our group6, 7.
Comparison between samples was performed using the effective electrophoretic mobility as migration parameter. The high concentration of AGP obtained after IAC was enough to distinguish by UV detection up to 13 peaks corresponding to groups of isoforms of intact
AGP by CZE. Different supervised and non-supervised statistical techniques were employed to discriminate and classify CE profiles of
AGP samples from healthy donors taken at different hospitals and from patients with either AAA or CTA. Preliminary results indicate
that, besides the CVD, other variables such as additional clinical data, sex, etc should be taken into account to study the potential role
of AGP electrophoretical profile as biomarker of AAA or CTA.
1 Fournier, T., Medjoubi-N, N., Porquet, D., Biochim. Biophys. Acta 2000, 1482, 157-171
2 Ceciliani, F., Pocacqua, V., Curr. Protein Pept. Sci. 2007, 8, 91-108
3 Mackiewicz, A., Mackiewicz, K., Glycoconj. J. 1995, 12, 241-247
4 Kremmer, T., Szollosi, E., Boldizsar, M., Vincze, B., Ludanyi, K., Imre, T., Schlosser, G., et al., Biomed. Chromatogr. 2004, 18, 323-329
5 Poland, D. C. W., Vallejo, J. J. G., Niessen, H. W. M., Nijmeyer, R., Calafat, J., Hack, C. E., Van het Hof, B., et al., J. Leukoc. Biol. 2005, 78, 453-461
6 Lacunza, I., Sanz, J., Diez-Masa, J. C., de Frutos, M., Electrophoresis 2006, 27, 4205-4214
7 Ongay, S., Neususs, C., Vaas, S., Díez-Masa, J. C., de Frutos, M., Electrophoresis 2009, in revision
P004:
Biofunctionalized magnetic microparticles for efficient biomarkers capturing adapted for magnetic force-based microfluidic device
Zuzana Bilkova1, Zuzana Svobodova1, Hermann Esselmann2, Barbora Jankovicova1, Jana Kucerova1
1 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
2 Department of Psychiatry and Psychotherapy, Rhine-State Hospital, University of Duisburg-Essen, Essen, Germany
The goal was to set together and validate a panel of bioaffinity-based microseparation methods for isolatiton and purification of specific
proteins/peptides. The analytical approach based on the combination of biofunctionalized magnetic micro/nanoparticles with microfluidic analytical device (μ-TAS) enables to capture and detect desired biomolecules occuring at trace amounts in a complex biological
material avoiding the time-consuming separation steps before final MS analysis. For quantitative capturing of target biomolecules we
developed carrier with site-specific immobilized antibodies. IgG molecules have a number of potential immobilization sites corresponding to particular functionalities on the molecules, these groups can be polypeptide-derived or associated with post-translational modification. Efforts were being exerted to prepare carrier with high binding capacity, steric accessibility of active sites, high operational and
storage stability and low non-specific sorption to minimize the contamination of the target analyte. The binding and elution conditions
for antigen were precisely adjusted considering the high complexity of applied biological samples. Based on described strategy we
have just prepared immunosorbent for specific capturing of PrP cellular isoforms from brain homogenate and cerebrospinal fluid1,2. The
epitope extraction technique based on magnetic microspheres self-organized in a channel of microfluidic device has also been applied
for isolation and identification of main immunogenic epitopes of the ovalbumin (OVA), a typical representative of food allergens3. Alzheimer disease (AD) is the most prevalent cause for dementia in the elderly population today. Binding efficiency and specificity of newly
developed immunosorbents with anti-Aß or anti-Tau IgG molecules were validated by Aß peptides or Tau protein immunoprecipitation.
Consequently the carriers were integrated into microfluidic device and applied for isolations of AD biomarkers from spiked or real ce63
Poster Abstracts
rebrospianl fluid (CSF). Based on this simple and versatile approach we can assess new candidates of biomarkers and study their role in
patogenesis of related disease. The miniaturization and integration of innovative detection technologies will greatly extend the sensitivity
of biomarker detection, and thus improve the validity of clinical outcomes.
This work was supported by the E.C. project NeuroTAS No. 037953 and partly by the Ministry of Education, Youth and Sports of the
Czech Republic (MSMT 0021627502). Great thanks go to long-standing research colaborator Dr. Jean-Louis Viovy from Institute Curie,
Paris (France) for PDMS chip providing.
1 Le Nel A. et al., Lab Chip. 2008 Feb;8(2):294-301
2 Bilkova Z. et al., Proteomics. 2005 Feb;5(3):639-47
3 Jankovicova B. et al., J Chromatogr A. 2008 Oct 3;1206(1):64-71
P005:
Study of metallothionein aggregation in dependence on redox conditions and zinc concentration by using a chip capillary electrophoresis
Sona Krizkova1, Michal Masarik2, Tomas Eckschlager3, Rene Kizek1
1 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University, Zemedelska 1, CZ-613 00 Brno, Czech Republic
2 Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Komenskeho namesti 2, CZ-662 43 Brno, Czech Republic
3 Department of Paediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University, V Uvalu 84, CZ-150 06 Prague 5, Czech Republic
Metallothionein (MT) is a ubiquitous cysteine-rich protein. One of the most known MT’s functions is heavy metals detoxification and
homeostating. Nowadays it is known, that MT is involved in regulation of gene expression, transcription, embryogenesis, immunomodulation and its antiapoptotic function was discovered recently. In cancer diseases, MT is associated with its diagnostics, course, prognosis
and metastating. Enhanced MT expression leads to the formation of cytostatics resistance in tumour cells. Molecular mechanisms of
these processes are still insufficiently known and understood. However it is known, that MT’s function is closely related to its secondary
and tertiary structure. Under physiological conditions, MT contains Zn(II) ions chelated in cysteine clusters. Single MT molecules differ in
zinc load and oxidation state. Recently it was found, that MT can form function aggregates in dependence on redox conditions and heavy
metals concentration in the cell. These aggregates are probably involved in fine regulation of Zn concentration and therefore to activation
or deactivation of subsequent cell processes. Compared to monomer MT forms, MT aggregates exhibit a higher binding capacity for heavy
metals. Determination and evaluation of aggregated and monomer MT form in heavy metals homeostating could bring a new insight into
biological processes linked to heavy metals.
To study MT aggregates sophisticated methods and procedures are necessary. Capillary electrophoresis seems to be suitable for separation of MT isoforms. In this work we used a capillary electrophoresis on chip with fluorescence detection. Rabbit liver MT was used as
standard with detection limit of 100 ng with RSD of 3.5 %.
In following experiments MT was oxidized with H2O2 in concentrations of 0, 0,1, 0.5, 1 and 3 % (v/v) for 2 h at 37°C. We observed aggregates at Mr of 52, 33, 22 and 12 × 1000. With increasing H2O2 concentration the proportion of aggregated MT increased. The same
aggregated were observed also after oxidation of MT with 0.5 % H2O2 in dependence on temperature (4, 10, 25, 30 and 37°C). Changes
in MT aggregation were successfully reversed with dithiotreitol (DTT). After incubation of MT with Zn2+ (0, 5 and 100 × 10-6 M) similar
results were observed in dependence on Zn concentration.
Experimental data were analyzed also by using SDS-PAGE. MT was visualized by western-blotting using chicken anti-MT antibodies. In
addition to this, aggregated MT forms were confirmed with electrochemical methods.
The obtained results suggest that MT aggregation can be studied with the used methods.
This work was supported from IGAMZ 10200-3, GACR 301/09/P436 and GAAV IAA401990701
P006:
Determination of the oxidative stress markers isoprostanes in sea animal tissues
Daniela Mácová*, Angela Helene Squier, Josef Čáslavský, Kenneth Boyd, Stuart Gibb
F2-isoprostanes (F2-IsoPs) are a group of prostaglandin-like compounds that are formed following the oxidation of arachidonic acid, through
64
Poster Abstracts
a free radical-catalyzed mechanism. They are considered to be sensitive and specific indicators of lipid oxidation both in vitro and in vivo.
Arachidonic acid is polyunsaturated fatty acid (20:4; n-6). Four groups of F2-isoprostanes are formed as a result of free radical attack at three
of these double bonds with 16 diastereoisomers in each of these groups. These groups can be distinguished on the basis of the characteristic fragmentations in tandem mass spectrometry. Lasting recent years a lot of methods for their determination in biological samples have
been developed. These usually include at least one sample clean-up step followed in most cases final analysis by GC-MS or to a lesser extent
by LC-MS; in exceptional cases a combination of both is applied. Due to IsoPs structure containing three hydroxyl groups and one carboxyl
acid group the GC-MS method requires a derivatization step. In our method we combined HPLC chromatographic separation to minimize
sample preparation with identification of each type of isoprostane regioisomer by tandem mass spectrometry. In addition, to date, F2-IsoPs
studies have in almost all cases focused on the evaluation of oxidative damage in mammals, and in particular, humans. Arachidonic acid is,
however, widespread in the animal kingdom, so there is potential for much wider application of IsoPs as indicators of oxidative stress. As part
of an ongoing effort to investigate the effects of oxidative stress on the phenomenon of coral bleaching, we have applied this method to the
phylogentically related sea anemone, Actinia equina. In the present study, sea anemones were oxidatively stressed under different laboratory
conditions. IsoPs formed from arachidonic acid were released by alkali hydrolysis, partially purified by solid phase extraction and quantified
by liquid chromatography-tandem mass spectrometry with electrospray ionization (HPLC-ESI MS2).
P007:
Acid phosphatase from potato tubers immobilized onto magnetic beads: efficient system for phosphoprotein analysis
Libor Kubosek1, Jana Frydlova2, Marie Ticha3, Zdenka Kucerova2
1 1) Institute of Pathological Physiology and CEH, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Praha 2, Czech Republic
2) Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, Universi
2 Institute of Pathological Physiology and CEH, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Praha 2, Czech Republic
2) Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 2030
Protein phosphorylation is involved in many important cellular events and abnormal phosphorylation is associated with many serious human
diseases, e.g. gastric cancer. The investigation and characterization of proteins involved in these processes as well as understanding a role of
the protein modification represent still a difficult task. Several experimental strategies can be employed in the phosphoproteomic studies.
The application of a phosphatase treatment and the comparison of the behavior of phosphorylated and dephosphorylated protein represent
one of such approaches. Contrary to proteinases immobilized to various materials that are often used in the proteome research, immobilized
phosphatases have been only rarely applied in the phosphoproteome analysis.
The use of enzymes immobilized to magnetic beads has several advantages as compared with an application of soluble forms of enzymes: an
increased stability of enzymes, an easy removal of a modifying enzyme from a reaction mixture, a possibility of direct use of enzyme reaction
products for MS analysis, or for a further reaction, e.g. proteolytic digestion. In our experiments, acid phosphatase from potato tubers was
immobilized to Glyoxal agarose magnetic beads (BioScience Bead Division of CSS, USA). Immobilized acid phosphatase was characterized in
comparison with the soluble enzyme by higher stability at increased temperatures and broader pH optimum. The activity of acid phosphatase coupled to magnetic beads was not significantly changed after repeated uses. Kinetic parameters were assayed using p-nitrophenyl
phosphate as a substrate for both the soluble and the immobilized enzyme. The value of Km (1.0 mM) for the immobilized enzyme was higher
than that for the soluble one (0.6 mM). Acid phosphatase from potato tubers immobilized to magnetic particles was successfully used for the dephosphorylation of gastric aspartic
proteinase - porcine pepsin A as a model phosphoprotein. Therefore, the prepared magnetic beads containing immobilized acid phosphatase
could be used in the analysis of phosphorylation state of human aspartatic proteinases that could serve as biomarkers of serious gastric
diseases.
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project CEH
LC 06044) and by the Czech Science Foundation (grant 203/09/0857).
P008:
Aptamer-Facilitated Biomarker Discovery (AptaBiD)
Maxim V. Berezovski1, Matthias Lechmann2, Michael U. Musheev3, Tak W. Mak4, Sergey N. Krylov3
65
Poster Abstracts
1 University of Ottawa
2 University of Erlangen
3 York University
4 University of Toronto
We introduce a technology for biomarker discovery in which (i) DNA aptamers to biomarkers differentially expressed on the surfaces of
cells being in different states are selected; (ii) aptamers are used to isolate biomarkers from the cells; and (iii) the isolated biomarkers
are identified by means of mass spectrometry. The technology is termed aptamer-facilitated biomarker discovery (AptaBiD). AptaBiD
was used to discover surface biomarkers that distinguish live mature and immature dendritic cells. We selected in vitro two DNA aptamer pools that specifically bind to mature and immature dendritic cells with a difference in strength of approximately 100 times. The
aptamer pools were proven to be highly efficient in flow- and magnetic-bead-assisted separation of mature cells from immature cells.
The two aptamer pools were then used to isolate biomarkers from the cells. The subsequent mass spectrometry analysis of the isolated
proteins revealed unknown biomarkers of immature and mature dendritic cells.
P009:
Generation and characterization of a comprehensive analyte library representing the human plasma proteome
András Kovács1, Edit Sperling1, László Takács3, János Kádas3, István Kurucz3, András Guttman6
1 Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Hungary
3 BioSystems International Ltd., Debrecen, Hungary
6 Horváth Laboratory of Bioseparation Sciences, University of Debrecen, Hungary; BioSystems International Ltd., Debrecen, Hungary
Characterization of the human plasma proteome is a highly challenging task due to the large dynamic range (up to 12 orders of magnitude) in the concentration of the individual proteins it contains. Therefore, methods that increase the detectability of low abundant
plasma proteins are of high interest. We have generated a comprehensive set of fractions, representing the entire human plasma
proteome, referred to as the analyte library, using combined chromatographic and precipitation techniques, resulting in hundreds to
thousands of well-defined fractions, each containing of some 10-100 intact proteins. First the large human serum albumin and immunoglobulin content were removed from 500 mL normal pooled human plasma by albumin and IgG specific partitioning methods and the
resulting depleted plasma was pre-fractionated by ammonium sulfate precipitation. Each fraction was then further separated by size
exclusion chromatography, followed by cation and anion exchange chromatography. The interim and final fractions were first analyzed
by SDS-PAGE and screened by ELISA and/or microarray techniques with antibodies specific for various diseases. Fractions that proved
positive are being used for the identification of the individual protein antigens as part of our biomarker discovery endeavor.
P010:
Magnetic IDA-modified hydrophilic methacrylate-based polymer microspheres for IMAC phosphopeptide enrichment
Tereza Emmerova1, Lenka Novotna2, Petr Prikryl2, Daniel Horak4, Zdenka Kucerova2, Marie Ticha1
2) Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 12
2 Institute of Pathological Physiology and CEH, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Praha 2, Czech Republic
4 Institute of Macromolecular Chemistry, AS CR, Heyrovsky Sq. 2, 162 06 Praha 6, Czech Republic
Protein phosphorylation-dephosphorylation is one of the major signaling mechanisms for modulating of protein functional properties.
Non-regulated phosphorylation is associated with many human diseases. Therefore, the detection and characterization of protein phosphorylation can contribute to better understanding of cellular processes and identification of disease biomarkers.
Phosphoprotein investigation is not an easy task, as these modified proteins mostly belong to low abundant ones. For this reason
many strategies used in phosphoproteome analysis involve an enrichment step based on different principles. Immobilized metal affinity
chromatography (IMAC) represents one of them.
The aim of our study was to apply magnetic nonporous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres modified with iminodiacetic acid (IDA) for the IMAC separation of phosphopeptides. Fe(III) and Ga(III) ions immobilized on mag66
Poster Abstracts
netic IDA-modified microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins
differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine a-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The adsorbed phosphopeptides
from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project
CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857)
P011:
Serum Biomarker Discovery by Quantification of Glycoproteins with Dimethyl Labeling of Non-glycopeptides
Rui Chen, Mingliang Ye, Hanfa Zou
CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences
A robust, reproducible and high-throughput method was developed for the quantitative analysis of concentration changes of glycoproteins in human serum. This method consists of the capture of glycoproteins in serum samples and the release of non-glycopeptides
by trypsin digestion of captured glycoproteins followed by dimethyl labeling the released non-glycopeptides. The labeled samples
from different state are mixed and simultaneously analyzed by two dimensional LC-MS and the mass difference of the dimethyl labels
is used to compare the peptide abundance in the different samples. This method showed almost the same specificity and sensitivity
in glycoprotein identification compared with capture at glycopeptide level. By combination with isotope labeling of the released nonglycopeptides, reproducible quantification of glycoproteins was carried out to study the serum biomarker discovery of liver cancer. The
differential abundance of proteins present at ng/ml levels was quantified with high confidence. The established method was applied
to the analysis of human serum samples from healthy people and patients with primary hepatocellular carcinoma and a number of
glycoproteins, including the currently used biomarker, alpha-fetoprotein were found with substantial concentration changes between
normal and disease serum sampleswhich suggested this method was a good way to find potential biomarkers and demonstrated the
potential application in the biomarker discovery for other diseases.
P012:
Biomarkers of gastric cancer – detection using magnetic sorbent with immobilized peptide inhibitors
Michaela Filuszová1, Marie Tichá2, Zdenka Kučerová1
1 Institute of Pathophysiology and CEH, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Praha 2, Czech Republic
2 1) Institute of Pathophysiology and CEH, First Faculty of Medicine, Charles University in Prague, U Nemocnice 5, 128 53 Praha 2, Czech Republic
2) Department of Biochemistry, Faculty of Science, Charles University in Prague, Albertov 30, 128 40 Praha
Aspartic acid proteinases present in gastric juice are secreted as pepsinogens and transformed to pepsins at acid pH. The presence
and relative concentration of pepsin A (EC 3.4.23.1, pepsin) and pepsin C (EC 3.4.23.3, gastricsin) vary according to vertebrate species,
genetic variation, and in human, with different gastric diseases. Content and changes in the ratios between individual human aspartic
proteinases and their zymogens were found to be important from the diagnostic point of view: e.g. low concentration of pepsinogen A
in serum was found to be a marker of gastric cancer. Affinity based separation technique is one of the possibilities to separate individual
proteinase forms and thus to make possible their level evaluation. Synthetic inhibitors as immobilized ligands are the best choice for
this purpose, as other possible substances either interact with aspartic proteinase weakly (e.g. compounds derived from substrates) or
too strong (e.g. natural inhibitors). Aim of the present study was to elaborate a simple and rapid method of affinity separation of gastric
aspartate proteinases using peptide-modified magnetic particles. Two synthetic heptapeptides containing D-amino acid residues were chosen (Val-D-Leu-Pro-Phe-Phe-Val-D-Leu and Val-D-Leu-Pro-TyrPhe-Val-D-Leu) differing in the substitution of one L-phenylalanine residue with L-tyrosine one. The effect of the synthetic heptapeptide
inhibitors on the activity of porcine pepsin A and rat pepsin C was investigated: a type of inhibition was evaluated and values of inhibition constants and were determined. Both heptapeptide inhibitors were immobilized to Glyoxal agarose magnetic particles (BioScience,
USA) and used to study the separation of pepsin A and pepsin C.
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Poster Abstracts
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project
CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857).
P013:
Peptide mapping by CZE of tau protein, a biomarker of Alzheimer’s disease
Céline Riviere1, Diego Bohoyo1, Myriam Taverna1, Hans Klafki4, Jens Wiltfang4, Barbora Jankovicova6, Marcela Slovakova6,
Zuzana Bilkova6, Florence Poirier9, Isabelle Le Potier1
1 Laboratoire des Protéines et Nanotechnologies en Sciences Séparatives, UMR CNRS 8612, Faculté de Pharmacie, Châtenay-Malabry, France
4 University of Duisburg-Essen, LVR-Klinikum Essen, Department of Psychiatry and Psychotherapy, Essen, Germany
6 Department of Biological and Biochemical Sciences, University of Pardubice, Czech Republic
9 Service protéomique, IFR-141, Faculté de Pharmacie, Châtenay-Malabry, France
Alzheimer’s disease is a neurodegenerative disorder that causes progressive loss of mental functions. Established biomarkers for this
disease are elevated levels of Tau protein and reduced amyloid-beta (1-42) peptides in cerebrospinal fluid. Tau belongs to the microtubule-associated proteins (MAP) family. In the human brain, six isoforms are expressed. Hyperphosphorylated tau isoforms are the
major components of the neurofibrillary lesions found in Alzheimer's disease and other tauopathies. Our project intends to develop an
analytical methodology based on CE to quantify in biological fluids, the ratio between phosphorylated and normal Tau protein. Considering the high number of potential phosphorylation sites, the existence of six tau isoforms in adult human brain and the possible
occurrence of truncations, the analysis of intact tau may become quite tricky. Thus, our approach has been to focus on fragments of
tau, obtained after an optimized procedure of chemical cleavage of tau. One of the six theoretically generated peptides is expected to
be common to all the Tau isoforms. We have used the lab on chip bioanalyser and developed CZE conditions to monitor not only the
cleavage efficiency on tau, but also to establish a high resolution peptide mapping of this protein. Both BGE and coating conditions
were optimized. Assignments of peaks were performed by combining CE to other analytical approaches such as immunoprecipitation,
HPLC, preparative SDS–PAGE and mass spectrometry techniques. Finally differences in the CE profile for normal Tau and phosphorylated Tau were highlighted.
P014:
Immunomagnetic Separation on Chitosan Coated Ferrite Nanoparticles: a Tool for Biomarkers Quantification
Kamila Syslová1, Petr Kačer1, Marek Kuzma3, Štěpánka Vlčková4, Zdeňka Fenclová4, Jindřiška Lebedová4, Daniela Pelclová4
1 Institute of Chemical Technology, Prague, Czech Republic
3 Institute of Microbiology Czech Academy of Science, Prague, Czech Republic
4 Department of Occupational Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic
A new analytical method combining immunomagnetic separation on chitosan coated magnetic particles with highly sensitive and
selective LC-MS detection for quantification of biomarkers derived from basic organism structure units was developed. The analytical
procedure covers monitoring of 8-iso-prostaglandin F2a (= 8-isoprostane), o-tyrosine and 8-hydroxy-2’-deoxy-guanosine as a group of
oxidative stress markers in various body-fluids (blood plasma, urine, exhaled breath condensate, cerebrospinal fluid). Oxidative stress
results from an imbalance between production of free radicals and antioxidant defenses. With the cells’ natural antioxidant mechanisms overwhelmed, the excess reactive oxygen species may cause radical-mediated damage to biomolecules. In humans, oxidative
stress is involved in many diseases, such as atherosclerosis, Parkinson's disease and Alzheimer's disease, but it may also be important
in prevention of aging. 8-isoprostane is produced by non-enzymatic direct oxidation of arachidonic acid on the cell surface by oxygen
radicals. It is generally well-established as the most significant biomarker of oxidative stress. Oxidation of amino acids and nucleic acids
produces other markers of oxidative stress like o-tyrosine and 8-hydroxy-2’deoxy-guanosine.
The separation method consisted in immunomagnetic extraction by chitosan coated ferrite nanoparticles with anchored monoclonal
antibodies (immunoglobulines) against particular biomarkers present in low concentrations in various biological matrixes. LC-ESI-MS/
MS detection method operated in single reactions monitoring (SRM) mode was used for its exceptionally high degree of selectivity, and
stable-isotope-dilution assay for its high precision of quantification. The developed method allowed unequivocal parallel determination
of oxidative-stress biomarkers at the same run. The method was optimized and validated. The precision was higher than 84.3 %, the
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Poster Abstracts
mean accuracy (relative error) was determined lower than 11.6 %. Finally, the method was tested on real samples collected from patients
with different oxidative stress induced diseases and on the control group of healthy subjects.
P015:
Diagnosis of enzyme defects in the second part of purine de novo synthesis by UHPLC-tandem mass spectrometry
Hana Krätschmerová, David Friedecký, Tomáš Adam
Laboratory for Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital and Palacký University, Olomouc, Czech Republic
Background: Enzyme defects of purine de novo synthesis (PDNS) (adenylosuccinate lyase deficiency, AICA-ribosiduria) known to date
are characterized by abnormal concentrations of substrates in cells and their excretion into body fluids in dephosphorylated forms. The
aim of this study was to develop a method for determination of all ribosides related to the second part of PDNS.
Methods: Reversed-phase UHPLC (ACQUITY UPLC®BEH C18 1.7 um; 2.1 × 100 mm; Waters) with electrospray ionization tandem mass
spectrometry (API 4000, Applied Biosystems) was used to analyse 6 ribosides of purine metabolism. Gradient elution using eluent
A - 20 mM formic acid - ammonium hydroxide (pH 4) and eluent B - a mixture of solvent A and methanol (1:1 v/v) was applied. Compounds were analysed by multiple-reaction monitoring in positive mode (m/z for 5-amino-4-imidazole riboside: 216->84; 5-amino-4imidazolecarboxy riboside: 260->128; 5-succinylamino-4-imidazolecarboxamide riboside: 375->243; 5-amino-4-imidazolecarboxamide
riboside: 259->127; 5-formamino-4-imidazolecarboxamide riboside: 287->155 and succinyladenosine: 384->252).
Results: Total time of analysis was 11.5 min. Limit of detection was in the range 0.1–1.7 umol/L. Reference ranges were established for
each riboside on 200 urine samples from healthy children. Analysis of ADSL deficient urines and urines spiked by ribosides revealed
major differences in tandem mass spectrometry profiles compared with those of control samples.
Conclusions: This method enables reliable and rapid detection of all the studied ribosides, and it can be used for diagnosis of known
and also not so far identified defects in the second part of PDNS.
Supported by grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism and the Norwegian Financial Mechanism (A/CZ0046/2/0011) and MSM6198959205.
P016:
Solid phase extraction of organic acids from Salicornia herbacea L. using ionic liquids-based monolithic column
Kyung Ho Row, Tao Zhu
Inha University, Korea
A solid phase extraction (SPE) method for determination of organic acids in Salicornia herbacea L. using ionic liquids-based monolithic
column was developed. An ionic liquids-based monolithic column was prepared by in situ polymerization technique, and the influence
of porogenic composition, monomer–ionic liquids ratio and polymerization conditions on the chromatographic performance were investigated. The composition of the mobile phase and column temperature were characterized. Based on the optimal separation condition,
the interfering substances and organic acids in natural plant samples could be separated successfully using monolithic column, and
C18 column was used to determinate the organic acids in in Salicornia herbacea L. Good linearity, high recovery and high extraction
efficiency of this method were obtained for these organic acids. The HPLC method, developed in this study, was proved to be acceptable
for drugs assay, and this ionic liquids-based monolithic column as the stationary phase was a potential tool for future HPLC separation.
P017:
Electrochromatographic retention of peptides on strong cation-exchange stationary phases
Ivo Nischang1, Alexandra Höltzel2, Ulrich Tallarek2
1 Johannes-Kepler-University Linz, Institute of Polymer Chemistry, Welser Strasse 42, A-4060 Leonding, Austria
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Poster Abstracts
2 Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany
We analyze the systematic electrical field-dependence of electrochromatographic retention for counterionic peptides on a strong
cation-exchange (SCX) stationary phase. Our experiments show that the retention behavior in the studied system depends on the
charge-selectivity of the stationary phase particles, the applied voltage, and the peptides’ net charge1. Retention factors of twice positively charged peptides decrease with increasing applied voltage, while lower charged peptides show a concomitant increase in their
retention factors. The observed behavior is explained on the basis of electrical field-induced concentration polarization (CP) that develops around the SCX particles of the packing and which has been successfully visualized by confocal laser scanning microscopy2, 3. The
intraparticle concentration of charged species (including buffer ions and charged analyte) increases with increasing applied voltage due
to diffusive backflux from the enriched CP zone associated with each SCX particle2. For twice charged and strongly retained peptides
the local increase in mobile phase ionic strength reduces the electrostatic interactions with the stationary phase, which explains the
decrease of retention factors with increasing applied voltage and CP intensity. Lower charged and weaker retained peptides experience
much stronger relative intraparticle enrichment than the twice charged peptides1. It results in a net increase of retention factors with
increasing applied voltage similar to small single positively charged drug molecules3. The CP-related contribution to electrochromatographic retention of peptides on the SCX stationary phase is modulated by the applied voltage, the mobile phase ionic strength, and
the peptides’ net charge and could be used for selectivity tuning in difficult separations beyond that achievable by pure electrophoretic
and chromatographic contributions alone.
1 Nischang I, Höltzel A, Tallarek U (2010) Electrophoresis in press
2 Nischang I, Reichl U, Seidel-Morgenstern A, Tallarek U (2007) Langmuir 23:9271-9281
3 Nischang I, Spannmann K, Tallarek U (2006) Anal Chem 78:3601-3608
P018:
Preparation of zirconia monolithic column for enantioseparation by capillary electrochromatography
In Whan Kim1, Avvaru Praveen Kumar2, Jung Hag Park2
1 Daegu University
2 Yeungnam University
This work reports preparation of monolithic zirconia chiral columns for separation of enantiomeric compounds by capillary electrochromatography (CEC). Using sol-gel technology porous zirconia monoliths of interconnected skeleton structure with through pores were
synthesized in the capillary column, and then the surface of the monoliths was coated with cellulose tris(3,5-dimethylphenylcarbamate)
(CDMPC) to obtain a monolithic chiral column (CDMPCZM). The process of the preparation of the zirconia monolithic column was investigated by examining the effects of concentration of the components of the sol solution that include zirconium n-butoxide, polyethylene glycol, water and acetic acid as a complexant on the formation of porous zirconia monolithic structure. The electroosmotic flow of
and enantioseparation behavior of a set of racemic analytes on CDMPCZM were measured in reversed-phase mobile phases of varying
pH, organic composition and applied voltage.
P019:
Preparation and use of porous polymer monoliths in micrometer-sized spatial confinement
Ivo Nischang, Oliver Brüggemann
Johannes-Kepler-University Linz, Institute of Polymer Chemistry, Welser Strasse 42, A-4060 Leonding, Austria
Porous polymer monoliths have emerged as material for a wide variety of applications including liquid chromatographic analyses at an
unrivaled speed, solid phase extraction, and enzyme immobilization in capillary and microfluidic chip format. We present the state of
the art in the preparation of monoliths in capillaries and microfluidic chips via free radical polymerization processes and their miniaturization under conditions of spatial confinement with a focus on morphological aspects1. Monolithic columns have been prepared
successfully in conduits with an inner diameter of 5 to 10 µ m using thermally initiated free radical polymerization with the aim to
maintain the desirable properties of their porous structure found in larger diameter capillaries2. The effect of confinement resulting
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Poster Abstracts
from the decreased size of the capillary on both the preparation and the properties of the monoliths can be significant1, 2. Our results
indicate that the downward scalability of the monolithic columns is greatly affected by the confining wall and its increasing surface
to volume ratio at decreasing capillary sizes. The most important variables that affect the downscaling process are the polymerization
temperature (determining polymerization kinetics), the diffusion of the propagating radicals, and the density of coverage of polymerizable groups on the inner walls of the capillary rendering it reactive. Under successful downscaling conditions, the formation of undesired
dense polymer layers attached to the capillary wall was minimized, which significantly introduces a radial density distribution of porous
polymer monoliths. Such wall effects are not desirable for their efficient application in nano-LC. The chromatographic performance of
monolithic columns with identical flow properties in a variety of shapes and sizes of the conduit was evaluated in the reversed phase
gradient separation of proteins2,3. Further, the suitability of the prepared monoliths for analysis of both large and small molecule is also
discussed. Therefore we address the origin of their poor performance in the separation of small molecules under isocratic equilibrium
elution conditions. Our results identify the most significant contribution to band broadening and provide guidance for the most promising routes enabling fabrication of porous polymer monoliths suitable in the separation of small analyte molecules1,4.
1 Nischang I, Brüggemann O, Svec F (2010) Anal Bioanal Chem submitted
2 Nischang I, Svec F, Fréchet JMJ (2009) Anal Chem 81:7390-7396
3 Nischang I, Svec F, Fréchet JMJ (2009) J Chromatogr A 1216:2355-2361
4 Nischang I, Brüggemann O (2010) manuscript in preparation
P020:
Fabrication of C18-silica hybrid monolithic capillary column using organic monomer and alkoxysilane
Minghuo Wu, Zhenbin Zhang, Ren‘an Wu, Fangjun Wang, Hanfa Zou
Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China
A C18-silica hybrid monolithic column was prepared via the “one-pot” process. In which, the alkoxysilanes precursors of tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS) was firstly hydrolyzed in a 0.01 M acetic acid solution, and then the organic monomer
of N-(2-(methacryloyloxy)-ethyl)-dimethyl- octadecylammonium bromide (MDOAB) for introducing the C18 moiety to the monolith along
with the initiator of AIBN were added into the hydrolyzed solution. After sonicated for 15 min, the mixture was introduced into a pretreated capillary. The polycondensation of alkoxysilanes and the copolymerization of organic monomers and as-precondensed siloxanes
were subsequently carried out at proper reaction conditions. The morphologies of the synthesized organic-silica hybrid monolithic
columns were characterized by scanning electron microscopy (SEM). The performances of the resultant C18-silica monolithic columns
were investigated by capillary electrochromatography (CEC) and the results confirmed that the MDOAB was successfully incorporated
into the silica monolithic matrix. Furthermore, the C18-silica hybrid capillary monolithic column was applied in the analysis of BSA
digest by μLC-MS/MS to demonstrate its potentials in proteome analysis.
P021:
Light propagation in coated fused silica capillaries in photo-initiated polymerization of monolithic stationary phases: a three-dimensional model and light intensity distribution
Tomasz Piasecki, Dermot Brabazon, Mirek Macka
National Centre for Sensor Research and School of Chemical Sciences, Dublin City University
Monolithic stationary phases have been made of organic polymers since their introduction by F. Svec and co‑workers in 1990. These have
developed through the last decade into a very popular and important element in separation science. Photo-initiated polymerisation is
arguably the most flexible and popular method for making monoliths. Coated fused silica capillaries and transparent microfluidic chips are
commonly used moulds for photo-polymerised monoliths with many examples in the literature and from this group.
Coated fused silica capillaries, as multilayered structures with cylindrical symmetry, provide a significant challenge for understanding the
penetration of light through the capillary coating and the capillary body. This light is attenuated through absorption in the capillary bore
filled with the photo-polymerisation mixture. Knowledge of light propagation and intensity distribution modelled from the first principles
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Poster Abstracts
should give a better understanding and control over the formed monoliths and thus help in optimising the photo-polymerisation process.
In this work, the previously presented theoretical models1 were extended to a new three-dimensional approach using newly developed
software in LabVIEW™ 9.0. A direct calculation of the three-dimensional light intensity profile in the polymerization mixture filled bore has
been performed. This model has taken into account light attenuation inside the capillary bore and all refraction effects of a multi-layered
capillary structure during the propagation of the light. This model reveals for the first time a quantitative image of light distribution inside
of such system. Examples using common initiators and experimental conditions show that the expected light penetration through the
polymerization solution in the capillary can have wide spatial variances widelyoften in an unexpected and counterintuitive fashion. This approach may be helpful in explaining some known phenomena such as mask under-illumination, fuzzy edges, uneven monolith morphology,
light waveguiding in the capillary or formation of a open-tubular monolithic layer structure at the capillary wall.
T. Piasecki, S. Abele, M. Oelgemoeller and M. Macka, Light propagation in capillaries filled with polymerisation mixture to form monoliths: Theoretical models based on
optics and photochemistry, Book of Abstracts, 32th ISCC, Riva del Garda 2008, p. 138
P022:
Direct macropore-scale simulation of mobile phase transport in reconstructed silica monoliths
Dzmitry Hlushkou, Stefan Bruns, Ulrich Tallarek
Philipps-Universität Marburg, Germany
This work presents an approach towards resolving hydrodynamic flow in real porous media by carrying out direct numerical simulations in
the reconstructed macroporous (flow-through) domain of a silica monolith. The macroporous domain of a 60 µm × 60 µm × 12 µm segment
of a 100 µm i.d. capillary silica monolith was reconstructed by confocal laser scanning microscopy. A 60 µm × 12 µm × 12 µm segment
of the reconstructed domain was then used as the 3D matrix for simulation of fluid flow by the lattice-Boltzmann method on a highperformance computing platform. Excellent agreement is observed between the experimental and simulated Darcy permeabilities. The
flow velocity field is analyzed in detail, including longitudinal and transverse velocity distributions, the occurrence of negative longitudinal
velocities, as well as the beginning transition to the viscous-inertial flow regime. The presented methodology promises great potential for
resolving the key relationships between morphology and band broadening in monolithic columns for HPLC applications.
P023:
CEC linked with a neutral hydrophobic monolithic column accomplish the separation of peptide diastereomers
Ronny Ludewig1, Jing Dong2, Hanfa Zou2, Gerhard K. E. Scriba1
1 Department of Pharmaceutical and Medicinal Chemistry, School of Pharmacy, Jena, Germany
2 National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Dalian, P. R. China
A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has been
evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. The
peptides are positive charged and migrate due to their own electrophoretic mobility caused by the acidic mobile phase. Hydrophobic
interactions with the stationary phase contributed to the separation. Compared to CZE little changes in migration order were obtained.
Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of the mobile phase.
Peak resolution increased with buffer pH and concentration. Di- and tripeptides composed only of L-configured amino acids migrated faster
than peptides containing D-amino acids. A mixture of isomeric Asp tripeptides that could not be completely resolved by CZE or HPLC could
also be separated by CEC on the hydrophobic monolith.
P024:
Design of monolithic nanoLC columns for ultra-high-efficiency LC-MS peptide mapping
Sebastiaan Eeltink, Evert-Jan Sneekes, Bjorn* de Haan, Remco Swart
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Poster Abstracts
Dionex Corp. Amsterdam
Introduction: Peptide sequencing by LC-MS/MS applied in proteomics research has made great advances over the past years in terms of
sensitivity and scan speeds. However, in order to truly tackle present-day proteomic samples, separation performance is still insufficient.
Different strategies have to be followed to increase separation power and this includes the development of novel column technologies.
Polymer monolithic columns have become an attractive alternative for packed columns. Especially for the nanoLC-MS analyses of complex samples they offer high-efficiency separations due to the absence of inter-particle mass transfer. The porosity of monolithic materials can be influenced to tune the permeability and to reach maximum separation efficiency. Optimization of the monolithic structure
allows preparation of very long columns. Method: In this study we optimized the morphology and column dimensions of monolithic
columns to maximize the LC performance for peptide separations with nanoLC-MS. The porous properties of the monolith were systematically altered and investigated with scanning electron microscopy. The performance and the permeability of nanoLC columns upto
1 m in length was tested by the gradient-elution of complex tryptic digests. Experiments were conducted at low and high pH mobile
phases with different ion-pairing agents. The nanoLC columns were coupled via electrospray to an ion-trap mass spectrometer operated
in positive and negative ion mode. Preliminary data: With the optimized monolithic structure, high-efficiency LC-MS peptide separations
were obtained on long high porosity monolithic columns yielding peak widths smaller than 2 s. The maximum peak capacity on long poly(styrene-co-divinyl benzene) monolithic nanoLC columns was obtained at much longer (shallower) gradients than typically applied for
conventional (5 cm long) monolithic columns. Peak capacities exceeding 1000 were achieved when using 1 m long monolithic columns
with optimized morphology and applying 1 -5 h gradients. Novel Aspect: Application of 1 meter long polymer monolithic columns for
very high efficiency LC-MS separations of complex peptide samples.
P025:
Seamless and Comprehensive Two-Dimensional HPLC System Using Methacrylate-Based Monolithic Microbore Columns
Tomonari Umemur1, Shin Shu, Hiroharu Kobayashi, Norihisa Kojima, Yoshinori Wakita
Nagoya University
A comprehensive two-dimensional liquid chromatography (LC×LC) system was constructed by making full use of monolithic column
technology. The system consists of strong cation-exchange (SCX) chromatography in the first dimension and reversed-phase (RP) liquid
chromatography in the second dimension. The two LC systems were connected by a ten-port two position valve equipped with two
storage loops. Electrospray ionization-mass spectrometer (ESI-MS) was also coupled on-line with the LC × LC system. The SCX and RP
columns were all custom-made in our laboratory by in situ copolymerization of functional monomer and cross-linker in a presence of
suitable porogenic solvent. In the present work, 1 mm i.d. microbore monolithic columns were exploited, considering improved sample
loadability and easy coupling to mass spectrometer. The optimization of the analytical run time, flow rate, mobile phase composition,
and gradient condition was carried out for both dimensions. The potential and versatility of this LC×LC system is demonstrated through
the two-dimensional chromatographic separations of the peptides obtained from a tryptic digest of cytochrome c and/or bovine serum
albumin.
This work was supported by Industrial Technology Research Grant Program in 2007 (No. 07C46215a) from New Energy and Industrial
Technology Development Organization (NEDO) of Japan.
P026:
Separation of peptide diastereomers using CEC and a hydrophobic monolithic column
Ronny Ludewig1, Jing Dong2, Hanfa Zou2, Gerhard K. E. Scriba1
1 Department of Pharmaceutical and Medicinal Chemistry, School of Pharmacy, Jena, Germany
2 National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Dalian, P. R. China
A neutral hydrophobic monolith prepared by radical in situ copolymerization of lauryl methacrylate and ethylene dimethacylate has
been evaluated for the CEC separation of diastereomers of small peptides using acidic mobile phases containing ACN as organic modifier. The peptides are positive charged and migrate due to their own electrophoretic mobility caused by the acidic mobile phase.
73
Poster Abstracts
Hydrophobic interactions with the stationary phase contributed to the separation. Compared to CZE little changes in migration order
were obtained. Peptide mobility and resolution increased with increasing the ACN content. Retention times increased with the pH of
the mobile phase. Peak resolution increased with buffer pH and concentration. Di- and tripeptides composed only of L-configured amino acids migrated faster than peptides containing D-amino acids. A mixture of isomeric Asp tripeptides that could not be completely
resolved by CZE or HPLC could also be separated by CEC on the hydrophobic monolith.
P027:
Separation of Infact Proteins with Porous Layer Open Tubular (PLOT) Columns
Steven R Wilson, Magnus Rogeberg, Elsa Lundanes, Tyge Greibrokk, Hanne K Hustoft
University of Oslo
Protein separations with HPLC can often be difficult, resulting in broad peaks and carry-over. However, we have found that Porous
Layer Open Tubular (PLOT) polystyrene divinylbenzene columns may be an attractive alternative. Standard proteins separated on PLOT
columns appeared as narrow and symmetrical peaks with good resolution and sensitivity. 10 µm I.D. × 3 m columns were easily coupled
to standard LC-MS instrumentation with recently available commercial fittings. PLOT columns are relatively easy to make, and the
within-and between column retention time repeatabilities were below 0.6% and below 2.5 %, respectively. Carry-over was not an issue
with the proteins investigated. Temperature can be an important separation parameter for PLOT columns.
We are currently developing an open tubular tryptic reactor, which will be connected to the outlet of the PLOT column, so that separated proteins are subsequently digested for easier detection and peptide mass fingerprinting.
P028:
Porous layer open tubular monoliths with immobilised enzyme: a new direction in enzymatic reactors for on-line digestion-mass
spectrometry
Petr Smejkal1, Oksana Yavorska2, Silvija Abele3, Tomasz Piasecky2, Frantisek Foret5, Mirek Macka2
1 National Centre for Sensor Research and School of Chemical Sciences, Dublin City University, Dublin 9, Ireland;
Institute of Analytical Chemistry of the ASCR, v.v.i., Veveří 97, 60200 Brno, Czech Republic; University of Pardubice, Faculty of Chemical
3 University of Latvia, 19 Raina Blvd., Riga, LV 1586, Latvia
A significant practical advantages of porous layer open tubular (PLOT) capillary columns is their lower backpressure compared to full
monolithic columns (across the full capillary lumen as opposing to PLOT) and larger surface when compared with open bore silica capillaries (without monolithic layer). The aim of this work was to provide an experimental comparison of these three columns as reactor
formats for on-line enzymatic protein digestion. For enzyme immobilisation Pepsin A was selected due to its ability to work at low pH
and an easy online coupling with MS in positive mode.
Poly(glycidylmethacrylate-co-ethyldimethacrylate (GMA-EDMA) PLOT monolithic columns and full monolithic columns were prepared
in silica capillaries with different inner diametres: 25 µm, 50 µm, and 100 µm. Pepsin A was immobilised through epoxy groups by
reductive amination directly on the surface of the monoliths or fused silica capillary. The reactors were then connected into a system
where a solution of Myoglobin as a model protein was pumped continuously through the column and the digested protein was on-line
sprayed into a Mariner ESI-TOF MS.
Pepsin is known for its low selectivity and therefore a library of reference MS spectra was first prepared for the tested substances. With
a purpose to compare MS spectra from digestion in solution with spectra obtained from on-line digests, a straightforward routine was
used based on counting matching peptides of identical mass with a help of a programme written in LabView.
The results showed that digestion of Myoglobin in only 6 cm long monolithic reactors (both PLOT and full monolith) was comparable
with digestion in a 30 cm long reactors prepared in plain silica capillaries. The OTC monolithic reactors compared to full monolithic
reactors have significantly lower backpressure and can be easily flushed. 74
Poster Abstracts
P029:
Hydrophilic interaction liquid chromatographic (HILIC) procedure for the separation of phenolic acids and amino acids
Jan Soukup, Pavel Jandera
1 University of Pardubice, Studentská 95, Pardubice 2, 532 10, Czech Republic Faculty of Chemical Technology, Department of Analytical Chemistry
In reversed phase liquid chromatography, polar compounds are too weakly retained whereas in non-aqueous normal phase adsorption
liquid chromatography polar compounds are retained too strongly and are often insoluble in purely organic mobile phases. HILIC method employing polar stationary phases and aqueous-organic mobile phases have become increasingly popular because of avoiding the
disadvantages of both reversed phase liquid chromatography and normal phase liquid chromatography.
In this work we investigated the influence of the HILIC conditions on the separation.We focused our attention on temperature, concentration of buffer and its pH. The mobile phases consisted of acetonitrile and 10, 20 mM aqueous amonium acetate buffer, 95:5, 90:10
(v/v) with pH adjusted to 2.5 – 3.5. Both isocratic and gradient elution were used for separation. Detection was carried out using a UV
detector at 220 – 280 nm. 10 µL samples were injected into the chromatograph and the separations were performed at a constant flow
rate of 0.5 mL/min and the temperature of column was set up to T = 40 – 80°C.
Three types of columns (XBridge HILIC, Atlantis HILIC and hydrosilated Hydride silica column) have been tested for separations of selected phenolic acids. The Xbridge column particles are prepared on the basis of a hydrid support containing both silica and organic
(1,2-bis(siloxy)ethane(O3SiCH2CH2SiO3)) components. In comparision with the Xbridge HILIC column, the Atlantis HILIC column contains
5 µm silica particles with 70 per cent porosity. The Hydride silica column (4.6 × 75 mm) consists of 5µm hydrosilated silica particles.
Both XBridge HILIC and Atlantis columns showed very low retention of phenolic acids and hence are not useful for their separations in
the HILIC mode. Considerably stronger retention was found on the Hydride silica column, where succesful separations some of phenolic
acids were achieved either in the isocratic or in the gradient modes, especially these containing two or more phenolic groups, which
are poorly retained in the RP systems.
The retention and selectivity in HILIC on the Hydride silica column are strongly affected by varying the fraction of organic solvent, the
concentration of buffer, the pH, and the temperature. The Hydride silica column was also chosen for separations of amino acids. The
attention was focused on the amino acids containing aromatic moilties, which show absorption in the UV region (tryptophan, tyrosine
and phenylglycin). Possibilitie of their separation at various operative conditions , like with the phenolic acids.
This work was financially supported by the Ministry of Education, Youth and Sports of Czech Republic under project MSM 0021627502
and by the Grant Agency of Czech Republic under project No. 203 / 07 / 0641.
Pesek J.J., Matyska M.T.: J. Sep. Sci. 32 (2009), 1-13
Jandera P.: J. Sep Sci. 31 (2008), 1421-1437
Alpert, A.J.: J. Chromatogr. 499 (1990), 177-196
P030:
Sulfonated Monodisperse Hypercrosslinked Microspheres Based on the Dispersion Polymerization of Styrene and Divinylbenzene
Petr Šálek1, Daniel Horák2
1 Brno University of Technology, Faculty of Chemistry, Institute of Materials Chemistry, Purkyňova 118, 612 00 Brno, Czech Republic;
Institute of Macromolecular Chemistry AS CR, Heyrovského Sq. 2, 162 06 Prague 6, Czech Republic
2 Institute of Macromolecular Chemistry AS CR, Heyrovského Sq. 2, 162 06 Prague 6, Czech Republic
Microspheres with high specific surface area (SBET > 1,000 m2 g-1) and high micropore volume (~ 0.6 ml g-1) are considered for various
applications including chromatography, storage of hydrogen, water purification and isolation of biological compounds (DNA, RNA, proteins,
etc.) from real samples. Such particles can be prepared by crosslinking of lightly crosslinked precursors swollen in thermodynamically good
solvents. The method was introduced by Davankov in 1980s involving Friedel-Crafts alkylation of aromatic rings using a catalyst. However,
the conventional precursors suffered from some disadvantages, such as large particle size (hundreds or tens of micrometers), broad particle
size distribution and lack of morphology control. Aim of this contribution was to obtain micrometer-size polymer particles of a narrow
size distribution by a single-step method of the dispersion polymerization, investigate their hypercrosslinking after chloromethylation with
different chloromethyl alkyl ethers and introduce sulfonic acid groups required by the subsequent chromatographic application.
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Poster Abstracts
In the first step, precursor particles were obtained by the dispersion polymerization of styrene and divinylbenzene in ethanol or in
ethanol/2-methoxyethanol mixture. Morphology, size and polydispersity of the particles were controlled by the concentration of hydroxypropyl cellulose stabilizer, 2-methoxyethanol/ethanol ratio, concentration of benzoyl peroxide initiator and mode of divinylbenzene
addition. Monodisperse 3–4 μm microspheres were thus successfully obtained depending on the reaction conditions.
The microspheres were then hypercrosslinked in anhydrous 1,2-dichloroethane using SnCl4 catalyst and different alkylation agents,
such as chloromethyl methyl ether, chloromethyl ethyl ether and chloromethyl octyl ether. Morphology, size and polydispersity of prepared microspheres were analyzed by scanning electron microscopy. Specific surface area was measured by the dynamic desorption
of nitrogen and pore volume and pore size distribution were determined by mercury porosimetry and cyclohexane regain using the
centrifugation technique.
In order the resulting microspheres were used as a packing material in high-performance liquid chromatography, they were functionalized by sulfonic acid groups. Sorption of various compounds on the microspheres is in progress.
The work has been supported by by the Grant Agency of the Czech Republic (project 203/09/0857) and EU (project NMP4SL-2009-228980).
P031:
Capillary Electrophoresis with Laser-Induced Fluorescence detection for quality analysis of plasmid DNA
Cynthia L. Boardman, Johannes A. Dewald
Beckman Coulter, Inc
A method providing rapid, sensitive, reproducible, automated and quantitative analysis of plasmid DNA isoforms using capillary electrophoresis and laser-induced fluorescence (CE-LIF) detection is described. The rapid analysis time (2 among plasmid isoforms (linearized,
supercoiled, nicked and dimers) in less than 10 minutes. Sets of 32 runs performed on three separate days yielded %RSD values of
the supercoiled isoform of 0.89 and 1.05 for migration time and Area % respectively. The application of the method to the analysis of
multiple commercially available plasmids is presented.
P032:
Detection of Single-Molecule DNA by Rolling Circle Amplification in Microchannels
Hui Xi1, Yo Tanaka1, Kae Sato3, Kazuma Mawatari1, Mats Nilsson5, Takehiko Kitamori1
1 Graduate School of Engineering, The University of Tokyo, Japan
3 Center for NanoBio Integration, The University of Tokyo
5 Department of Genetics and Pathology, Rudbeck lavoatory, Uppsala University, Sweden
Introduction: Ultra sensitive analytical systems capable of single molecule detection is a much desired goal since it represents the
endpoint of analysis; when achieved sensitivity cannot be improved any further. We have identified two crucial factors, under which we
believe we could achieve this goal. Firstly, ligands must be high affinity and selectivity, and each single molecule must give rise to a
countable signal. Rolling Circle Amplification (RCA) is a powerful methodology to detect single DNA as a fluorescent dot. However, the
detection efficiency in liquid bulk is only 0.1 %. Secondly, a system that is capable of processing all components in this minute volume
must be devised. By decreasing channel dimensions below the micrometer scale, all analyzes flowing over a surface be allowed to interact with the channel wall and any ligands immobilized to it. We conceived that highly efficient single DNA molecule detection by utilizing RCA and micro space both. Here we propose new approach to detect single DNA in micro space with immobilized DNA as primer.
Theory: We developed UV-mediated immobilization techniques of DNA oligos in micro channels. Here, we combine UV-mediated
immobilization, examine the concept for immobilization, and RCA amplification. At first, we utilizes an amine surface modification
where a carboxyl group of MS(PEG)12 is coupled, followed by UV-mediated benzophenone activation as BP-NCS covalently coupled as
patterning. Then, we demonstrate a new principle for DNA sequence detection that the target DNA is recognized after linear padlock
probe through probe hibridization and ligation, followed by RCA. Then, fluorescence labeled DNA hybridization can be detected by a
microscope as fluorescence dots.
Experimental: DNA Oligos were introduced into a 40 µm deep and a 100 µm wide channel in a fused silica microchip, the channel was irradiated by
76
Poster Abstracts
UV 365 nm. Irradiation time is 90 s, at 250 mW cm-2. We examined this method by DNA immobilization first, then took experiments
of RCA with the same size of microchip.
Results and Discussion: A 10 µM target solution of complementary DNA was introduced after DNA immobilization, followed by padlock
probe and RCA reagents for detection and amplification. Briefly, by ligating this circle and using the immobilized DNA as a primer for
DNA polymerase, a long strand of repeating DNA is formed. We succeed in immobilizing of DNA as first, capture of complementary target oligo and subsequent detection and amplification of complementary DNA using padlock probes and RCA. Detection efficiency was
increased to 5.0% which is centuplicated comparing to our conventional method [i] by circumventing the need for beads as solid support
in microchips as in first time. Furthermore, we calculated the calibration for micro-RCA at varying DNA target concentration from 1 fM.
Conclusion: We developed DNA immobilization and RCA in microchannels.
[i] Björn Renberg et.al. Lab Chip, 9, 1517-1523 (2009).
P033:
Manipulation of DNA Molecules on Electro-osmotic Flow Microfabricated Fluidic Bio-chip
Ho-Chien Lin1, Hao-Ting Chang2, Yu-Cheng Lin*1
1 Department of Engineering Science, National Cheng Kung University, Taiwan
2 Institute of Nanotechnology and Microsystems Engineering, National Cheng Kung University, Taiwan
The study proposes a potentially effective method using microfluidic bio-chip, mechanism of Electro-Osmotic Fluid (EOF) and modifying
surface of chip to enhance stretching and Immobilizing DNA. The research includes two main parts: first, the surface of chip is modified
with different hydrophilic materials for stretching and immobilizing DNA. Second, the optimal voltage was determined to stretch DNA.
In this research, the U937 (RPMI 1640 medium + 10% F.B.S.) cell was used to extract DNA as a sample. The microfluidic bio-chips were
fabricated on glass substrates by soft lithography process. To enhance ability of stretching and immobilizing DNA, this study used O2 plasma, acetone and nano material: YCC-805, to change the hydrophobicity of chip surface. After modifying chips, the contact angle between
the chip surface and margin of bead were measured and analyzed as the experimental results.
Based on the suitable environment to stretch and immobilize DNA, the optimal and limit voltage to stretch DNA could be obtained. The
power supply was used to drive EOF. DC voltage was set from 5 V and gradually increased until the maximum voltage was obtained. And
the stretching DNA is maximum length could be obtained during increasing the voltage.
The results indicate that the chip modified using different materials were corresponding to contact angle. The contact angle of glass
surface was 66.7°. We define hydrophobic is when the contact angle is more than 66.7°, and hydrophilic is when it is smaller than 66.7°
is hydrophilic. The contact angle of O2 plasma surface was 19°, indicating it was hydrophilic. The contact angle of acetone surface was
79.7°, indicating it was hydrophobic. Finally, the contact angle of nano material surface was 90.9°, which was hydrophobic. Based on the
experimental data, the hydrophobic surface has a good effect of stretching DNA, especially acetone surface. It has the optimal effect at
Immobilizing DNA. In addition, the optimal operating condition to stretch and immobilize DNA is to use a voltage of 20V to drive EOF. The
stretching length of DNA from 44 to 230 μm and the average stretching length was 120.8 μm. In additional, we compare the stretching
lengths of DNA in voltages at 5 V, 10 V, 15 V and 20 V respectively.
We have successfully developed a novel technique to enhance DNA stretch and immobilization. The microfluidic bio-chip was fabricated
and DNA solution was extracted in the research. It shows that the surface of chip could be successfully modified and obtained the optimal
results to stretch and immobilize DNA by Acetone. Using the method of EOF, it is found that the maximum voltage to stretch DNA is 20 V,
and the maximum stretching length is 230 mm. This method has many potential usages for DNA structure research and genetic diagnosis.
P034:
High-resolution single-stranded DNA separations in polydimethylsiloxane (PDMS)-glass microfluidic chips
Thomas P. Niedringhaus1, Ryan E. Forster2, Annelise E. Barron1
1 Stanford University
2 Northwestern University
Second-generation sequencing technologies offer ultra-high throughput but not long read lengths, which remain uniquely in the
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Poster Abstracts
domain of capillary and microchip electrophoresis. Previously, we developed EOF-suppressive polymer coatings and DNA separation
matrices for microchip electrophoresis in commercially available borosilicate glass microchips. Optimized materials of this class
can reduce separation times for accurate, long-read (600-base) DNA sequencing from hours (in CE) to minutes. Transitioning from
borosilicate glass to a hybrid polydimethylsiloxane (PDMS)-glass chip substrate can reduce manufacturing cost of microchips for
sequencing. Aside from material cost, the ability of PDMS to flow prior to curing and the use of a reusable master mold to generate channel features could enable cost-effective, scaled up manufacture of PDMS-glass chips. The fabrication of PDMS-glass chips
requires minimal clean room time and no harsh chemicals. We compare quantitative metrics of ssDNA separations in PDMS-glass
chips to the same separations carried out in a borosilicate glass chip with similar channel designs, and the same coating and separation matrices, using a fluorescently labeled Sanger sequencing ladder. The resolution per base and sequencing read lengths are
presented. Strategies for modifying the hydrophobic PDMS channel surface prior to the coating procedure in order to increase the
resolution per base are presented and discussed.
P035:
Mobility and Diffusion Regimes in Field Inversion Gel Electrophoresis of DNA in the Sub-35 Kilobase Size Range
Airong Li1, Victor M. Ugaz2
1 Sichuan University
2 Texas A&M University
Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications, butut
many aspects of DNA migration phenomena under these conditions are not well understood. One of the main reasons for this
deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (~ 10 – 15 hours) and the
need to perform gel analysis by post-staining after completion of the run. We have developed an easy to build miniaturized slab gel
apparatus that addresses these issues by enabling large DNA fragments up to 35 kb in length to be separated using field inversion
gel electrophoresis (FIGE) in 60 – 90 min. The compact size of the device combined with the use of quartz as the substrate material
permit the gel to be continuously illuminated with UV light so that the separation processes can be recorded in real time using a
CCD camera.
These capabilities allow us to probe size dependence of fundamental physical parameters associated with DNA migration (mobility,
diffusion, and separation resolution). These data reveal a surprising regime where separation resolution increases with DNA fragment size owing to a favorable interplay between mobility and diffusion scalings, and highlight the important role of diffusion (a
seldom quantified parameter). In addition to the practical benefit of separation times that are an order of magnitude faster than
conventional instruments, the results of these studies provide a previously unavailable rational basis to identify optimal separation
conditions and contribute new insights toward understanding the underlying physical processes that govern DNA electrophoresis
in pulsed fields. Monday, March 22nd, 2010
P036:
Microfluidic generation of hollow nanoporous capsules for DNA encapsulation
Matthew B. Kerby, Harald Nuhn, Annelise E. Barron
Stanford University
We present a microfluidic device for encapsulating genomic DNA fragments in a hollow, nanoporous, protective capsule. Unlike
emulsion PCR, the capsule is designed to retain DNA and a polymerase, while permitting the cross-wall exchange of smaller dNTPs,
metal co-factors and salts. An on-chip emulsification process envelops condensed DNA in a spherically shaped, thin layer of PEG
solution, which is then crosslinked. Because the capsules will have controlled porosity, undesired salts can be “dialyzed” away if the
capsules are placed in a different buffer or in distilled, deionized water, allowing DNA molecules can “re-expand” to fill the capsule.
The microfluidic chip design is being evaluated using fluorescently labeled dsDNA molecules to demonstrate emulsion generation
and capsule properties.
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Poster Abstracts
P037:
A novel strategy for the simultaneous detection of alternatively spliced transcripts: A study of MEF2C exon covariation
Kathryn Sciabica, Handy Yowanto, Jeff Chapman
Beckman Coulter, Inc.
Myocyte Enhancer Factor 2 (MEF2) proteins are transcription factors that exist in all metazoans and play pivotal roles in development
and differentiation of tissues. There are 4 vertebrate Mef2 genes, Mef 2A, B, C and D, and these have different temporal and spatial
expression patterns. Mef2A, C and D encode protein variants by virtue of alternative splicing of primary transcripts, and these genes
have similar structures and alternative splicing patterns that are conserved across evolution. The alternative splicing involves mutually
exclusive exons (alpha1 and alpha2), a cassette exon (beta), and alternative splice acceptors that flank a short region (gamma). The
corresponding short polypeptide domains encoded by these alternative segments are nested within the Mef2 carboxy-termini and are
structurally conserved across isotypes. These domains confer specific functions, important for binding co-activators, transactivation
and transrepression. In an effort to elucidate the roles of Mef2 alternative splicing variants, we have developed an RT-PCR long fragment assay in which all eight Mef2C mRNA isoforms are simultaneously monitored in cell and tissue samples. Capillary electrophoresis
with laser induced fluorescence (CE-LIF) was performed using the GenomeLabTM GeXP to separate and detect these fragments. CE-LIF
successfully resolved all eight fragments, the longest of which was 1049 nucleotides and only six nucleotides longer than the next
largest fragment. This assay was used to confirm and extend prior observations of regulated Mef2 alternative splicing among tissues,
during development and during muscle differentiation. The technique is well suited for rapid qualitative evaluation of splicing variant
expression, and could be effectively used for candidates with established splicing variants or for the validation of findings observed with
sequencing or fragment analysis. Importantly, this strategy uniquely allows for the evaluation of co-variations in multiple alternative
splicing events for primary transcripts of a given gene.
P038:
Multiplex gene expression analysis identifies and characterizes induced pluripotent stem cells derived from human somatic cells
Kathryn Sciabica1, Akitsu Hotta2, Knut Woltjen2, Jeff Chapman1
1 Beckman Coulter, Inc.
2 Ontario Human Induced Pluripotent Stem Cell Facility
Recent methods to reprogram human somatic cells, in order to create induced pluripotent stem cells (iPSCs), intend to provide important tools for drug discovery and models for the study of disease. The ultimate goal is to create an alternative to controversial embryonic stem cells (ESCs) and generate patient-specific pluripotent cells for autologous regeneration. Reprogramming methods include the
transfer of genetic material, protein transduction and/or application of chemicals to promote epigenetic modification and the expression of key pluripotency markers. Stable reprogramming results from the upregulation of endogenous pluripotency-associated genes and
repression of differentiation-associated genes. However, the mechanistic progression and timing of complete reprogramming are generally accepted as being highly stochastic. Somatic cell reprogramming often results in a heterogeneous population in which some cells
remain in a partially reprogrammed state, while others are fully competent iPS cells. A technical challenge faced by researchers is the
ability to rapidly and accurately predict the pluripotent capacity of these cells. A novel, multiplex RT-PCR method in combination with
capillary electrophoresis laser induced fluorescence (CE-LIF) surmounts this challenge by offering simultaneous, quantitative detection
of gene expression for 2 to 40 genes with a minimal requirement for template RNA input. In this study, a multiplex panel of 27 genes
was used to generate an expression profile that, when compared to human ESCs, characterizes and defines fully reprogrammed human
iPSCs derived from adult fibroblasts. Similar types of gene panels can be used to efficiently monitor the quality of iPSC maintenance
cultures and subsequent differentiation into specific cell lineages. Additionally, this method can be effectively applied to all studies that
involve gene expression research and biomarker detection.
P039:
Combination of multiplex PCR and capillary gel electrophoresis for detection of genetic mutations with high sensitivity
Daniel Lehmann, Margareta König, Nicole Lokmer, Udo Roth, Friederike Wilmer, Holger Engel
79
Poster Abstracts
Qiagen GmbH
Mutation analysis is performed in several areas of research, such as identifying mutations for diagnostics, typing of disease loci, and investigating relationship and paternity patterns. Reliable multiplex PCR assays are essential for such experiments. This includes specific
and sensitive co-amplification, even of low abundant targets or targets with high GC content and secondary structures. However, establishment of sensitive multiplex PCR assays can be challenging and often require laborious optimization of experimental parameters or
remain unsuccessful at all. At the same time the resulting multiplex PCR fragments are typically analyzed by agarose gel electrophoresis
or CE-based DNA sequencers. Both technologies possess drawbacks with respect to high acquisition and maintenance costs (DNA
sequencer) or lack of standardization for routine applications (agarose gel). Here we present data showing the successful application of
the Type-it® Mutation Detect PCR Kit in combination with the capillary gel electrophoresis QIAxcel® System to overcome these limitations. With the help of the PCR kit a mutated cancer related gene (4 copies per total template DNA) could be detected within a 1000×
fold excess of wild-type DNA as demonstrated by agarose gel-electrophoresis and CGE (Qiaxcel). However, in comparison to the results
of classical gel electrophoresis the QIAxcel system provided a highly standardized solution exhibiting superior band resolution (down
to 3-5 bp in the 500 bp range) and precise peak quantification at only 10 min analysis time, thus being an ideal detection platform for
PCR-based multiplex mutation assays.
P040:
Production of Long DNA Probes by Ligation Reaction by Capillary Gel Electrophoresis with Laser-Induced Fluorescence Detection
Virginia Garcia-Cañas, Monica Mondello, Alejandro Cifuentes
Institute of Industrial Fermentations (CSIC)
Many novel DNA amplification methods are being developed for sensitive detection and quantification of specific DNA target sequences for clinical, environmental and food testing. These new molecular methods often require the use of long DNA oligonucleotides as
probes for hybridization to the target sequences. Depending on the molecular technique, the length of DNA probes ranges from 40
to 450 nucleotides. Chemical synthesis is the general strategy adopted for oligonucleotide production. However, there is a decrease
in the fidelity of chemical synthesis of DNA with distance from the first position at the 3’-end of each oligonucleotide. Defects in the
oligonucleotide sequence result in the loss of hybridization efficiency of the DNA probe, affecting the sensitivity and selectivity of the
amplification method.
In this work, an enzymatic procedure has been investigated as alternative to solid-phase chemical synthesis for the production of long
oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Each long probe was
ligated together from two or more smaller oligonucleotides using short sequences that act as bridges stabilizing the molecular complex
for DNA ligation. The ligation reactions were analyzed by capillary gel electrophoresis with laser-induced fluorescence detection (CGELIF) using a bare fused silica capillaries. The effect of some parameters on the separation and detection of ligation products, such as
temperature of analysis and the pretreatment of the sample with formamide, was studied. The CGE-LIF method demonstrated to be
very useful and informative for the characterization of the ligation reaction, as well as for the optimization of the ligation conditions,
such as temperature and enzyme concentration, aimed at increasing the yield of ligation products. In-lab prepared DNA probes were
used in a novel Multiplex Ligation-Dependent Genome Dependent Amplification (MLGA) method for the simultaneous detection of three
genetically modified maize lines in maize samples. The quality of the produced probes was demonstrated by the specific and sensitive
detection of four genomic DNA targets, namely, one reference and three sequences from three varieties of transgenic maize.
P041:
Apollo 200TM: An Integrated Platform for Rapid DNA Identification
Helen Franklin, William Nielsen, Stefanie Pagano, Omar El-Sissi, Jacklyn Buscaino, Greg Bogdan, Jim Knittle, Roger McIntosh,
Joe Carrow, Iuliu Blaga, Stevan Jovanovich, Daniel Qi, Richard Belcinski
Microchip Biotechnologies Inc.
Short Tandem Repeats (STRs) are adjacent regions of repeated nucleotides (2 to 16 base pairs) typically found in the non-coding intron
80
Poster Abstracts
region of genome. STR analysis is routinely used in forensic identification. By examining enough STR loci and counting the number of
repeats for a given locus, it is possible to create a unique genetic profile and to identify an individual with a probability un-matched
by other forensic tests. This analysis is done by well trained persons, in specialized labs, and may take as long as 2 days to complete.
The demand for rapid STR analysis instruments for identification of criminals, immigration, diagnostics and agriculture is growing fast.
We report a four-channel sample-to-answer automated apparatus for DNA profiling, commercially known as Apollo 200™. The system
only requires an input of a buccal swab and it will output CODIS (American DNA databank format for 13 loci) data in less than three
hours. The instrument has three modules (nucleic acid extraction, PCR reaction and separation/detection), uses commercially-ofthe-shelf (COTS) reagents, and can be operated by non-specialist personnel. The key to the systems capability is the use of patented
Microscale On- Valve (MOVe™) technology for pumping and controlling volumes in the sub-microliter range. MOVe™ valves use a flexible
membrane sandwiched between two aligned microstructured glass or plastic plates. In either case a plastic manifold is bonded to the
MOVe™ array for handling a large range of volumes (up to 1 ml).
The DNA extraction device performs cell lysis and DNA extraction with magnetic bead purification. Reagents are supplied by a MOVe™
distribution device that is part of the instrument. The purified samples are pumped into the STR amplification module mixed with PCR
reagents, sealed, and thermocycled. The injection module dilutes the PCR products in a loading solution containing a size ladder, mixes
and pumps the four samples into four injection tubes. Four fused silica separation capillaries are inserted into the injection tubes,
mounted on a heated aluminium plate, and aligned with the detection system. The detector uses an excitation beam from a diode laser
split into four beams, one for each capillary, with prismatic diode array detection. All process steps were controlled by the proprietary
DevLinkTM software and a proprietary trace processing software performs baseline removal, noise removal, spectral separation, data
export to allele calling program and CODIS file generation. The paper shows data with all alleles called correctly in all four channels, fine
tuning of DNA extraction and purification chemistries, and optimization of PowerPlex 16HS* amplification protocol. Data with other
type of samples (including blood) are also discussed.
*PowerPlex 16HS is a trademark of Promega Corporation, Madison Wisconsin.
P042:
Experimental determination of dimerization risk for DNA barcode development software
Samantha M. Desmarais1, Thomas K. Leitner2, Steven M. Wolinsky3, Annelise E. Barron1
1 Stanford University
2 Los Alamos National Labs
3 Northwestern University
DNA barcodes are short, unique ssDNA primers that can “mark” individual biomacromolecules. DNA barcodes are required by numerous
methods for DNA and protein detection, e.g. nanoparticle-based bio-barcode-amplification and 454 sequencing. Reliable disease discovery with DNA barcodes requires well-designed DNA barcode primers. We aim to develop, and subsequently test, bioinformatics software for accurate primer and probe design that includes parameters such as read length and alignment length, and allows user input
to optimize primer design. As part of achieving this goal, we must calculate the dimerization risk amongst potential primer pairs. We
show that capillary and microchip electrophoresis can be used to determine, quantitatively, the extent of primer dimerization, allowing
our model to be tested. Both free-solution and matrix–based electrophoresis are used to separate fluorescently tagged oligonucleotide
pairs designed with complimentary regions from 5 -30 base pairs, yielding characteristic electropherograms revealing the extent of
heterodimerization. These data will assist with our prediction of the formation of unwanted homo- and hetero-dimer in primer or probe
constructs, and should allow us to define, experimentally, a parameter required in the primer design software, the dimerization risk. Tuesday, March 23rd, 2010
P043:
Microchip-based Investigation of DNA Mobility and Diffusion During Gel Electrophoresis in the Entropic Trapping Regime
Nan Shi, Victor M. Ugaz
The design of miniaturized DNA electrophoresis systems imposes demanding requirements that are challenging our knowledge about how the
81
Poster Abstracts
nanoscale pore morphology of the sieving matrix influences the physical mechanism of DNA migration through the gel. But a detailed understanding of this interplay is limited by a general lack of experiments probing fundamental parameters like mobility and diffusion coefficients
that are key descriptors of electrophoretic transport. We have recently developed an innovative way to address this need through the use
of a microfluidic platform that allows continuous whole channel scanning of DNA separation progress so that the size dependence of these
parameters can be quickly and accurately measured. When combined with material characterization methods we have developed that enable
both the mean pore size and pore size distribution of the gel to be quantified, we are able to establish a direct link between sieving matrix
morphology and DNA migration in photopolymerized crosslinked polyacrylamide gels cast under different UV intensities and concentrations. We have applied this approach to examine electrophoresis of double-stranded DNA in the 100 to 1000 bp size range, and have identified gel polymerization conditions that induce a transition in the physical mechanism of DNA migration from reptation to entropic
trapping. Furthermore, we have identified a range of conditions where a favorable interplay exists between these migration mechanisms that results in improved separation performance. These counterintuitive results have led us to reconsider the applicability of some
scaling relationships and theories widely used to describe electrophoretic migration of DNA, and explore the link between macromolecular transport and the gel pore architecture (particularly the role of diffusion, a seldom characterized parameter). This knowledge
can be applied to better predict separation performance in different sieving gels and operating conditions, and ultimately provide
new insights that can help enable rational selection of optimal matrix materials and polymerization conditions that deliver enhanced
separation performance.
P044:
Development and validation of a nonaqueous capillary electrophoretic method for the enantiomeric purity determination of a synthetic intermediate of new R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans using a single-isomer anionic cyclodextrin derivative
and an ionic liquid
Anne Rousseau1, Xavier Florence2, Bernard Pirotte2, Anne Varenne4, Pierre Gareil4, Didier Villemin6, Patrice Chiap7, Jacques Crommen1,
Marianne Fillet1, Anne-Catherine Servais*1
1 Laboratory of Analytical Pharmaceutical Chemistry, Dept. of Pharmaceutical Sciences, CIRM, University of Liege, CHU, B36, B-4000 Liege, Belgium
2 Laboratory of Medicinal Chemistry, Dept. of Pharmaceutical Sciences, CIRM, University of Liège, CHU, B36, B-4000 Liege, Belgium
4 Laboratory of Electrochemistry and Analytical Chemistry, UMR CNRS 7575, ENSCP, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France
6 Laboratory of Molecular and Thio-organic Chemistry, UMR CNRS 6507, ENSI Caen, 6, Boulevard du Maréchal Juin, 14050 Caen Cedex, France
7 Advanced Technology Corporation (ATC), CHU, B36, B-4000 Liege, Belgium
A series of diversely 4-arylthiourea-substituted R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans structurally related to (±)-cromakalim
has been recently synthesized1. The new compounds were examined as putative potassium channel openers on rat pancreatic islets
as well as on rat aorta rings1. The development of chiral drugs in their single-isomer forms requires the use of sensitive and selective
analytical methods to control the chiral purity. The usefulness of nonaqueous capillary electrophoresis (NACE) for the enantiomeric
separation of a synthetic key intermediate of these compounds has been investigated. In order to obtain high resolution and efficiency
values, the addition of a chiral ionic liquid to the background electrolyte (BGE) containing a single-isomer anionic CD was found to be
essential. After optimization of the BGE composition, the best enantioseparation of the key intermediate was achieved using a methanolic solution of 10 mM ammonium formate and 0.75 M formic acid containing 1.5 mM heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD and
5 mM ethylcholine of bis(trifluoromethylsulfonyl)imide. Levamisole was used as internal standard. The selected conditions allowed the
determination of 0.1% of each enantiomer in the presence of its stereoisomer using the method of standard additions. The NACE method
was then validated with respect to selectivity, response function, precision, accuracy, linearity, limits of detection and quantification.
1 X. Florence, PhD thesis, University of Liege, 2009
P045:
Chiral separation and quantitation of hydroxyzine and its main metabolite cetirizine by maltodextrin mediated capillary electrophoresis
Ali Reza Fakhari Zavareh, Saeed Nojavan
Department of Chemistry, Faculty of Sciences, Shahid Beheshti University, G. C., P.O. Box 19396-4716, Evin, Tehran, Iran
82
Poster Abstracts
Cetirizine hydrochloride, an antihistamine, is a major metabolite of hydroxyzine, and a racemic selective H1 receptor inverse agonist
used in the treatment of allergies, hay fever, angioedema, and urticaria.In the present study, a simple capillary electrophoresis method
using high concentration of maltodextrin solutions as separation medium has been developed. Several parameters affecting the separation were studied, including the dextrose equivalent of maltodextrin, pH and the concentration of phosphate buffer and temperature of
capillary column. The best resolution was obtained when maltodextrin with higher dextrose equivalent (16.5-19.5) was used as a chiral
selector. The best buffer conditions for cetirizine and hydroxyzine enantiomers were optimized as 100 mM sodium phosphate buffer
at pH of 2.0, containing 10% (w/v) maltodextrin. Under optimized conditions, a baseline separation of enantiomers was achieved in less
than 30 min. The linear range of the method was over 1.0 to 50.0 µg/mL for all enantiomeres of hydroxyzine and cetirizine. The detection limit (S/N 3) of each enantiomer was 0.02 µg/mL. The method allowed the enantioseparation of these drugs in bulk samples and
enantiomeric purity evaluation in pharmaceutical tablets, and it was also found to be suitable for enantioseparation in human plasma1-3.
1 M. P. Curran, L. J. Scott, C. M. Perry, Drugs 64 (2004) 523.
2 A. V. Eeckhaut, Y. Michotte, Electrophoresis 27 (2006) 2376.
3 Y.W. Chou, W.S. Huang, C.C. Ko, S.H. Chen, J. Sep. Sci. 31 (2008) 845.
P046:
Enantioseparation using sulfated-cyclodextrins in aqueous and nonaqueous capillary electrophoresis: Study of the mechanisms of
enantioselective intermolecular interactions
Anne-Catherine Servais1, Anne Rousseau1, Bezhan Chankvetadze3, Jacques Crommen1, Marianne Fillet1
1 Laboratory of Analytical Pharmaceutical Chemistry, Dept. of Pharmaceutical Sciences, CIRM, University of Liege, CHU, B36, B-4000 Liege, Belgium
3 Department of Physical and Analytical Chemistry, School of Exact and Natural Sciences, Tbilisi State University, Chavchavadze Ave 1, 0128 Tbilisi, Georgia
The interest of using single-isomer highly charged derivatives of ß-cyclodextrin (CD) for the enantioseparation of basic drugs in nonaqueous capillary electrophoresis (NACE) is now well established. Heptakis(2,3-di-O-methyl-6-O-sulfo)-ß-CD (HDMS-ß-CD) and heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD (HDAS-ß-CD) were more particularly studied for the separation of ten ß-blockers and, in most cases,
HDAS-ß-CD was found to lead to a higher selectivity compared with HDMS-ß-CD1. Nevertheless, even in a pharmacological class of drugs
having similar structures such as ß-blockers, a higher enantioresolution was obtained with HDMS-ß-CD for two compounds. Therefore,
the nature of substituents (methyl or acetyl groups) in the positions 2 and 3 of the CD derivatives seems to strongly influence the intermolecular interactions between the analyte enantiomers and the chiral selector. For example, the enantiomer migration order (EMO) of
propranolol was inverted using HDMS-ß-CD instead of HDAS-ß-CD as chiral selector. This might be the consequence of different complex
structures. The same study was undertaken in aqueous media. The EMO of propranolol was found to be inverted when an aqueous BGE
was replaced by a nonaqueous BGE in the case of HDMS-ß-CD but remained the same in the case of HDAS-ß-CD. This suggests that
for CE enantioseparations with chiral buffer additives, the interaction mechanisms might be very different in aqueous and nonaqueous
BGEs. The possible molecular mechanisms leading to this reversal of EMO were then studied by using nuclear magnetic resonance
(NMR) spectroscopy in both aqueous and nonaqueous BGEs. The results indicate important differences between the analyte-CD complexes formed in aqueous CE and in NACE, related to the presence or not of an inclusion complex but also to the nature and position
of the analyte moiety included in the CD.
1 A. Rousseau, P. Chiap, R. Oprean, J. Crommen, M. Fillet, A.-C. Servais, Electrophoresis, 30(16) (2009) 2862-2868
P047:
Enantiomer separation and determination of helix inversion barrier of helical dications by capillary electrophoresis
Dušan Koval, Louis Adriaenssens, Lukáš Severa, Filip Teplý, Petra Sázelová, Václav Kašička
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Flemingovo nám. 2, 166 10 Praha 6 - Dejvice, Czech Republic
A novel family of helical dications (helquats) was recently achieved via a facile three-step synthetic route. Pyridine disubstituted acetylene reagent prepared via Sonogashira coupling is subjected to bisquarternization with a triflate reagent to form triyne intermediate. Finally, the triyne is converted to helquat product by [2+2+2] cycloisomerization1. Helquat products are obtained as racemates containing
83
Poster Abstracts
M- and P-helical forms. Helquats as ditriflate salts are well soluble in water. Reversible electrochemical properties suggest the potential
of electron-accepting helquats in the field of nanoscience, for example, molecular electronics.
From a point of view of chemical analysis, water soluble dicationic helquats are ideal candidates as analytes for capillary electrophoresis.
Notably for analytical separation of racemic mixtures, capillary electrophoresis offers flexibility appreciated in method development
of novel compounds such as helquats. Additionally, capillary electrophoresis can be used to determine barrier to helix inversion of
helquats, i.e. activation energy for mutual interconversion of M- and P-helical forms of helquats.
For separation of enantiomeric helical forms of helquats, a sodium-phosphate buffer of pH 2.5 was used. Sulfated ß- and γ-cyclodextrins
turned out to be effective chiral selectors unlike their neutral derivatives. Interaction between dicationic helquat and sulfated cyclodextrin provided a complex with negative net charge. The helquats were, therefore, analyzed as anions at reversed voltage polarity in
untreated fused silica capillaries.
Helix inversion barrier measurements were performed on a home-made capillary electrophoresis apparatus equipped with a liquid
thermostating system of capillary allowing a rapid switch between two temperature levels in the 5 – 98°C interval. It was observed
in a series of compounds that some helquats are configurationally labile and, consequently, cannot be resolved. Enantiomers of more
hindered helquat structures were successfully resolved and free energy of helix inversion barrier was estimated by capillary electrophoresis to be in the 106 - 120 kJ/mol interval. Helix inversion barrier of rather configurationally stable helquats was > 120 kJ/mol and,
consequently, out of range of the capillary electrophoresis method.
The authors acknowledge a support from Czech Science Foundation (203/08/1428, 203/09/0705, P207/10/2391, 203/09/P485) and
IOCB (Z4 055 0506).
1 Adriaenssens L., Severa L., Šálová T., Císařová I., Pohl R., Šaman D., Rocha S.V., Finney N.S., Pospíšil L., Slavíček P., Teplý F., Chem. Eur. J. 15, (2009), 1072-1076
P048:
Enantioseparation in Single and Multi Chiral Selector Systems - Is there any difference?
Jana Svobodová, Pavel Dubský, Eva Tesařová, Bohuslav Gaš
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 128 43, Prague 2, Czech Republic
Commercial mixtures of chiral selectors, namely highly sulfated cyclodextrins, are well known for their good separation performance.
Several authors1, 2 proved better enantioseparation abilities of multi chiral selector (multi-CS) systems than single chiral selector ones
(single-CS). Single-CS enantioseparation is primarily controlled by thermodynamic mechanism. It means that enantiomer-CS complexes
differ in their distribution constants, while their complex mobilities are almost the same. In multi-CS systems, the situation is more
complicated. It can be shown that not only overall distribution constants but also overall complex mobilities considerably differ there
and thus contribute to the enantioseparation.
Distribution constants and complex mobilities were determined for a model analyte, lorazepam, using two different chiral selector
systems by ACE-like (affinity capillary electrophoresis) experiments. Heptakis(6-O-sulfo)-ß-cyclodextrin was used as well defined single
isomer chiral selector, while commercially available ß-cyclodextrin, sulfated sodium salt represented related multi-CS system. Excellent
fits, based on the equation proposed in our previous publications3, 4 for multi-CS systems, confirmed the predicted behavior. The single
isomer system showed different distribution constants and statistically the same mobilities for enantiomer-CS complexes. In the multiCS system, overall distribution constants as well as overall complex mobilities differ significantly. The thermodynamic selectivity (ratio
of distribution constants) is almost the same for the both systems, the additional difference in overall complex mobilities ensures better
enantioseparation in the multi-CS system.
In the next step an optimal concentration of the chiral selector was calculated according to a simplified Wren and Rowe equation5. A
theoretical value for the single-CS system is in a good agreement with the value determined experimentally. In the case of our multi-CS
system, no optimal CS concentration exists. The higher the CS concentration, the higher the effective mobility difference. The simplified
Wren and Rowe equation cannot predict such behavior. Using this equation to calculate optimal concentration might be inappropriate
in multi-CS systems and its full quadratic form should be used instead.
We gratefully acknowledge funding from the long term research plan of the Ministry of Education of the Czech Republic MSM 0021620857,
the Grant Agency of Charles University GAUK 101309, and the Grant Agency of the Czech Republic, grant No. 203/08/1428.
1 M. J. Rocheleau, Electrophoresis 26 (2005) 2320
2 D. J. Skanchy et al., Electrophoresis 20 (1999) 2638
84
Poster Abstracts
3 P. Dubsky, J. Svobodova, B. Gas, J. Chromatogr. B 875 (2008) 30
4 P. Dubsky, J. Svobodova, E. Tesarova, B. Gas, J. Chromatogr. B 875 (2008) 35
5 S. A. C. Wren, R. C. Rowe, J. Chromatogr. 603 (1992) 235
P049:
Association of two single-isomer anionic CDs in NACE for the chiral and achiral separation of fenbendazole, its sulphoxide and
sulphone metabolites. Application to their determination after in vitro metabolism
Anne Rousseau1, Florian Gillotin2, Patrice Chiap2, Jacques Crommen1, Anne-Catherine Servais1, Marianne Fillet1
1 University of Liège
2 ATC
A NACE method was developed for the separation of fenbendazole (FBZ), a prochiral drug giving rise to chiral (oxfendazole or OFZ) and
non chiral (FBZ sulphone or FBZSO2) metabolites. First, the effect of the nature and the concentration of CD as well as that of the acidic
BGE on the enantiomeric separation of OFZ were studied. OFZ enantiomers were completely resolved using a BGE made up of 10 mM
ammonium formate and 0.5 M TFA in methanol containing 10 mM heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD and 10 mM heptakis(2,3-di-Omethyl-6-O-sulfo)-ß-CD. Moreover, the NACE method was found to be particularly well suited to the simultaneous determination of FBZ,
OFZ enantiomers and FBZSO2. Thiabendazole was selected as internal standard. The CD-NACE potential was then evaluated for in vitro
metabolism studies using FBZ as a model case. OFZ enantiomers and FBZSO2 could be detected after incubation of FBZ in the phenobarbital-induced male rat liver microsomes systems.
P050:
Application of Capillary Electrophoresis with Ionic Liquid on Chiral Separation
Shu-Ping Wang, Jie-Bi Hu
Providence University
It has growing interest in ionic liquid for their potential in various field such as electrochemistry, catalysis, organic chemistry, and analytical
chemistry. With the last one, it has proven especially useful in separating determination. Generally, ionic liquid has versatile physico-chemical properties and really attractive as so called green solvent. In recent years, application of ionic liquid on separation of pharmaceutical,
agricultural industry and food additives by capillary electrophoresis has been reported. It also indicated ionic liquid has powerful potential
for separation. It was investigated for using chiral ionic liquid based on trimethylammonium and imidazolium as chiral selector for separating enantimers of flavonoid and quinolones by dynamic coating capillary electrophoresis in this study. (R)-3-(3-hydroxy-2-methylpropyl)1-methylimidazolium bromide (IM*) and (R)-3-hydorixy-2-methylpropyl-trimethylammonium bromide (TMA*) were synthesized separately
and applied to separate enantiomers of naringenin, hesperetin, salbutamol, terbutaline. It is important to prove that the ionic liquid has
potential for separation enantiomer by comparing the results with previous paper and may be applied to pharmaceutical industry.
P051:
Sample preparation for capillary zone electrophoresis-based separation and quantification of cellulases in sugarcane juice
Ruchi Gupta, Sara J. Baldock, Peter R. Fielden, Bruce D Grieve
University of Manchester
Integration of gene sequences expressing cellobiohydrolases (CBH) and endoglucanase (EG) in Trichoderma and ß-glucosidase (BG) in Aspergillus Niger with sugarcane genome can provide lower cost cellulases for ethanol production from bagasse1. But the concentration of
expressed enzymes is likely to be dependent upon cane variety and cultivation conditions. It is also necessary to obtain a concentration
profile of individual enzymes to assess the efficiency of a mixture in converting cellulose (in bagasse) into fermentable sugars.
85
Poster Abstracts
The high separation efficiency of capillary zone electrophoresis (CZE) imparts it an ability to separate BG, CBH and EG in a single run.
Preliminary CZE separations of a mixture of cellulases in de-ionized water using a P/ACETM MDQ (Beckman Coulter) in 50 μm internal
diameter neutral capillaries showed promising results. Basically, the separation of enzymes was accomplished within 15 minutes after
a separation length of 11 cm, using 10 mM sodium phosphate buffer (pH 7.75) and under an applied electric field of ~240 V/cm at
25ºC. The limits of detection for BG, CBH and EG using the above mentioned conditions were found to be 11 μg/ml, 28 μg/ml and
17.4 μg/ml respectively at a detection wavelength of 214 nm.
Initially the effect of sugarcane juice (sample matrix) on migration time and limits of detection of cellulases was investigated. The contribution of sugarcane juice to the current at the above mentioned separation voltage was negligible. But changes in the migration time
of BG, CBH and EG suggested that various components in the sample matrix interact either with the enzymes or with the capillary wall.
In addition, co-extractives (such as sucrose, glucose, fructose, salts, proteins, starch, dextran, waxes, fats and phosphatides) present
in sugarcane juice introduced additional peaks, thereby degrading the signal-to-noise ratio of the enzyme peaks and hampering the
limit of detection of cellulases. This necessitates enzyme-enrichment and removal of interfering sample components before cellulases
in sugarcane juice can be subjected to CZE analysis.
Ideally, it should be possible to transfer the prepared sample automatically (on-line coupling) for subsequent investigation, thereby
reducing the time for analysis. Although packed liquid chromatographic (LC) columns have been used for sample preparation, on-line
interfacing of LC‑CZE is problematic due to buffer incompatibilities and differences in sample volumes as well as pumping mechanisms2.
Therefore, we have investigated isotachophoresis- and membrane-based separation techniques for our purpose.
1 Cellulosic ethanol: huge potential but challenging, www.farmacule.com/news/news10/AusbioBioethanol.ppt, Last Accessed: 14/12/2009
2 T. Stroinka, et. al. J CHROMATOGR B (2004) 817 p49
P052:
A Rapid and Sensitive Genetic Identification Method for Detecting Beer-Spoilage Bacteria and Wild Yeast
Yong Wu, Manami Saha, Beena Lee, Lily Nan, Handy Yowanto, Jeff Chapman
Beckman Coulter, Inc
Although beer has been recognized as a beverage with high microbiological stability, many microorganism species have been reported
to spoil beer. To improve the hygienic status of breweries and perform quality control of beer products, it is important to rapidly detect
the presence of microorganisms and determine their beer spoilage ability. The most commonly used method is to culture the samples
under either aerobic or anaerobic conditions. However, this method generally takes a few days for detecting aerobic bacteria and up to
a few weeks for detecting anaerobic bacteria. Another drawback of this method is that it cannot determine the beer spoilage ability.
Therefore a new approach that can rapidly and reliably detect the microorganisms and determine their beer spoilage ability has long
been desired in the brewing industry. Here we present a multiplexed capillary electrophoresis method that can simultaneously identify
six major genera of beer-spoilage bacteria and their potential to spoil beer by detecting five hop-resistant markers. Equipped with an
8-channel capillary array and advanced polyacrylamide gel, the GenomeLab™ GeXP Genetic Analysis System is able to identify beerspoilage bacteria and determine their beer-spoilage potential from sample collection to data report within 24 hours. This multiplexed
approach also detects more than 10 wild yeast species that are commonly discovered during beer brewing. The capacity to detect
all major beer-spoilage bacterial genera and wild yeast, along with the information to determine their beer-spoilage ability in a timely
manner, will greatly help brewers to improve the quality control procedure and significantly reduce the inventory cost.
P053:
Determination of cis-and trans- resveratrol by HPCE, in Armenian wine -making residues to develop new dietary resveratrol based
supplements
Gayane U. Khachvankyan, Simak S. Martirosyan, Armine P. Avagyan, Emil S. Gabrielyan
Scientific Center of Drug and Medicinal Technology Expertise, MOH, Armenia
86
Poster Abstracts
The viticulture was known in Armenia, a country of ancient civilization, for thousands of years. Today vine plantations occupy in our
country about 16 thousands ha and the vines’ variety includes several hundred sorts. Wine-making is a well-developed branch of the
national industry. Today the abundant residues of wine-making are used poorly. There is no scientific based research on Armenian
grape and wine. Resveratrol is a phytoalexin produced naturally by several plants when under attack by pathogens such as bacteria or
fungi. Resveratrol is found in grapes, mulberry, cranberry and blueberry, white hellebore, Japanese knotweed, and peanut. In grapes, it
mainly accumulates in the skin. Grapes are known to contain, besides resveratrol, a number of biologically and pharmacologically active
constituents. Resveratrol is highly biologically and pharmacologically active compound. Various beneficial effects of resveratrol were
demonstrated both in vivo and in vitro: life span prolongation; anti-cancer, anti-inflammatory, cancer-preventing, and cardiovascular
action; high cytotoxicity for cancer cells; preventing of chemical and radiation mutagenesis; protection from free-radical damage; protection of blood vessels, etc. Resveratrol prevents obesity even at fat- and calories-rich nutrition (so called “French paradox”). Thus, the
significance of this compound has spawned numerous methods for its analysis. These include GC/MS, HPLC, LC/MS with SPE for sample
preparation. We developed sensitive, fast and reliable CE method for determination of both cis and trans- resveratrol.We achived good
recovery with SPE and CE separation. Runs were completed in ~ 8 min with good LOD and LOQ. Each year about 15,000 ton resveratrol
rich grape residues are discarded without any utilization. This huge amount of raw material is practically unlimited source of resveratrol.
It seems reasonably to develop the extraction of resveratrol and other valuable grape components from waste wine-making products
in Armenia to offer them to the market as biologically active food supplement. Resveratrol as a food additive, is present at the market
in developed countries. At the same time there is a huge undeveloped market where resveratrol offer is poor. It is Russia and other
new independent countries of the former USSR. Existing offers for resveratrol and other grape components are very scanty and cannot
satisfy demands. Because of geographic location of Armenia very attractive for any kind of fruits, so there is a reason for developing
Armenian dietary supplements, containing resveratrol.
P054:
Analysis of charge heterogeneities in wheat high-molecular-weight glutenins using capillary isoelectric focusing
Boleslaw P. Salmanowicz
Institute of Plant Genetics, Polish Academy of Sciences, Salmanowicz B.P., Institute of Plant Genetics, Polish Academy of Sciences, PL 60-479 Poznan, Strzeszynska
Str. 34, Poland
The high molecular weight glutenin subunits (HMW-GS) are important storage proteins in wheat endosperm, and these proteins have
a major role in determining some dough characteristics and forming the viscoelastic properties. HMW-GS are determined by genes at
Glu-A1 loci, with multiple alleles present in this species.
A two-step capillary isoelectric focusing (cIEF) method for separation of wheat HMW-GS using dynamically coated fused silica capillaries
was developed. Focusing and mobilization were carried out using the mixture of different polymers (in particular cellulose derivatives).
Resolution between particular HMW glutenin isoforms was increased by utilizing chemical mobilization and improving protein solubility
by incorporating urea into the carrier ampholyte solution. The invert of the pH gradient and addition of a high concentration of isoelectric acids to the sample solution resulted in the formation of the acidic portion of the pH gradient adjacent to the capillary window.
The used conditions reduced migration times during a flowing anodic mobilization step and all glutenin isoforms were separated in
fifteen minutes. Because repeatability of migration time, peak area, and linearity (pI vs. mobilization time) is a problem often encountered in cIEF and is mainly caused by protein precipitation and protein-wall interactions, the capillaries were rinsed with high (1.5–3
M) concentration of hydrochloric acid for five minutes after each run, followed by water for 15 minutes. Performing consecutive runs
with rinsed capillary improved the reproducibility of peak area and migration times (RSD, 8 % and 4 %, respectively). The optimization
of focusing times, and ampholyte and anodic stabilizer concentrations were critical for the development of the presented method with
high reproducibility in the acid pH range. Additionally, the use of quasi-isoelectric anolytes and catholytes was investigated to improve
CIEF performances. Statistical screening of the significance of the effects of pressure, voltage, polymer concentration, and molecular
weight on the efficiency of dynamic coating and repeatability of migration times and peak areas were performed.
P055:
Capillary electrophoretic analyses of tryptic digests of water-soluble proteins from Bt-transgenic and non-transgenic maize species
in strongly acidic volatile background electrolytes
Petra Sázelová*1, Václav Kašička1, Carlos Leon3, Elena Ibañez3, Alejandro Cifuentes3
87
Poster Abstracts
1 Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
3 Institute of Industrial Fermentations (CSIC), Juan de la Cierva 3, Madrid, Spain
Transgenic Bacillus thuringiensis (Bt) maize belongs to insect-resistant genetically modified organisms (GMOs), which are expanding
rapidly worldwide. Bt hybrids with the event MON 810 were constructed by introducing the cry1Ab gene from Bacillus thuringiensis,
in order to produce its own insecticide, δ-endotoxin, for European corn borer (Ostrinia nubilalis). Due to the public concern on GMOs
role in the environment and food and feed safety, the development, growth and use of GMOs are under regulation in many countries,
especially in the EU. In order to fulfill these regulations, reliable and powerful analytical methods to detect and quantify GMOs and/or
their products in foods are needed. Besides the detection of transgenic DNA, protein analysis represents an alternative way to detect
GMOs. Protein fractions from transgenic Bt and non-transgenic maize varieties were analyzed by perfusion and monolithic RP-HPLC1.
Capillary zone electrophoresis (CZE) is being increasingly used for protein analysis and peptide mapping2-4, but up to now, rather limited
research has been oriented on the development of CZE methods for separation of the water-soluble proteins from crops5, 6.
The aim of this study was to test separation conditions for analysis of tryptic digests of water-soluble proteins (albumin fraction) from
the maize flours of transgenic Aristis-Bt and non- transgenic Aristis and Coventry maize varieties by CZE with UV-absorption detection
and potentially with subsequent mass spectrometry detection. Hence, strongly acidic volatile background electrolytes (BGEs) composed of highly concentrated weak acids, 0.5–2.0 mol/L acetic and formic acids and their mixtures were tested. CZE analyses were
performed in a home-made CE analyzer equipped with UV-absorption detector at 206 nm7. Significant qualitative and quantitative
differences among CZE-UV profiles of tryptic digests of water-soluble proteins from different maize species have been found and will
be in detail discussed in this contribution.
The work was supported by the GACR, the grants no. 203/08/1428 and 203/09/0675, by the Research Project Z40550506 of the ASCR,
and by the project 2009-10/7 of the cooperation between ASCR and CSIC.
1 J. M. Rodriguez-Nogales, A. Cifuentes, M. C. Garcia, M. L. Marina, J. Agric. Food Chem. 55 (2007) 3835
2 V. Dolník, Electrophoresis, 29, (2008) 143
3 E. C. Rickard, J. K. Towns, Method. Enzymol. 271 (1996) 237
4 M. Herrero, E. Ibañez, A. Cifuentes, Electrophoresis 29 (2008) 2148
5 A. R. Piergiovanni, J. Agric. Food Chem. 55 (2007) 3850
6 P. Sázelová, V. Kašička, E. Ibañez, A. Cifuentes, J. Sep. Sci. 32 (2009) 3801
7 V. Kašička, Z. Prusík, P. Sázelová, E. Brynda, J. Stejskal, Electrophoresis 20 (1999) 2484
P056:
Determination of chlorophenols by capillary electrophoresis with dual electrode detection for peak purity assessment
Fuying Du, Yingsing Fung
Department of chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
Determination of chlorophenols in environmental samples is important for pollution and health assessment of water bodies. Procedure
using the high efficient capillary electrophoresis for separation has been successfully developed for environmental analysis of phenols
in water. However, the demand of extremely high detection sensitivity for detecting chlorophenols in drinking water required by World
Health Organization1 leads to the need to improve method sensitivity as well as assessment of peak purity. Electrochemical detection
provides a suitable high-sensitivity detector for chlorophenols. In order to improve sensitivity to ppb detection, procedure coupling
solid phase extraction for sample cleanup prior to capillary electrophoresis for separation and electrochemical detection is developed
in the present work. Peak purity is assessed by a pair of dual electrochemical detectors placing at the end of the capillary column to
provide a sensitive, reproducible and stable electrochemical detector for chlorophenol compounds which are prone to form a polymeric
film on solid electrode when chlorophenols are electrooxidized2. The modification of the carbon fibre electrode surface with ionic liquid
is found to improve the detection performance by offering good resistance to surface fouling. In addition to ppb detection capability,
the dual electrode detection scheme can provide information for peak purity assessment. The analytical performance and applicability
of the procedure developed will be discussed at the meeting.
1 Guidelines for drinking-water quality. World Health Organization
2 Wang J., Ruiliang L. Anal. Chem. 1989, 61, 2809
88
Poster Abstracts
P057:
HILIC – Effect of mobile phase composition on separation of phenolic and flavone compounds
Veronika Škeříková, Pavel Jandera
Department of Analytical Chemistry, Faculty of Chemical-Technology, University of Pardubice, Studentská 573, 532 10 – Pardubice, Czech Republic
Columns containing zwitterionic sulfobetaine groups have been earlier applied for the separation of polar compounds by HILIC1, 2. This
work investigates separation of phenolic and flavone natural antioxidants on a zwitterionic organic polymer capillary column designed
for Hydrophilic Interaction LIquid Chromatography. The monolithic column was prepared by “in-situ” radical polymerization in a fusedsilica capillary (id = 320 µm). The polymerization mixture consist of 20 % [2‑(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium
hydroxide as monomer, 15 % ethylene dimethacrylate as cross-linker, 25 % 1-propanol, 25 % 1,4-butanediol and 15 % water. 2,2´-azo-bisisobutyronitrile was used as the initiator of the polymerization reaction which performed at 60°C for 20 hours 3, 4.
The polymethacrylate monolithic zwitterionic capillary column was used for separation of phenolic and flavone compounds as the
alternative to conventional ZIC-HILIC column1. The separation of phenolic and flavone compounds depends on the pH of the mobile
phase and weakly acidic buffers as the mobile phases are generally required for their separation. Due to differences in the dissociation
constants of the individual compounds, even small changes in the mobile phase pH may cause significant differences in resolution. The
relationships between the retention and the structures of sample compounds were investigated with various combinations of aqueous
buffers and organic modifiers.
All experiments were carried out at lab. temperature; the flow rate was set in the range 3–30 µl.min-1, according to the actual composition of the mobile phase. The capillary column was connected to UV-detector. Mixtures of aqueous ammonium acetate with organic
modifiers were used as mobile phases. To find optimum separation conditions, the concentration of ammonium acetate was changed
in the range 10–20 mmol.l-1 and pH of aqueous buffers was varied from 2.6 to 4.0. Methanol and acetonitrile were used as organic modifiers. Isocratic conditions and gradient conditions were optimized to separate maximum number of phenolic and flavone compounds
in the shortest separation time. The best separation was observed using a linear gradient with initial isocratic step or using gradients
comprised of two or three subsequent linear steps. The monolithic column shows dual HILIC-RP mechanism, therefore isocratic and
gradient separations were optimized also in RP-mod.
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project MSM 0021627502
and by the Grant Agency of Czech Republic under the project No. 203/07/0641. 1. Hemström P., Irgum K., J. Sep. Sci. 29, 1784 (2006) 2. Jiang Z., Smith N.W., Ferguson P.D., Tailor M.R., Anal. Chem. 79, 1243 (2007)
3. Jandera P., Urban J., Škeříková V., Kubíčková R., Pospíšilová M., J. Sep. Sci. 32, 2530 (2009)
4. Jandera P., Urban J., Škeříková V., Langmaier P., Kubíčková R., Planeta J., J. Chromatgr. A 1273, 22 (2010)
P058: Enantioseparation of agriculture toxins by means of cyclodextrin-modified microemulsion electrokinetic chromatography
Ali Reza Fakhari Zavareh, Saeed Nojavan, Kobra Hashemi Nasab
Chiral compounds based on 2-aryloxypropionic acids include a number of important herbicides that have been widely used for the
pre- and post-emergent control of broadleaf weeds in field grass, turf grass and cereal crops1. It was recognized that for these chiral
compounds only the (R)-enantiomers show herbicidal activity. Also, the biological transformation of chiral compounds can be stereoselective, and the uptake, metabolism and excretion of enantiomers may be very different2. The goal of this work is to evaluate the
capability of cyclodextrin-modified microemulsion electrokinetic chromatography for the separation of some of these agrochemical
toxins such as: haloxyfop-methyl, fenoxaprop-pethyl and indoxacarb. Enantioseparation of these toxins was optimized by using a standard oil-in-water microemulsion and varying the nature and concentration of cyclodextrin additives whilst keeping the applied voltage
of 20 kV and capillary temperature of 30ºC constant. The standard oil in water microemulsion BGE solution consisted of 0.8 % w/w
octane, 6.6 % w/w 1-butanol, 2.0% w/w SDS and 90.6 % w/w 80 mM sodium phosphate buffer (pH 8.0). Enantioseparations with high
resolution and reasonable migration times was achieved by using hydroxyl propyl-ß-cyclodextrin and carboxymethyl- ß-cyclodextrin in
the microemulsion background electrolyte.
89
Poster Abstracts
1 J.M. Schneiderheinze, D.W. Armstrong, A. Berthold, Chirality 11 (1999) 330
2 H.R. Buser, M.D. Muller, Chimia 51 (1997) 694
P059:
Caleosin in native and artificial plant oil-bodies
Martina Vermachova1, Zita Purkrtova1, Jiri Santrucek1, Pascale Jolivet4, Thierry Chardot4, Milan Kodicek1
1 Department of Biochemistry and Microbiology, ICT Prague, Czech republic
4 JRU 206 Chimie Biologique, INRA Thiverval - Grignon, France
Oil-bodies are lipoprotein particles used by plants for lipid storage, especially in the oil seeds. They are composed of triacylglycerols surrounded by a phospholipid monolayer with many integrated proteins that stabilize the structure and may play the crucial role in formation of the
bodies and also in lipid metabolism during embryo germination. Due to the high hydrophobicity of these proteins, only little information is
available about their steric arrangement and specific functions. They are supposed to be the main source of high stability of oil-bodies, which
embarrasses oil production from plant oil seeds.
Artificial oil-bodies can be prepared from the mixture of lipids, phospholipids and recombinant oil-bodies' proteins and are considered a good
model for studying these proteins. They can be used as delivery vesicles for hydrophobic drugs or as an expression system. It's supposed
that the protein's arrangement within artificial oil-bodies resembles the native structure, but they exhibit lower stability than the native ones.
The main aim of this work is to compare the arrangements of proteins (namely caleosin) within the native and artificial particles. We chose
the method of surface mapping based on selective chemical modifications of individual amino acids residues and their identification by mass
spectrometry. It enables identification of protein regions that are in direct contact with aqueous environment.
Native oil-bodies were isolated from seeds of Arabidopsis thaliana. This model organism can well demonstrate the properties of oil crop-plant
Brassica napus, because they are members of the same plant family (Brassicaceae). Recombinant His-tagged caleosin was expressed in
Escherichia coli and purified by affinity chromatography. Artificial oil-bodies were prepared by sonication. Both types of particles were modified by reagents specific for arginine, lysine and tyrosine. We tried to investigate the presence of disulphide bridges by modifications of cysteine residues. Proteins were separated by SDS-PAGE and in-gel digested by trypsin or chymotrypsin. Mass shifts, characteristic for individual
modifications, were searched in mass spectra of peptide mixtures by MALDI-TOF or LC-MS/MS method.
Several modifications of arginine, lysine and tyrosine were identified reliably. The set of obtained results showed certain difference between
the natives and artificials particles. It could be caused by the different mechanism of insertion of the protein into the lipid core. Unexpected
caleosin isoforms were identified.
Financial support from the Czech Ministry of Education (grant number 6046137305) is kindly acknowledged. Monday, March 22nd, 2010 P060:
Lipidomic analysis of plant and animal tissues using HPLC/MS
Eva Čáňová, Miroslav Lísa, Michal Holčapek
University of Pardubice, Faculty of Chemical Technology, Department of Analytical Chemistry, Studentská 573, 53210 Pardubice, Czech Republic
Lipids are an important part of human diet and they play an essential role in many biochemical processes in the human organism. The disruption of lipid metabolism can lead to serious diseases (i.e., obesity, atherosclerosis, cancer, cardiovascular problems, etc.) and the characterization
of lipid composition is necessary to describe their roles. For this purpose, lipidomic analysis using HPLC/MS was optimized. The total lipid
extract containing all lipid species was prepared using chloroform/methanol/water extraction according to Folch procedure. The total lipid
extract was fractionated into individual lipid classes according to their polarity using the hydrophilic interaction liquid chromatography. The
isolated fractions of individual lipid classes were separated using reversed-phase high-performance liquid chromatography with C18 porous
shell particles column. Individual lipid species were separated according to the composition of fatty acids, i.e., their acyl chain lengths, number and position of double bonds. Individual lipids were identified using mass spectrometry. Electrospray ionization was used for polar lipids
(phospholipids) and atmospheric pressure chemical ionization for non-polar lipids. The lipid composition of various plant and animal tissues
was characterized using optimized HPLC/MS method. The retention behavior and important fragment ions observed in mass spectra and their
use for the identification of lipids was described.
90
Poster Abstracts
This work was supported by the grant project No. MSM0021627502 sponsored by the Ministry of Education, Youth and Sports of the
Czech Republic and projects Nos. 203/09/P249 sponsored by the Czech Science Foundation. Tuesday, March 23rd, 2010
P061:
ANALYSIS OF ANTHOCYANIN DYES IN LONICERA CAERULEA EXTRACTS
Renáta Myjavcová1, Petr Marhol2, Vladimír Křen2, Vilím Šimánek4, Jitka Ulrichová4, Karel Lemr1, Barbora Papoušková1, Petr Bednář1
1 Department of Analytical Chemistry, Faculty of Science, Palacký University, Olomouc, Czech Republic
2 Institute of Microbiology Academy of Sciences of the Czech Republic, Praha, Czech Republic
4 Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University, Olomouc, Czech Republic
Anthocyanins are important plant dyes belonging to the group of flavonoids. They are formed as secondary metabolites during biochemical processes occuring in plant cells. These substances are long time objects of research due to their antioxidant, antimicrobial
and other positive effects on human health. This contribution deals with their investigation in Lonicera caerulea fruits as a promising
source of flavonoids for human diet. Optimization of extraction of anthocyanins from Lonicera fruits was done. Several extraction solutions (i.e. various mixtures of methanol, ethanol, acetone and water) were tested. It was found out that the highest extraction yield is
achieved using a mixture of methanol and acetone (50/50) acidified with 0.1 % phosphoric acid (v/v). A combination of semipreparative
chromatographic fractionation with microcolumn high performance liquid chromatography and mass spectrometry (μLC/DAD/ESI-MS2;
QqTOF as analyzer) was used for the analysis of obtained extracts.
Among anthocyanin dyes, cyanidin glycosides dominate (i.e. 3-hexoside, 3,5-dihexoside and 3-rutinoside) followed by peonidin, pelargonidin and delphinidin glycosides. Surprisingly high yield of 5-methylpyranocyanidin-3-glucoside was observed which seems to rise
during the contact with acetone as component of extration media. Beside, several more complex dyes were also identified (i.e. cyanidin-3-hexoside-ethyl-(epi)catechin, dimer of cyanidin-3-hexoside etc.) were identified in prepared fractions. Identity of those dyes was
further confirmed using exact mass measurement and interpretation of MS/MS spectra. Authors gratefully acknowledge the support of the Ministry of Education, Youth and Sports, Czech Republic (MSM6198959216).
P062:
The influence of extraction conditions on total phenolic content and anthocyanins profile of grape skins
Lenka Šťavíková*1, Martin Polovka2, Barbora Hohnová1, Michal Roth4
1 Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Veveří 97, 602 00, Brno, Czech Republic;
Institute of Food Science and Biotechnology, Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00 Brno
2 Department of Chemistry and Food Analysis, VUP Food Research Institute, Priemyselná 4, P.O. Box 25, SK-824 75 Bratislava, Slovak Republic
4 Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Veveří 97, 602 00, Brno, Czech Republic
The improvement of human health by the consumption of food or food supplements rich in antioxidants have been increasingly recognized in recent years. Phenolic compounds of grape berries have attracted much interest due to their antioxidant properties and their
beneficial effects for human health, e.g., reduction of coronary heart disease, antimutagenic, anti-inflammatory and anti-carcinogenic
properties. Therefore, rapid and efficient extraction technique prior to chromatographic analysis is of primary interest. The liquid extraction at elevated temperature and pressure – Pressurized fluid extraction (PFE) presents fast, effective and environmentally friendly
extraction method for the analysis of grape pigments.
In this contribution, PFE in static mode was utilized for the extraction of 3-O-monoglucoside anthocyanins malvidin (Mv), delphinidin
(De), peonidin (Pn), cyanidin (Cy) and petunidin (Pt) from grape skins of two grape varieties Alibernet and St.Laurent. Extracts were prepared from 0.5g of lyophilized grape skin powders using the solvent extraction to methanol, ethanol and water at different temperature
from 40 up to 120°C. In addition, total phenolic content (TPC) of individual extracts was determined using the Folin-Ciocalteu assay.
The identification and quantification of the above compounds by high-performance liquid chromatography with diode array detection
(HPLC-DAD) was performed. The wavelength was set at 520 nm. All compounds were separated and identified during 50 minutes. Depending on the extraction solvent and temperature, the amount of anthocyanins ranged from 0.44 to 267.84 mg/g for Alibernet and
from 0.21 to 178.27 mg/g for St. Laurent. The limit of detection for malvidin-3-O-glucoside, delphinidin-3-O-glucoside was 488 ng/l and
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Poster Abstracts
244 ng/l for peonidin-3-O-glucoside, cyanidin-3-O-glucoside and petunidin-3-O-glucoside. For TPC of individual extracts a double-beam
UV-VIS spectrometer with appropriate equipment was used. The TPC in Alibernet extracts is approximately two-times higher than that
in St. Laurent.
HPLC analysis of anthocyanins content revealed their presence at significant level, but their presence only partly contribute to the
properties of extracts conventionally related to TPC. The experimental data were evaluated by comparing with the results obtained by
standard Soxhlet extraction. This work was supported by the Research project of Ministry of Agriculture (08W0301), by the Slovak Research and Development Agency (APVV SK – CZ 0072 – 07), by the Czech Science Foundation (GA203/08/1536), by the Academy of Sciences of the Czech Republic
(Institutional Research Plan AV0Z40310501), by the Czech Academy of Sciences (KAN400310651) and by the Ministry of Education of
the Czech Republic (LC06035).
P063:
Hyphenated technique for the extraction and determination of isoflavones in algae: ultrasound-assisted SFE followed by RRLC MS/MS
Bořivoj Klejdus1, Lea Lojková1, Merichel Plaza de Moral3, Dagmar Štěrbová1, Marie Šnóblová1
1 Department of Chemistry and Biochemistry, Mendel University in Brno
3 Departamento de caracterizacion de alimentos, Instituto de fermentaciones incustriales, Madrid, Espana
Isoflavones are widely studied because of their phytohormonal functions, that influence health positively and are used as additives to functional food. Higher concentrations of isoflavones can harm the reproduction abilities of cattle. These compounds are present in large concentrations in Fabaceae (soya, red clover) and in smaller concentrations in 20 other families.
Thanks to new extraction methods and RRLC, monitoring of trace amounts of isoflavones in real samples became possible as well as determination of their precursors along the methabolic pathway. Sea algae were chosen as the samples: Cystosteia abies, Chondrus crispus, Sargassum
muticum, Undaria pinnatifida, Halophyllis incurva, Porphyra sp., Hypnea spinella and Sargassum vulgare, with several freshwater algae and
cyanobacteria used as control samples: Spongiochloris spongiosa, Scenedesmus and Nostoc.
Ultrasonic-assisted superitical fluid extraction is excellent sample preparation hyphenated technique. SFE optimisation was carried on with a
sonication bath, but it was found out during the study, that the instrumentation mode with a thorn (Sonoplus, Bandeln electronic, Germany)
put directly in the sample gives the sonication much higher boost, and the optimisation of sonications parameters led to even better results.
The hypothesis is, that sonication enables the transport of analytes out of the matrix and destructs the analyte-matrix bonds. Because of
that, SFE is no longer driven by diffusion, where the mass transfer from matrix to extraction fluid is the most important factor, but it is more
driven by solubility – and although the solubility of polar analytes in supercritical fluid is quite low, this problem can be solved via modifier
and high flow-rate.
Rapid resolution liquid chromatography 1200 Series with MS-MS detector 6460 Triple Quad with a Jet Stream technology (both Agilent Technologies) and the chromatographic column Zorbax SBCN (2.1 x 100 mm, particle size 3,5 μm) was used for the determination of isoflavones
in the extracts. New analytical method was developed for this instrument configuration, with excelent parameters from the viewpoint of
determination of very low concentration of isoflavones, and still requiring only 9 min per one analysis.
Thanks to the combination of US-SFE and RRLC MS-MS, isoflavones were determined for the first time in real algae samples. New extraction
methods are crucial in the methabolomic area.
Daidzin, genistin, ononin, daidzein, sissotrin, genistein, biochanin A and large amounts of formononetin were found in the samples. The most
rich sample was Chondrus, but all sea algae samples showed comparable or much higher content of isoflavones than freshwater samples.
LOD varied in the range 1 – 16 pg/μl, RSDs of HPLC were in range 0.8 – 3.1 %, RSDs of sonication/SFE method were in range 2.9 – 7.4 % for
real samples.
This study was supported by grants No. 525/07/0338 and 525/08/P540 from Grant Agency of the Czech Republic.
P064:
New SPE/SFE combinatory approach for the extraction of phenolic compounds from real microalgae samples
Lea Lojková1, Jiří Kopecký2, Jiří Vlček1, Bořivoj Klejdus1
92
Poster Abstracts
1 Ústav chemie a biochemie, Mendelova universita v Brně
2 Ústav autotrofních mikroorganismů, Mikrobiologický ústav AVČR
SFE is known as a well-proven tool for the extraction of non-polar compounds from complex matrices. Thanks to the continuous modifier addition, supercritical fluids can extract well also very polar analytes, like isoflavones or aminoacids.
Combined with SPE, the method is able to process large volumes of liquid samples containing trace concentrations of target analytes
mixed with large amount of ballast compounds. The analytes of interest are pre-concentrated in the SPE cartridge, which is small enough to fit in the extraction cell. During this step, some ballast compounds are rinsed from the cartridge so the sample is cleaner before
the SFE step. This helps to prevent the restrictor plugging and the structure of SPE cartridges keeps tiny particles in the SFE column.
The aim of this work was to develop a method for the extraction of polar phenolic compounds from freshwater microalgae: Chlorella
vulgaris, Chloridella, Euglena gracilis, Porphyridum cruentum, Chlamydomonas rainhardii, Spongiochloris spongiosa, Spirulina platensis,
Anabaena doliolum, Pleurastrum sarcinoideum, Nostoc sp. and Cylindrospermum sp.. Following target analytes were investigated: gallic
acid, protocatechuic acid, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic, chlorogenic, vanillic, caffeic and syringic acid, 4-hydroxybenzaldehyde, p-cumaric, ferulic, sinapic and o-coumaric acids.
Two extraction methods were developed for two different types of extraction cartridges. It was found out, that in both cases the most
efficient modifier composition is close to the composition of solutions used in standard SPE for a given type of cartridge. If this will
prove to be a common trend, it could simplify a lot the modifier selection in development of new SPE/SFE methods.
A method using Oasis MCX 3cc cartridge (60 mg, Waters) requires 400 µL of 2M HAc as entrainer. The extraction conditions were
optimised to 40 MPa, 80°C, 60 min, 12 cm restrictor 50 µm i.d., 20 ml MeOH as trapping solvent and two different modifiers, MeOH/
H2O 1:1 (50/50 v/v) and with a 5 % ammonia solution (20 % MeOH and 5 % ammonia in ultra-pure water).
The method using Spe-ed Advanta cartridge (100 mg, Applied Separations) has slightly increased recovery and a bit shorter extraction
time, and requires modifier mixture containing 50 % MeOH and 5 % ether, the use of corrosive substances like HAc and ammonia is not
necessary. Recoveries varied from 86 % to quantitative.
All extractions were carried out in SE-1 extractor (SEKO-K, Brno) equipped with a device for continual modifier addition.
Reversed phase liquid chromatography-electrospray mass spectrometry analysis was used for the analyte determination: the chromatograph was HP 1100 with single quadruple mass-selective HP MSD detector (G1946A, both Hewlett-Packard) and chromatographic
column Zorbax SB – C 18 (75 mm × 4.6 mm, 3.5 μm, Agilent Technologies). LOD varied in the range 1 – 4 ng/l, combined RSDs for both
SFE and HPLC were in range 1.2 – 5.8 %.
This study was supported by grants No. 525/07/0338 and 525/08/P540 from Grant Agency of the Czech Republic. Wednesday, March 24th, 2010
P065:
HPLC/MS Characterization of Regioisomeric Triacylglycerols
Hana Velínská, Miroslav Lísa, Michal Holčapek
Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic
Triacylglycerols (TGs) are the main compounds of animal fats and plant oils. Each TG can differ from other TG in the length of acyl chain
of fatty acids, number and position of double bonds (DB) in acyl chains, cis-/trans- configuration of DB and stereochemical position
(sn-1, 2 or 3) of fatty acid on the skeleton of glycerol (regioisomers). TGs regioisomers were analyzed by optimized silver-ion normalphase high performance liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry.
An optimisation of separation conditions included the mobile phase composition, column temperature and gradient steepness. The
separation conditions were optimized with the emphasis on the separation of triacylglycerol regioisomers R1R2R1 and R1R1R2. Abundant
peaks of [M+H-RiCOOH]+ fragment ions in APCI mass spectra were used for the identification of individual fatty acids. The regioisomeric
position of each acyl was determinated on the basis of relative ratio of fragment ions [M+H-RiCOOH]+, because the neutral loss from
the middle sn-2 position is less energetically favoured than from the position sn-1(3). The chemical interesterification (randomization)
was used for the generation of standard mixture series of TGs regioisomers. This relatively simple process allows preparing selected TGs
regioisomers from single-acid TG standards. The degree of changes is based on the reaction temperature, reaction time and catalyst
used. The most common catalyst in this process is sodium methoxide, because of its better reactivity. First, it was necessary to optimize
the reaction conditions to achieve sufficient reaction robustness and reproducibility. In our work, eleven randomization mixtures of
different standards of TGs were prepared. The ratios of fragment ions from the saturated, mono/di/untrisatured TG regioisomers were
summarized in the table.
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Poster Abstracts
This work was supported by project MSM0021627502 sponsored by the Ministry of Education, Youth and Sports of Czech Republic and
project 203/09/0139 sponsored by the Czech Science Foundation.
P066:
Investigation of a Novel Electrophoretic Exclusion Technique for the Concentration and Differentiation of Proteins and Small Molecules
Michelle M. Meighan, Michael W. Keebaugh, Jared Vasquez, Mark A. Hayes
Arizona State University
Electrokinetic separations juxtaposed by flow fields represent a valuable tool for separating complex mixtures. A novel separations
technique that can dynamically capture specific species in bulk solution from a complex mixture without molecular recognition elements is presented here. The basic premise of the new device is based on electrophoretic principles and exploits differential transport
near the capillary entrance by employing a large contraction ratio and setting flow and electric field gradients opposite one another. Initial experiments examine the interface where potential is initiated near a channel by examining the fundamental parameters of flow
dynamics, applied potential, and electrophoretic mobility. Initial published results demonstrate the successful exclusion, concentration,
and differentiation of small dye molecules in solution. Using a 75 µm i.d. capillary that was 13 cm in length, positively-charged Neutral
Red (MW 288.8) was isolated from a solution of 5 mM aspartic acid buffer (pH 2.8) containing cationic Methyl Green dye (MW 458.5).
The Neutral Red dye was successfully concentrated while allowing the Methyl Green to evacuate the main chamber at 2.5 kV. The
results from these experiments enabled the study of proteins using the electrophoretic exclusion method. Several parameters were
altered in order to examine myoglobin, including buffer, capillary, and pH. The new system contained an EOF- reducing capillary and
1 mM tris-HCl buffer, pH 8.0. Preliminary results indicate over 500 times concentration of myoglobin in 45 s. This simple approach of
countering electric field and hydrodynamic flow to exclude species has the potential to improve upon electrophoretic separations and
the analysis of complex biological solutions. Tuesday, March 23rd, 2010
P067:
Inherent peak compression of charged analytes in electrochromatography
Ivo Nischang1, Ulrich Tallarek2
1 Johannes-Kepler-University Linz, Institute of Polymer Chemistry, Welser Strasse 42, A-4060 Leonding, Austria
2 Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany
We resolve peak compression effects of charged analytes in capillary electrochromatography using columns packed with strong cationexchange stationary phase particles1. For this purpose, we combine electrochromatographic peak shape analysis with the results of
numerical simulations and confocal laser scanning microscopy in the packed capillaries. We successfully identify electrical field-induced
concentration polarization as the key physical phenomenon responsible for the inherent existence of local electrical field gradients on
the scale of an individual support particle. As a result, positive and negative field gradients exist between and inside the particles along
the whole packing. These field gradients are an inherent property of hierarchically structured porous media under typical conditions
in electrochromatography2. Their intensity depends on the particles’ cation-selectivity (governed by the particles volume charge density and the mobile phase ionic strength) and the applied field strength. The interplay of these local field gradients with the analytes’
retention based on intraparticle adsorption determines whether fronting, tailing, or spiked analyte peaks are observed. It provides a
mechanism by which strongly retained analytes can be eluted over long migration time scales with little zone dispersion. These unique
stacking phenomena, which cannot be related to different injection histories, can explain the “anomalous” peak compression effects
with strong cation-exchange particles, which have been reported more than a decade ago and since then remained largely unresolved3.
1 Nischang I, Tallarek U (2009) J Sep Sci 32:3157-3168
2 Nischang I, Chen GF, Tallarek U (2006) J Chromatogr A 1109:32-50
3 Smith NW, Evans MB (1995) Chromatographia 41:197–203
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Poster Abstracts
P068:
A new electrophoretic focusing principle: Focusing of non-amphoteric weak ionogenic analytes using inverse EMD profiles
Zdeňka Malá, Petr Boček, Petr Gebauer
Institute of Analytical Chemistry of the ASCR, v.v.i., Brno, Czech Republic
This contribution presents a new separation principle in capillary electrophoresis that offers focusing of weak non-amphoteric ionogenic
species and their inherent transport to the detector and brings another dimension into electrophoretic separations since its mechanism
is different from that of CZE and IEF. Preliminary results indicate that the new focusing technique will be useful especially for resolution
of low analyte concentration levels in the μM range and that its separation power is sufficient to provide baseline resolution of analytes
differing by only 0.05 pKa units. The principle of the method is the existence of an inverse electromigration dispersion (EMD) profile,
where pH is decreasing towards the anode or cathode for focusing of anionic or cationic weak analytes, respectively. The course of velocity, pH and conductivity along such a profile allows capturing and focusing analytes of certain properties (ionic mobilities and pKas).
An analyte is focused at a point where its velocity is equal to the velocity of the EMD profile, and, at the same time, its velocities behind
and in front of that point are higher and lower than those of the EMD profile, respectively. The width of a focused zone depends on the
slope of the analyte velocity against the profile velocity at the focusing point. A focused analyte is bound to the focusing point during
the entire run and is broadened together with broadening of the EMD profile. The time dependence of this broadening is described
by equations that are formally the same as those for simple diffusion. The new principle and its theory presented are supplemented
by convincing computer simulations and experiments, and electrolyte systems for focusing of various kinds of analytes according to
velocity and pH range are recommended.
We gratefully acknowledge support by the Grant Agency of the Academy of Sciences of the Czech Republic (Grants No IAA 400310609
and IAA 400310703), by the Grant Agency of the Czech Republic (Grant No 203/08/1536) and by the Institutional Research Plan
AV0Z40310501 of the Academy of Sciences of the Czech Republic.
P069:
Determination of acid-base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis
Sille Ehala, Anastasiya A. Grishina, Andrey E. Sheshenev, Ilya M. Lyapkalo, Václav Kašička
1 Institute of Organic Chemistry and Biochemistry, v.v.i., Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10, Prague 6, Czech Republic
Knowledge of acid-base dissociation constants (pKa) of ionogenic compounds is essential in a wide range of their applications, because
the neutral and ionic forms of these compounds may exhibit very different physicochemical and biological properties. Traditionally,
potentiometric and spectrophotometric methods are used for the pKa determination. The main drawback of these classical methods
is the difficulty to handle impure compounds. In recent years, capillary zone electrophoresis (CZE) has been recognized as an effective
method for pKa determination1, 2 because of some intrinsic advantages, such as a high resolving power, low analyte and solvent consumption and the ability to handle impure compounds.
In this study, CZE was employed to determine thermodynamic pKa values of seven newly synthesized enantiopure zwitterionic bases
pertaining to hitherto unknown family of heterocyclic compounds, 2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2‘,1‘-f]1, 3, 2 diazaphos-phinin-4-ium-5-olate 5-oxides, originally designed as chiral Lewis base catalysts for enatioselective reactions. Determination of their pKa
values was essential for assessment of their activity and efficiency3. To achieve this goal, the pH dependence of the effective electrophoretic mobilities of these compounds was measured by CZE in bare fused silica capillaries in strongly acidic background electrolytes
(BGEs), within pH range 0.85 – 2.80. The effective electrophoretic mobilities, measured by CZE at variable temperatures and ionic
strengths (due to different ambient temperatures, Joule heating and very acidic BGEs), were first corrected to reference temperature,
25°C, and constant ionic strength, 25 mm. Only then, these corrected mobilities were used for determination of the pKa values. A
non-linear regression analysis was employed to estimate the pKa values of the studied heterocyclic bases from the dependence of their
effective electrophoretic mobilities on pH. Thermodynamic pKa values of the investigated heterocyclic bases were found to be very low,
in the range 0.48 – 0.62.
In conclusion, the applied CZE method has proved to be a suitable and efficient technique for determination of the exceptionally low pKa
values of seven newly synthesized very weak zwitterionic heterocyclic bases in aqueous medium. Despite the measurements of effective
electrophoretic mobilities in BGEs at fairly low pH range (0.85 – 2.80), the pKa determination with reasonable precision was attained.
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Poster Abstracts
This work was supported by the Czech Science Foundation, grants no. 203/08/1428 and 203/09/0675, and by Research Project
no. Z40550506 of the Czech Acad. Sci.
1 S.K. Poole, S. Patel, K. Dehring, H. Workman, C.F. Poole, J. Chromatogr. A 1037 (2004) 445-454
2 D. Koval, V. Kašička, J. Jiráček, M. Collinsová, Electrophoresis 27 (2006) 4648-4657
3 S.E. Denmark, G.L. Beutner, Angew. Chem. Int. Ed. 47 (2008) 1560–1638
P070:
High-resolution electrophoretic simulations: Performance characteristics of one-dimensional simulators
Richard A. Mosher1, Michael C. Breadmore2, Wolfgang Thormann*3
1 RAM Software Solutions, Tucson, AZ, USA
2 University of Tasmania, Hobart, Australia
3 University of Bern, Bern, Switzerland
The development of electrophoretic computer models and their use for simulation of electrophoretic processes has increased significantly during the last few years. Our interest is focused on one-dimensional transient electrophoresis models which allow high-resolution, real-power simulations of complex buffer systems in zone electrophoresis, isotachophoresis and isoelectric focusing1-6. These
models are based on equations that are derived from the transport concepts in solution under the influence of a d.c. electric field
together with user-inputted experimental conditions, including initial concentrations, pKa values, mobilities, field strength or current,
column length and column segmentation. Simulations provide detailed insight into the processes involved in the separation allowing
the researcher to understand the result of a particular combination of experimental conditions, and with this information design new
superior systems. Simulations can also be employed for educational purposes. Three comprehensive simulators, SIMUL5 of Hruška et
al.5, SPRESSO of Bercovici et al.6 and our GENTRANS code, were used to simulate the dynamics of different configurations, including
an isotachophoretic boundary, a migrating hybrid boundary and the zone electrophoretic separation of dipeptides. Simulation data
were produced at real power, i.e. at electric field strengths that are comparable to those used experimentally. The simulators generate
identical data when identical initial and boundary conditions are used. The simulators, however, differ in the numerical schemes used
and other features. The performance characteristics of the simulators, including execution time, number of required column segments
and ease of performing a simulation, were investigated via execution of the simulation examples on the same PC.
1 R.A. Mosher, D.A. Saville, W. Thormann, The Dynamics of Electrophoresis, VCH, Weinheim, Germany, 1992
2 W. Thormann, C.-X. Zhang, J. Caslavska, P. Gebauer, R.A. Mosher, Anal. Chem. 70, 549, 562 (1998)
3 R.A. Mosher, W. Thormann, Electrophoresis, 23, 1803-1814 (2002)
4 W. Thormann, J. Caslavska, M.C. Breadmore, R.A. Mosher, Electrophoresis 30, S16-S26 (2009)
5 V. Hruška, M. Jaroš, B. Gaš, Electrophoresis 27, 984-991 (2006)
6 M. Bercovici, S.K. Lele, J.G. Santiago, J. Chromatogr. A 1216, 1008-1018 (2009)
P071:
Focusing of metals in ligand field step gradient
Eliška Šišperová, Eliška Glovinová, Jan Pospíchal
Dept. of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemědělská 1, 613 00 Brno, Czech Republic
The capillary electrophoretic method for the focusing and selective pre-concentration of metal chelates with subsequent on-line ITP
analysis was developed and verified.
The ions of alkali earth metals (Mg, Ca, Sr, Ba) were selectively pre-concentrated from the mixture and analyzed. Focusing of metals
was carried out in ligand field step gradient, which was realized by an addition of a convenient ligand agent to the regular stationary
pH step gradient1. During the first step, the metal ions were continuously dosed into the column, where they were selectively trapped on the stationary
ligand field step gradient in the form of non moving zones of chelate complexes with effective zero charge. After accumulation of de96
Poster Abstracts
tectable amount of analyte, the dosing was stopped and accumulated zones were mobilized to the analytical column, where they were
analyzed e.g. by ITP method with conductivity or photometric detection.
The proper electrolyte system for the dosing (mode IEF), mobilizing (mode MBE) and analytical step (mode ITP) were developed and
realized.
The selectivity of the trapping can be regulated by a choice of the pH and convenient complexing agents. As a sample analytes served
model real mixtures of alkali earth metals. The proposed method enable increase of detection limit is 5-29x in comparison to classical
methods – e.g. ITP.
P072:
Shannon and Rényi entropy in microscope image processing
Tomáš Náhlík, Dalibor Štys, Jan Urban, Petr Císař
Institute of physical biology
Shannon and Rényi entropy express a measure of uniqueness of information contained in the image. A parameter alpha in equation of
Réniy entropy defines what part of the image information will be highlighted. Shannon entropy represents correctly normal distribution.
Coefficient alpha smaller than 1 represents distribution more narrow than normal (light tails) coefficient higher than the opposite, heavy
tails. We have shown experimentally that each alpha parameter emphasizes different features of the image. There is not one universal
solution to optimal visualization of the image information content for human eye and brain. In addition, several colour enhancement
methods may be utilized when distinct structures only are sought, essentially without impact on the information content.
Instead of examining the response of human brain, we examined the objective information content of the image. We used the most
common constructive phase contrast images of standard balls and living cells. We found that different color channels carry different
information, in many cases, the information differed dramatically. For each of the channels, we tested the dependency of the entropy
value for the whole image on alpha parameter. We performed the calculation for different positions of the standard ball with respect to
the focal plane. If the image of the living cell was composed of objects behaving as standard balls and if they would behave sufficiently
dissimilar, we should be able to de-construct the cell image into elements showing optimally in these traces. Instead, we found that
images of balls in various distances from the focal plane behaved similar except, perhaps, for few traces of balls placed close to focal
plane. In any case, the alpha-dependency of the entropy of living cell image could not be reconstructed from combination of that of
standard balls.
In conclusion we confirmed that the living cell contains objects of shapes distinct from standard balls which are transmitted by
microscope optics in a manner different from that of standard ball. The resulting image, and most obviously also the origin, is of multifractal character, most probably both in physical and in chemical space. We report also initial experiments on determination of biological
cell states as the map of asymptotically stable cycles. Tuesday, March 23rd, 2010
P073:
Influences on protein properties
Sabine Redweik, Xi Deng, Hermann Wätzig
Institute of Pharmaceutical Chemistry, University of Technology Braunschweig, Beethovenstrasse 55, D-38106 Braunschweig, Germany
Understanding the unique properties of proteins is essential. However, the knowledge about their physico-chemical properties and their
real affecting size is still insufficient. Recently, findings that small proteins do not fit into large pores have been reported1. Their hydrodynamic radius can be measured by various methods, e.g. by Dynamic Light Scattering (DLS) or Atomic Force Microscopy (AFM) where
similar values for the radius are achieved. The concept of a hydrodynamic radius assumes a sphere-like protein. This it is influenced
by the viscosity of the solution. Because this hydrodynamic radius is much smaller than the above mentioned pores there must be an
expanded affecting size of the proteins. Therefore the influence and behavior of the solvent layers and of various analytes on protein
properties have to be thoroughly discussed. Our so-far obtained results about this topic will be presented.
1 Edward Yeung, presentation on HPLC 2009 in Dresden
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Poster Abstracts
P074:
Optimization of peak capacity in one- and two-dimensional nanoLC
Sebastiaan Eeltink, Evert-Jan Sneekes, Bas Dolman, Bjorn* de Haan, Remco Swart
Dionex Corp. Amsterdam
Introduction: The higher peak capacity of two-dimensional (2D) LC separations is often sacrificed for the ease of use of a one-dimensional (1D) approach. This has resulted in extremely long gradients (up to 4 hours) under conditions that waste analysis time, without
significantly improving the separation. In order to obtain the best compromise between peak capacity and analysis time for highly
complex proteomic samples it is necessary to optimize column technology and LC conditions in one- and two-dimensional LC. We use
the “peak-capacity-to-analysis-time ratio” to characterize the efficiency of a chosen separation method. Method: The effects of gradient
time, column temperature, flow rate, and column length were studied to maximize the peak-capacity-to-analysis-time ratio for peptide
samples in one-dimensional RP gradient nanoLC. These findings were compared to an offline 2D-LC approach using SCX and RP in which
effects of 1D gradient time, sampling time, and 2D gradient time on performance were studied. The 2D-LC approach was developed
with a microfractionation option of the autosampler allowing automated fractionation and reinjection of the separated peptides providing maximum ease of use. Preliminary data: Optimal 2D-LC conditions at undersampling conditions were obtained applying a short
(10 min) 1D gradient and 20 min 2D RP gradients. For separations requiring a maximum peak capacity of 360 1D-LC was superior to
the off-line 2D-LC approach in terms of analysis time. For separations requiring a peak capacity higher than 360 it is more profitable to
perform a 2D experiment. With the right conditions this will provide a higher peak-capacity-to-analysis-time ratio. Finally, the potential
of the optimized offline 2D-LC coupled to tandem mass-spectrometric detection is demonstrated by the analysis of a tryptic digest of
a 20 protein mixture and an E. coli tryptic digest. Novel aspect: Automation of off-line 2D-LC and maximizing separation resolution per
time for peptide mapping
P075:
New Resolution Equations For CZE and Micellar Electrokinetic Capillary Chromatography, MECC
Kiumars Ghowsi1, Hosien Ghowsi2
1 Islamic Azad University Majlesi Branch,86315/111 Isfahan Iran
2 Department of Mathematics NaJaf Abad Payam Noor University Isfahan Iran
Abstract
The fundamental equation for the number of theoretical plates is given by Jorgenson1.This equation for the number of theoretical plates
has been used to obtain resolution eqution by Jorgenson.It is shown by us2, the number of theoretical plates equation by Jorgenson is
wrong, automatically makes the resolution equation wrong.
A new model based on effective length migrated on a similar to tread mill case for various modes of operation in Capillary Zone
Electrophoresis has been constructed. New resolution equation for Capillary Zone Electrophoresis has been proposed. This equation
proposed by us is compared with Jorgenson resolution equation.These two resolution equations have similarities and differences.Our
resolution equation could be used further for discussing optimization as function of variables such as electrophoretic and electroosmotic mobilities.
Similar treatment has been applied to obtain number of theoretical plates for effective length for Micellar Electrokinetic Capillary
Chromatography, MECC Terabe‘s equation for resolution without including effective length3 and proposed equation by us for resolution
with including effective length are compared.The new resolution equation by us could be used as continuation of further work in our
previous work in optimization4.
1 Jorgenson , J; Lukacs,K.D. Anal. Chem. 1981,53,1298.
2 Dunn, C.D; Hankins,M.G;Ghowsi, K. Sep.Sci.Technol,1994,29(18)2419-2433
3 Terabe,S; Otsuka, K ; Ando, T.Anal. Chem. 1985,57,834-841
4 Ghowsi, K; folly, Joe P; Gale , R.G. Anal. Chem.1990,62,2714-2721
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Poster Abstracts
P076:
Blank measurement based time-alignment in LC-MC
Jan Urban1, Pavel Hrouzek2, Jan Vaněk1, Jiří Kopecký2, Dalibor Štys1
1 Institute of Physical Biology
2 Institute of Microbiology
We are presenting the blank based time-alignment (BBTA) as a strong analytical approach for treatment of non-linear shift in time occurring
in HPLC-MS data. Need of such tool in recent large dataset produced by analytical chemistry and so-called omics studies is evident.
Our approach is based on measurement and comparison of blank and analyzed sample evident features. In the first step of BBTA procedure, the number of compounds is reduced by max-to-mean ratio thresholding, which extensively reduce the computational time. Simple
thresholding is followed by selection of time markers defined from blank inflex points which are then used for the transformation function,
polynomial of second degree, in our example. BBTA approach was compared on real HPLC-MS measurement with Correlation Optimized
Warping (COW) method. It was proved to have distinctively shorter computational time as well as lower level of mathematical presumptions. Finally, BBTA does not required usage of extra internal standards and due to its simplicity it has a potential to be widespread tool in
HPLC-MS data treatment.
We describe in details, mathematically and experimentally justify approach for time alignment of LC-MS spectra using blank measurement
data as (inherent) internal standards (BBTA). BBTA utilizes solvent contaminants and other important events (inflex points) detectable both
in blank run and the compared experiment for alignment of multiple 2D chromatograms. Addition of internal standards may increase number of data points available for calculation but is not necessary for general laboratory practice. Obvious advantage of BBTA is its readiness
and essentially low expenditure level of its application. All mathematical descriptions are derived
immediately from the system based description of the measurement data sets with respect to the common used definitions.
P077:
Investigations of the Sialic-acid Linkages of Glycans Derived from Blood-serum Glycoproteins
William R. Alley, Jr., Milos V. Novotny
Department of Chemistry, Indiana University, Bloomington, IN, USA 47405
Advances in the sensitive measurements of glycans by either permethylation mass spectrometry (MS)-based methodologies or capillary
electrophoresis (CE)-laser-induced fluorescence (LIF) detection have enabled us to profile routinely and quantitatively dozens of N-linked
oligosaccharides released enzymatically from blood-serum glycoproteins. As a number of glycan-concentration changes were observed in
association with different types of cancers, there is a significant medical importance to study aberrant protein glycosylation. As one of the
cases previously studied in our laboratory, we were able to distinguish the serum-glycan profiles of cancer-free patients from those diagnosed with breast cancer. These profiles indicated that a number of highly-sialylated N-linked oligosaccharides were more abundant in the
pathological samples, suggesting the importance of sialic acid residues in cancer. However, several highly-abundant sialylated structures
did not pass diagnostically-relevant statistical tests. Reasoning that linkage isomerism, i.e. biological interactions favoring either α2,3- or
α2,6-sialylated glycans, could be involved in disease recognition, we developed here an analytical method for monitoring the ratios of α2,3to α2,6-linked isomers in the glycans derived from serum glycoproteins. After activation of their carboxylate, the sialic acid residues were
treated with ammonium chloride, amidating those that were linked α2,6, while α2,3-linked structures spontaneously generated lactones. A subsequent permethylation replaced the hydrogen atoms of the amide nitrogen with methyl groups for α2,6-linked sialic acids, while
for those linked α2,3, the lactone was cleaved and esterified the resulting carboxylate. The resulting mass difference was observable by
matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (MS). When this methodology was applied to blood-serum samples
derived from control patients and those diagnosed with late-stage breast cancer, a number of glycans that were not diagnostic of breast
cancer were now capable of indicating the presence of the disease based on the analysis of the sialic-acid linkages. The glycans included
in this study showed a trend towards higher levels of α2,6-sialylation.
Increased levels of α2,6 sialylation of the glycoproteins expressed on the cell surfaces of tumors have been reported for a number of cancers, including breast cancer, and increases of the metastatic potential of tumor cells. Much of the information about the sialylation of
cell surfaces has resulted from the analyses of the extracts of cell lines or tissues based on enhanced glycoprotein binding to Sambucus
Nigra agglutinin (SNA), a lectin with an affinity for α2,6-linked sialic acids, or by an analysis of the mRNA expression levels of ST6Gal-1, the
enzyme responsible for catalyzing the addition of an α2,6-linked sialic acid to a galactose residue. While effective in their own right, these
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Poster Abstracts
methods do not provide information about the individual glycan structures possessing the enhanced α2,6 sialylation. Consequently, the
methodology reported here has a potential for additional glycomic screening.
P078:
Separation of Fucosylated and non-Fucosylated Carbohydrates Associated with Monoclonal Antibodies using Capillary Electrophoresis
Sushma Rampal, Mark Lies
Beckman Coulter Inc.
In order to gain a comprehensive understanding of therapeutic glycoprotein function, it is necessary to critically characterize glycosylation associated with these molecules. Carbohydrates, and therefore glycosylation, are known to play an important role in the structure, function, and clearance of many therapeutic proteins and have been shown to be responsible for invoking immune responses in
humans. Changes in carbohydrate composition or concentration can significantly impact the overall efficacy of a therapeutic and may
lead to side effects. Presence of fucose is a notable glycan modification on monoclonal antibody - associated N-linked oligosaccharides
and has been linked to a decrease in antibody dependent cellular cytotoxicity (ADCC) of IgG therapeutics. Capillary electrophoresis (CE)
technology has been successfully used to separate major IgG N-linked oligosaccharides G0, G1, G2, and mannose structures from one
another. The basis for this separation relies on electrophoresis of oligosaccharides labeled with amino pyrene trisulfonic acid (APTS).
The complexity of glycans associated with many molecules, however, calls for high resolution separation strategies in order to assess
heterogeneity among carbohydrate isomers. Since CE is already an established technology for automated and quantitative analysis of
N-linked oligosaccharides, we set out to develop a methodology by which fucosylated oligosaccharides can be differentiated from nonfucosylated species. Optimization of chemistry and CE methods enabled separation of fucosylated and non-fucosylated carbohydrates
from one another as well as from co-migrating glycan species.
P079:
Universal tool for fast in-depth glycan analysis
Carolin Huhn1, L. Renee Ruhaak2, Manfred Wuhrer2, André M. Deelder2
1 Forschungszentrum Jülich, Germany
2 Leiden University Medical Center, The Netherlands
A large number of analytical techniques for the characterization of glycoproteins have been developed. Recent years have also shown a
rise in publications on sample preparation strategies for glycosylation analysis. This can be ascribed to the complexity, resulting from
both glycan composition and protein diversity, when glycoproteins derived from complex mixtures like plasma or cell cultures in clinical
or biopharmaceutical applications are to be analyzed.
A hexapeptide library (marketed as Proteominer beads®1 originally introduced for proteomics applications2 is applied for the first time
for glycosylation analysis. By varying the pH of the loading solution we show that the binding of the proteins to the beads is dominated
by the protein backbone; differences in glycosylation do not significantly change adsorption.
Proteins were eluted from the beads using relatively harsh conditions and were subsequently analyzed with gel electrophoresis to show
the relative concentrations of proteins at different loading and elution conditions.
However, for N-glycan analysis we show that the elution of proteins from the beads is not necessary; instead on-bead digestion is
possible using PNGase F. Glycans will thus be released into the supernatant and they can then easily be collected and labeled with
derivatization agents such as 2-aminobenzoic acid and 8-aminopyrene-1,3,6-trisulfonic acidiii. We here used multiplexed gel electrophoresis with LIF detection for analysisiv. The repeatability for on-bead digestion and labeling is similar to the classic in-solution labeling.
Several advantages of this approach can be stated:
1) The beads are easy to use for a broad range of proteins. Simultaneous protein enrichment and purification is possible as salts and
other matrix components, which do not bind to the hexapeptide library, are washed away. Very diluted protein samples can easily be
enriched due to the non-selective nature of the beads.
2) Glycan release can be achieved as an on-bead digestion without disturbance from the original sample matrix.
3) The overall repeatability is similar to the in-solution approach.
4) For plasma samples, there is an equilibration of protein concentrations; a depletion of high-abundant proteins as well as an enrichment of low-abundant proteins is possible. Thus an in-depth profiling of plasma glycosylation is possible.
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Poster Abstracts
5) The use of the Proteominer beads can be regarded to be ideal for biomarker studies if proteins are eluted and subsequently fractionated via different separation techniques.
1 V. Thulasiraman, S. Lin et al., E. Boschetti, Electrophoresis 2005, 26, 3561-3571
2 P.G. Righetti, E. Boschetti et al., Proteomics 2006, 6, 3980-3992
3 L.R. Ruhaak, C. Huhn et al., Anal. Chem. 2008, 80, 6119-6126
4 L.R. Ruhaak, R. Hennig et al., in preparation
P080:
Identification of Unusual Hyperfucosylated Glycan Structures and the Related Glycoproteins in Pancreatic Cyst Fluids of Cancer Patients
Benjamin F. Mann1, John A. Goetz1, Michael G. House3, Milos V. Novotny1
1 Department of Chemistry, Indiana University
3 Department of Surgery, Indiana School of Medicine
While performing a thorough glycomic and glycoproteomic investigation of several disease conditions pertaining to pancreatic cancer,
we identified a number of unusual glycan structures in morphologically different pancreatic cysts. These structures feature heavily
fucosylated N-glycan branched entities, which occur prominently in the oligosaccharide profiles recorded through MALDI mass spectrometry after solid-phase permethylation. While a degree of fucosylation has been associated with cancer for some time, such “hyperfucosylated” structures were not, to our knowledge, previously seen in a biological fluid aspirated directly from the diseased pancreas. In
addition to profiling the cyst fluid glycome components, the data were analyzed through the principal component analysis (PCA) and
the relative abundance of individual glycans for the different disease states has been evaluated through a series of statistical methods.
These highly unusual glycomic analytical results led us to a glycoproteomic studies, in which lectin enrichment was employed to target
fucosylated glycoproteins. The enriched glycoproteins were then tryptically digested and subjected to bottom-up proteomic analysis
with LC-MS/MS at high MS resolution. All samples were analyzed using a label-free quantitative strategy developed in-house, and the
relative abundance of confidently identified proteins was determined for each sample. The interrelation of glycoprotein profiles from
each sample will be illustrated using a dendrogram as well as PCA, while the degree of glycoprotein overlap has been depicted through
Venn diagrams for different disease states. Targeted analyses are in progress to establish whether the hyperfucosylated glycans and
their corresponding glycoproteins could be significant markers of pancreatic cancer progression. Monday, March 22nd, 2010
P081:
Separation of glycopeptides and their glycoforms using HILIC columns and UPLC-MS(E) system
Martin Gilar, Hongwei Xie, Thomas E. Wheat, Beth Gillece-Castro
Waters Corp
Peptide mapping is typically performed in reversed-phase liquid chromatography mode (RP-LC). Resolution of tens to hundreds of
peptides in peptide maps remains difficult even when using ultra performance liquid chromatography (UPLC) systems and columns.
An especially challenging problem is the separation and relative quantitation of glycosylated peptides (the same peptide sequence,
different glycoforms). Because RP columns retain peptides based on hydrophobicity, they are rather insensitive to the presence of
hydrophilic glycans. Consequently, the different forms of glycopeptides elute at similar times, and baseline resolution of all glycoforms
is rarely achieved. We have evaluated a novel HILIC column packed with 1.7 um particles for peptide mapping of monoclonal antibody
(Herceptin). Due to the greater affinity of HILIC sorbents towards hydrophilic compounds, the separation mode appears to be promising
for separation glycans and glycopeptide glycoforms. The glycopeptides are more strongly retained on HILIC sorbent compared to nonglycosylated peptides. In addition, glycoforms of peptides are separated with sufficient resolution, so they can be quantified. Data-independent mass spectrometry acquisition MS(E) was utilized to highlight the retention times of glycopeptides using the characteristic
glycan ions (204.1 and 366.1 Da). In addition to UV detection, the MS signal of peptide precursors was utilized for relative quantitation
of glycoforms, producing comparable results.
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Poster Abstracts
P082:
Studying the glycosylation of Francisella tularensis lipoprotein FTH_1071 through a combination of glycoprotein enrichment techniques and mass spectrometry
Lucie Balonova*1, Lenka Hernychova2, Marek Link2, Benjamin F. Mann4, Zuzana Bilkova5, Milos V. Novotny4, Jiri Stulik2
1 Institute of Molecular Pathology, FMHS UD, Hradec Kralove, Czech Republic
Department of Analytical Chemistry and 3Biological and Biochemical Sciences, FCHT UPa, Pardubice, Czech Republic
2 Institute of Molecular Pathology, FMHS UD, Hradec Kralove, Czech Republic
4 Department of Chemistry, NCGG IU, Bloomington, IN, USA
5 Department of Biological and Biochemical Sciences, FCHT UPa, Pardubice, Czech Republic
Introduction: The presence of glycosylation in bacteria has been shown to impact the function of proteins modified by glycans in terms
of their implication in adhesiveness and invasion to host cells1. Investigation of Francisella tularensis glycoproteome revealed the lipoprotein FTH_1071, together with other proteins, as being possibly glycosylated. In a present study, we draw attention to the protein
FTH_1071 that was recently found to be essential for virulence of both F. tularensis subspecies, holarctica and tularensis2,3.
Methods: The membrane-enriched protein fraction of the F. tularensis subsp. holarctica strain FSC200 was investigated for the presence
of glycosylation by combining two distinct glycoprotein detection/enrichment approaches with mass spectrometry: (1) Hydrazide as
an agent reacting with oxidized carbohydrates was used for detection of 2D-separated glycoproteins as well as for the glycoprotein
enrichment, in which the glycoprotein identification is based on the mass-spectrometric analysis of eluted nonglycosylated peptides
originating from hydrazide-imobilized glycoproteins (the glycopeptides were irreversibly bound to the hydrazide resins). (2) Lectins, proteins that specifically recognize particular carbohydrate motifs present on glycoproteins, were used for the detection of 2D-separated
and electroblotted glycoproteins as well as for the glycoprotein enrichment via lectin affinity chromatography.
Results: The protein FTH_1071 was identified as being glycosylated by both hydrazide detection and enrichment techniques, and also
in eluted fractions from lectins ConA, SNA, DSA, and SBA. Bioinformatic studies have suggested the presence of six potential O-glycosylation sites, Thr34, Thr45, Thr46, Ser32, Ser37, and Ser41. In addition, all of the potential O-glycan occupancy sites were found to
occur within a low-complexity region (LCR) of protein. Further, the sequence of FTH_1071 was manually inspected for the occurrence
of prokaryotic D/E-Xa-N-Xb-S/T sequon with a negative result.
Conclusion: The protein FTH_1071 appears to be modified by O-glycosylation. Further studies on the glycosylation of this protein are
under way.
This work was financially supported by Ministry of Education No. MSMT0021627502 and No. ME08105, Ministry of Defence, Czech
Republic No. FVZ0000604, and Czech Science Foundation No. GA203/09/0857. These studies were facilitated through collaborative
research with the National Center for Glycomics and Glycoproteomics at Indiana University, which has been supported by grant
No. RR018942 from NCRR, U.S. Department of Health and Human Services.
1 Karlyshev, A. V.; Everest, P.; Linton, D.; Cawthraw, S.; Newell, D. G.; Wren, B. W. Microbiology 2004, 150, 1957-64.
2 Qin, A.; Scott, D. W.; Thompson, J. A.; Mann, B. J. Infect Immun 2009, 77, 152-61.
3 Straskova, A.; Pavkova, I.; Link, M.; Forslund, A. L.; Kuoppa, K.; Noppa, L.; Kroca, M.; Fucikova, A.; Klimentova, J.; Krocova, Z.; Forsberg, A.; Stulik,
J. J Proteome Res 2009, 8, 5336-46.
P083:
Novel Bioinformatics Approaches to Model Carbohydrate Structures and Predict Their Capillary Electrophoretic Mobility
Stefan Mittermayr1, Andras Guttman2
1 Horvath Laboratory of Bioseparation Sciences, University of Debrecen, Hungary / University of Innsbruck, Austria
2 Barnett Institute, Northeastern University, Boston, MA
Structural determination of complex oligosaccharides is a difficult task mainly due to the large diversity of sugar structures found in nature, the
complex biosynthetic path variations and the physical and chemical similarities of carbohydrates. Development of new high-resolution separation methods is crucial to provide comprehensive analysis of glycan pools containing positional and/or linkage isomers. Capillary electrophoresis (CE) holds the promise to fulfill this quest offering high separation efficiency in very short separation times compared to chromatography
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Poster Abstracts
techniques. However, the computational tools are still missing to assist glycan structure elucidation directly from electrophoretic mobility data.
The immense heterogeneity of glycans impedes an exhaustive accumulation of experimentally obtained structure-mobility relation data. This is
mainly due to a large number of time consuming experiments, and also due to the limited availability of pure glycan standards. Application of
model-based approaches to predict the electrophoretic mobilities of complex glycans from known physicochemical properties can substitute
this tedious experimental work. This endeavor requires determination of structural descriptors derived from glycan molecular modeling followed by the application of appropriate separation predicting algorithms. Therefore, structural descriptors such as hydrodynamic volume and
molecular radius, derived from molecular modeling of glycans, have been utilized to compute their electrophoretic mobility in silico through
predictive models. Successful application of contemporary bioinformatics tools, encompassing molecular modeling and separation prediction,
enabled direct deduction of glycan structural data from experimental electrophoretic separations of unknown carbohydrate mixtures.
P084:
Performance Characteristics of the New Agilent 7100 Capillary Electrophoresis System
Christian Wenz, Hans Brunnert, Gerard Rozing, Martin Greiner
Agilent Technologies R&D and Marketing GmbH & Co KG, Waldbronn, Germany
Capillary Electrophoresis (CE) offers separations with exceptional efficiency and resolution for charged substances such as biomolecules, low
molecular weight basic or acidic drugs and ions – separations that are often difficult to achieve with HPLC. The last years have seen a growing
interest for CE based separations, e.g. for the analysis of biopharmaceuticals, and for the coupling of CE with mass spectrometric detection.
Here, the analytical capabilities of Agilent’s 2009 newly introduced 7100 CE System are illustrated with a multicomponent sample analyzed
by CZE with UV-vis spectrophotometric detection. Main focus is the sensitivity of the system and improvements compared to the standard
capillary that can be obtained with an extended light path capillary (bubble cell) and the Agilent high sensitivity cell. Furthermore, the CE
system was coupled with a mass spectrometer via Agilent’s tri-axial sheath flow CE-MS sprayer. The characteristics of both UV/vis and mass
spectrometric detection systems are highlighted.
P085:
Sensitive determination of indoleamines and catecholamines using end-column amperometric detection with boron-doped diamond
electrode
Lin Zhou, Fengjun Shang, Jeremy D. Glennon
Analytical and Biological Research Facility (ABCRF), Department of Chemistry, University College Cork, Cork, Ireland
A simple and efficient approach for the simultaneous separation and determination of catecholamines and indolamines by capillary electrophoresis with amperometric detection was developed. A fused silica capillary (45 cm × 50 µm i.d.) was dynamically coated with poly (diallyldimethylammonium chloride) (PDDA) modified gold nanoparticles (AuNPs) and employed for the separation of 3-indoxyl sulfate (3-IXS), vanillylmandelic
acid (VMA), homovanillic acid (HVA), L-tryptophan (L-Trp), epinephrine (EP), normetanephrine (NMN), 5-hydroxytryptamine (5-HT), tryptamine
(TA), 4-hydroxy-3-methoxybenzylamine (HMBA), 3,4-dihydroxybenzyl amine (DHBA) and isoproterenol (IP). The positively charged PDDA modified AuNPs formed a stable coating which significantly improved the selectivity of the solutes via ionic interaction. The effects of separation
voltage, buffer pH and concentration were investigated. Under the optimal stacking condition, the eleven bioamines were baseline separated
within 20 min in 50 mM H3PO4-Tris buffer (pH 2.8). Compared to UV detection, amperometric detection with a boron-doped diamond (BDD)
electrode has been proved more sensitive and effective for the detection of electroactive catecholamines and indolamines. The detection limit
is about 1 mM for those compounds. The results show that the PDDA/AuNPs-coated capillary with BDD electrochemical detection provides
superior resolving power and sensitive detection of electroactive bioamines. Wednesday, March 24th, 2010
P086:
Chemiluminescently Labeled Aptamers as Affinity Probes for Capillary Electrophoresis
Hong-Yi Li, Qin-Pei Deng, De-Wen Zhang, Yin-Ling Zhou, Xin-Xiang Zhang*, Jia-Xiang Xu
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Poster Abstracts
College of Chemistry, Peking University
Interaction analysis between biomolecules is important for revealing their function in organism. Affinity evaluation is a common way to
realize the interaction analysis involving separation method such as capillary electrophoresis. Aptamers are nucleic acid oligonucleotides which can recognize targets with high affinity and specificity. They are adopted as ligands in affinity probe capillary electrophoresis
(APCE) by labeling aptamers with fluorescein [1]. Besides fluorescence detection, chemiluminescence (CL) detection is another ultrasensitive detection method for capillary electrophoresis. In this work, aptamers were labeled with Isoluminol isothiocyanate (ILITC) at
their 5’ ends, which was the first chemilunimescently covalent labeling for aptamers according to our knowledge. 29-mer anti-thrombin
aptamers were chosen as the model. HPLC-ESI-MS was used to separate and characterize the labeled aptamers from unlabeled ones
and pure labeled aptamers were collected. Affinity constant between aptamers and thrombin was measured after the labeling and compared to that of aptamers without the labeling. The results proved that there was no severe influence of labeling on affinity of aptamers,
which means that it is practical to label aptamers chemiluminescently and use them as affinity probes in capillary electrophoresis.
P087:
Optimization of Anion Analysis with Capillary Electrophoresis
François de l‘Escaille, Fabrice Benoit, Jean-Bernard Falmagne
Analis R&D Diag
Ions are highly charged species by nature and lend themselves well to analysis by capillary electrophoresis. We usually deal with small
organic and inorganic anions and small cations or aliphatic amines.
This technique is often used as an alternative to ion chromatography (IC). However, CE offers a number of advantages over IC: It is
associated with simplicity, great separation efficiency, and unique selectivity. Other advantages are limited sample pre-treatment and
speed of analysis and reduction of solvent consumption.
A strategy was developed to optimize the analysis for speed: up to 96 analysis overnight and dynamic range: from 0.1 to 100 µg/mL.
With this strategy sample preparation could be done with an automatic workstation.
Analytical results for ion analysis on water and waste water will be presented showing repeatability, linearity, LOD/LOQ and recovery.
P088:
A fully automated capillary electrophoresis instrumentation for separating zone electrophoresis as joined with isotachophoresis
Miroslava Halašiová*, Róbert Bodor, Dušan Kaniansky
Department of Analytical Chemistry, Faculty of Science, Comenius University, Mlynska Dolina CH-2, SK-842 15 Bratislava, Slovakia
CZE combining with ITP for the column-switching operation with the closed separation system was introduced some time ago1. Clearly, its CZE stage is applicable for selectivity and/or sensitivity of the CZE analytes. Surprisingly, CZE for the ITP-CZE combination is
employed still rather rare in practice. Anyway, just compare for CZE with the single-column technology along with 50-75 μm I.D.s or
so and the opened separation system. This work, introducing fully automated capillary electrophoresis (CE) instrumentation with the
column-coupling technology, is focused to increase further potentialities of ITP-CZE. Importantly, this is employed for a closed separation system as eliminating both the laminar and electroosmosis transports while operating almost exclusively for the electrophoresis
transport. In addition, such an approach is applicable in ITP-CZE: (A) to include the ITP stage, concentrating the analytes between the
leading and terminating zones, and transferred the analytes to the CZE stage while separable in CZE (ITP-CZE), (B) to mix the analytes
with the discrete spacers (DSs) as these are separating in the ITP stage while included analytes as focused in boundary layers between
the ITP zones; subsequently, these are transferred for a particular fraction to the CZE stage (in fact, well-defined by a pair of the discrete
spacers) and separating for CZE; taking a set of the CZE stages is summing-up to reach a two-dimensional CE separation as based on
ITP(DS)-CZE. The electrolyte solutions were chosen, (1) for the ITP stage [the leading electrolyte (LE) with the terminating electrolyte
(TE)] and (2) for the CZE stage as using the background electrolyte (BE) solution. The ITP-CZE runs (see, (A), in the previous paragraph),
loading for 50 anionic analytes, were shown to resolve for 35-37 analytes in the CZE stage. These could be attributable for the analytes
of closer migration properties. Clearly, considering an elution model as proposed by Giddings with Davis, it is setting overlaps of the
analytes. We were investigated in details to use discrete spacers (see, (B), as shown above) as operating for ITP [ITP(DS)] and, in addition,
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Poster Abstracts
provided a set of fractions of the analytes (migrating isotachophoretically in the boundary layers between the ITP zones of the discrete
spacers). Such ITP configurations and transferred to the CZE stages were running automatically to show as enhanced resolutions of
the analytes. Reproducibilities for these 50 analytes were found very good as linked with ITP(DS)-CZE. Apparently, we can state as our
ITP(DS)-CZE operation is analytically very beneficial by suppressing for the laminar and electroosmotic transports.
This work was supported by grants from the Slovak Grant Agency for Science (VEGA 1/0672/09 and 1/0882/09), the Slovak Research
and Development Agency (VVCE-0070-07).
1 D. Kaniansky, J. Marák, J. Chromatogr., 498(1990)191
P089:
Non-aqueou CE of Eu(III) complexes of alkylphenol-derivatives with time resolution fluorescence detection leading to highly sensitive analysis
Yukihiro Esaka1, Mari Mizutani2, Yumi Isida2, Bunji Uno1, Keiko Sumitomo5, Yoshinori Yamaguchi6
1 Gifu Pharm. Univ. and Grad. School of Gifu Univ., Japan
2 Gifu Pharm. Univ.
5 Waseda Univ.
6 Grad. School of Osaka Univ.
In the present method, an analyte undergoes precolumn derivatization with a reagent, 5-(4"-chlorosulfo-1',1"-diphenyl-4'-yl)-1,1,1,2,2,pentafluoro-3,5-pentanedione (CDPP, developed by Matsumoto, K et al.). The corresponding derivative (DPP-analyte) forms a complex
with Eu3+ in non-aqueous running solutions of CE. The antenna moiety of the regand, the diphenyl group, absorbs light (lmax=350 nm)
and the energy of the light transfers to Eu3+ within the complex and then, Eu3+ produces luminescence with its inherent wavelengths
(lmax=615 nm). The energy transfer involves an intersystem crossing process and thus, the emission possesses relatively a long lifetime.
Delayed detection by a time-resolution method enables us to detect the DPP-analyte after attenuation of background fluorescence by
the matrices and the detection cell, leading to much more sensitive detection compared with conventional fluorescence detections.
Here, non-aqueous media is essential for sufficient formation of the Eu3+-complex and thus, not only for the efficient emission but also
for electrophoretic migration of the neutral DPP-analyte for its separation analysis. Four alkylphenols (APs) of endocrine disturbing
chemicals, 4-nonylphenol, 4-octylphenol, 4-tert-butylphenol and bisphenol, were analytes in the present work. Acetonitrile (AN) and
dimethylsulfoxide (DMSO) were studied as solvents of the non-aqueous media. Generally, addition to hydrophobic circumstances, some
supporting agents such as triphenyl phosphine oxide (TPPO) in this work are used for increasing quantum yield of emission. The supporting agents will form multi-component complexes with the complex of Eu3+ and DPP-analyte, binding to unoccupied coordination positions of Eu3+. The four DPP-APs could not separated completely in the pure AN solutions. This would be interpreted that whole sizes of
the complexes in the separation systems were so large compared with those of analytes themselves that difference of the sizes between
the analytes could not be reflected to the CZE separations well. DMSO was found to be a much effective modifier in both supporting
emission and improvement of separation. DMSO will occupy the coordinating sites on Eu3+ with its S=O group in the same manner of
TPPO. Additionally, Increase in concentration of DMSO increased electrophoretic mobilities of the DPP-APs remarkably (up to 30 %) and
resulted in obvious difference in migration velocities between the DPP-APs and thus, complete separations were given eventually. This
will be interpreted that replacement of “large” TPPO by DMSO would occur gradually in the multi-component complexes and the sizes
of the complexes decreased with the increase in DMSO concentration. As a result, the difference in sizes of analytes itself could reflect
to their CZE separation. The sensitivities of the four DPP-APs with the present system without a laser-light source were observed to be
ca. 5 × 10-10 mol/L as detection limits.
P090:
Effect of the pH of the anolyte and catholyte solutions in CIEF analysis with sandwich injection set-up
Csilla Páger*1, Andrea Vargová2, Ferenc Kilár1
1 Institute of Bioanalysis, Faculty of Medicine, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary
2 Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Ifjúság útja 6, H-7624 Pécs, Hungary
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Poster Abstracts
Isoelectric focusing within a fused silica capillary (cIEF) is a useful separation technique for the analysis of amphoteric compounds, such as
peptides or proteins. The analytes are separated according to their isoelectric point (pI), in a pH gradient established by ampholytes under
the influence of an electric field.
The sandwich injection set-up (in which the analysed sample compounds were injected between two ampholyte zones) offers an efficient
separation of amphoteric compounds having pI values outside the pH range of the applied ampholyte.
When cIEF is coupled to MS, the presence of the ampholyte compounds might suppress the signals of the sample components. Our aim
was to improve the detection with experimental conditions, in which after the isoelectrophoretic separation the substances migrate out
from the zone of the ampholytes. Changing the pH of the anolyte and catholyte solutions offers an option to this. In our experiments, ampholyte with narrow pH range (covering pH range 7-9) was applied in uncoated capillary and aminomethylated nitrophenol dyes (with pIs
5.3, 6.4, 6.6, 7.9 and 10.4) were used as the pI marker samples. Changing the pH of the anolyte in the pH range between 2.5 and 7.5 and
altering the pH of the catholyte in the pH range between 11 and 8, the dye with pI=10.4 was effectively excluded from the ampholyte zone.
The work was supported by the grants GVOP-3.2.1-0168, OTKA-K75717 and OTKA-NKTH-NI-68863.
P091:
Divergent flow IEF operated without carrier ampholytes for separation of biological samples
Miroslava Stastna, Karel Slais
Institute of Analytical Chemistry of the ASCR, v.v.i., Veveri 97, 602 00 Brno, Czech Republic
The important part of identification and characterization of the components creating the complex biological sample is their efficient separation. Recently, we introduced preparative electrophoretic method named divergent flow IEF (DF IEF) as well as we constructed the basic
DF IEF instrument and verified its function in practice. In this study, we have tested the modified DF IEF device operated without the use of
background carrier ampholytes. We have shown that during separation and focusing of biological samples consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be formed by autofocusing of the sample components
without any addition of carrier ampholytes. In suggested DF IEF device, the proteins of sample are separated, concentrated and desalted in
one step. At the DF IEF device output, the fractions have been collected, subjected to polyacrylamide gel IEF and the protein bands in fractions
visualized by CBB staining. The fractions of yeast lysate sample contained the bands of proteins covering gradually the pI range from 3.7 to
8.5. In our arrangement, the majority of proteins have been found in the fractions with pI values around 5.3. In these fractions, identified
numbers of protein bands and their separation have shown relatively good separation efficiency of DF IEF instrument operated without the
carrier ampholytes. During DF IEF, the selected protein bands have been concentrated up to 16.8-fold. The suggested DF IEF instrument can
be used as a pre-fractionation technique for highly complex biological samples and might serve as an excellent tool in field of proteomics.
Moreover, as DF IEF is free of carrier ampholytes, the proteins in collected fractions can be consequently analyzed, e.g. by mass spectrometry
or to examine for protein biological activity.
P092:
Preparative two dimensional separation by on-line coupling of reversed-phase flash chromatography with divergent flow isoelectric
focusing
Karel Slais
Institute of Analytical Chemistry, AV CR, Brno,Czech Republic
The increasing complexity of analyzed objects implicates the use of number of consecutive separation steps involved in the sample cleanup.
The sophisticated approaches lead to multidimensional separations namely in proteomics. The combined separation principles should be
orthogonal and the resulting method should be comprehensive. The critical parameter is also the sampling from one dimension to the next
one. Among frequent combined methods in proteomics are reversed-phase liquid chromatography and isoelectric focusing (IEF). At the initial
steps of multi dimensional separations, the simple disposable devices including SPE cartridges and chips are often incorporated. Recently the
principle of divergent flow isoelectric focusing (DF IEF) was outlined1. The long term stability of performance was demonstrated2. The suitability for the separation and focusing of proteins was also verified2–4. The on-line monitoring of DF IEF dynamics was enabled by use colored
low-molecular-mass pI markers1–4. Here, a new two dimensional (2D) separation method based on coupling of gradient reversed-phase liquid
chromatography (RP-LC) on SPE cartridge as the first dimension with divergent flow isoelectric focusing DF IEF as the second dimension was
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Poster Abstracts
developed. The feasibility of the method was verified with a mixture of 14 model colored ampholytes with different pI’s and colors. Sample was
loaded onto disposable SPE cartridge that was subsequently connected to DF IEF inlet. The continuous gradient of organic modifier was generated in situ by mixing in SPE barrel. The developed 2D method combines both separation dimensions on-line, comprehensively and without
need of extra parts or activities. During 2D separation run, both LC and DF IEF separations are running simultaneously and continuously, and
it allows introducing another sample on a fresh SPE cartridge as well. Both SPE cartridge and DF IEF separation space are simple and cheap
enough to be disposable which could make the method suitable for raw protein sample analyses.
1 Šlais, K., Electrophoresis 2008, 29, 2451 - 2457
2 Šťastná, M., Šlais K., Electrophoresis 2008, 29, 4503 - 4503
3 Mazanec, K., Bobálová, J., Šlais, K., Anal. Bioanal. Chem. 2009, 393, 1769-1778
4 Šťastná, M., Šlais, K., Electrophoresis, # elps.200900484, accepted
P093:
Pre-separation of biological samples using of preparative divergent flow isoelectric focusing
Marie Vykydalová, Dana Moravcová, Vladislav Kahle, Karel Šlais
UIACH, Brno, Czech Republic
Isoelectric focusing (IEF) is important method in proteome analysis. IEF appears to be very suitable method for separation of complex biological
samples in which proteins or peptides are the major components. Divergent flow isoelectric focusing (DF-IEF) is based on planar divergent flow
and control of local electric field by conductivity of electrolytes. A polyester based nonwoven fabric was used for creation of flow and electric
manifold, making the assembled device cheap, flexible and easy to set up and operate1. Along the margins of the separation space are two pairs
of electrodes for applying appropriate voltage. Proteins migrate through the fabric and they are separated according to their isoelectric points
(pI). Proteins are separated, desalted and concentrated in a single step. This method was used for the pre-separation of samples extracted from
wheat flour. Individual fractions were collected in vials and separated using gradient elution on reversed-phase monolithic capillary column.
The column was prepared inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and
stearyl methacrylate in the presence of 1,4-butandiol and cyclohexanol acting as a porogen system. The elution was achieved using a linear
gradient of acetonitrile from 0 to 100 % v/v in water with 0.1 % trifluoroacetic acid formed in a microsyringe. Individual LC fractions of separated proteins were collected on the MALDI target plate and they were analyzed using MALDI-TOF-TOF mass spectrometry. The advantages of
this method include good concentrating effect and avoiding the presence of additional reagents in separated fractions of proteins. It leads to
improvement in the sensitivity of mass spectrometry measurements and finally to the increase in protein sequence coverage. The described
technique is cheap, flexible and easy method for handling with biological samples and it is ideal for treatment of large sample volumes.
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic by grants No. LC06023, by the Grant Agency of
the Academy of Sciences of the Czech Republic by grant No. IAAX00310701, and by Institutional Research Plan No. AV0Z40310501.
1 K. Šlais: Divergent flow isoelectric focusing, Electrophoresis, 29 (2008) 2451–2457
P094:
Two-dimensional separation of peptides combining divergent flow isoelectric focusing and capillary liquid chromatography
Filip Duša1, Marie Vykydalová2, Dana Moravcová3, Vladislav Kahle3, Karel Šlais3
1 Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Kotlářská 2, 611 37, Czech Republic
Institute of Analytical Chemistry of the ASCR, v.v. i., Veveří 97, 602 00 Brno, Czech Republic
2 Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Kotlářská 2, 611 37, Czech Republic
Institute of Analytical Chemistry of the ASCR, v.v. i., Veveří 97, 602 00 Brno, Czech Republic
3 Institute of Analytical Chemistry of the ASCR, v.v. i., Veveří 97, 602 00 Brno, Czech Republic
High resolution separations play crucial role in many scientific applications nowadays. This study is focused on the combination of
two separation techniques applied in proteomic research with accent on good resolution of peptide complex mixtures originated
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Poster Abstracts
from tryptic digests of biological samples.
Bovine serum albumin was used as a model protein for preparation of peptide mixture using tryptic digest protocol. The offline combination
of divergent flow isoelectric focusing and reverse-phase liquid chromatography was used for peptide separation after digestion. Mixture was
loaded dropwise on home-made preparative divergent flow isoelectric focusing instrument1 and separated with and without addition of carrier
amfolytes. Fractions were collected continuously from separation bed channels to micro test-tubes. After the first dimension, each fraction
was injected on reversed-phase column 1.0 × 75 mm Poroshell 300SB-C18 and eluted with water/acetonitrile gradient containing 0.1 % v/v of
trifluoroacetic acid on capillary liquid chromatograph AGILENT 1200 series. UV diode array detector was used for detection of separated analytes. The two dimensional peptide map was constructed from obtained data. Our separation results confirmed that 2D offline combination of
divergent flow isoelectric focusing and RP-LC is effective tool for peptide mixture preparation and thus has potential for processing of complex
biological samples. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic by grants No. LC06023, by the Grant Agency
of the Academy of Sciences of the Czech Republic by grant No. IAAX00310701, and by Institutional Research Plan No. AV0Z40310501.
1 K. Šlais: Divergent flow isoelectric focusing, Electrophoresis, 29 (2008) 2451–2457
P095:
Enhanced resolutions of analytes in capillary zone electrophoresis as preceded by capillary isotachophoresis with discrete spacers
Róbert Bodor, Vladimíra Jánošová, Dušan Kaniansky
Department of Analytical Chemistry, Faculty of Science, Comenius University, Mlynska Dolina, SK-842 15 Bratislava, Slovakia
Capillary zone electrophoresis (CZE) with capillary isotachophoresis (ITP), as based for a column-coupling technique (CC), is operated
by ITP-CZE. Our efforts were studied to benefit for using the discrete spacers (DSs) along with the analytes. In fact, such a common
ITP-CZE approach1 is loading for the sample constituents to which appropriately chosen DSs are added. (A) The ITP stage for ITP-CZE
is separated as reaching for the ITP steady state. Importantly, the DSs as forming for the ITP zones and, in addition, “filling-up” the
ITP boundary layers for the analytes (particular ITP layers as defined by concomitant ITP zones). For obvious reasons the leading ions
are transported electrophoretically to get outside from the separation system. In fact, the DSs (ITP zones) are acting as a set of “serial
sub-columns” while each of these sub-columns is transferring electrophoretically to the CZE stage. (B) Subsequently, the ITP stage is
transferring to the CZE stage (an electrophoretic path via the bifurcation channel2) and running along the CZE stage. In this stage is
destacking for the ITP configuration and, in fact, CZE separating (resolving) analytes (a) for the particular ITP boundary layers and (b)
migrating some analytes as getting out from the original (concomitant) ITP zones. In our simulation study (SIMUL program, B.Gaš with
co-workers) was included for ITP-CZE (without using the DSs) and ITP(DS)-CZE (with the DSs). For example, these simulations were taken
for 40 model analytes with three DSs and without these spacers. In fact, these model analytes clearly documents for ITP(DS)-CZE as
enhanced for the CZE resolutions, especially, when compared for ITP-CZE (without any DSs). Our experiments for ITP(DS)-CZE and ITPCZE, in the cationic mode of separation, were carried out for human urine samples. Importantly, these experiments were documented
very clearly to enhance the CZE resolutions in ITP(DS)-CZE while compared for ITP-CZE without any DSs. In fact, our simulation and
experimental studies, increased the use for the second separation dimensions by ITP(DS)-CZE. However, the ITP(DS)-CZE simulations, as
operating for very differing migration properties of model analytes, showed some logical limits for these second dimensions.
This work was supported by grants from the Slovak Grant Agency for Science (VEGA 1/0882/09) and the Slovak Research and Development Agency (VVCE-0070-07). Our deep thanks are expressed for Bob Gaš with his co-workers at Charles University in Prague (Prague,
Czech Republic).
1 D. Kaniansky, J. Marák, J. Chromatogr., 498(1990)191
2 D. Kaniansky, J. Marák, M. Masár, V. Vaváková, Capillary Electrophoresis Analyzer with Coupled Columns, Omega Info, Bratislava, 2004
P096:
RP-HPLC with off-line maldi MS detection of selected sterol
Blanka Hégrová*1, Ondřej Peš1, Edward S. Yeung3, Jan Preisler1
108
Poster Abstracts
1 Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
3 Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 500 11, USA
Plant sterols (phytosterols) are bioactive components found in all vegetable foods; they resemble cholesterol in vertebrates in term of
both function and structure. In the group of the most abundant phytosterols belong ß-sitosterol, campesterol and stigmasterol. Phytosterols shows potency in decreasing the levels of low-density lipoprotein (LDL) cholesterol in the serum and thus in protecting against
cardiovascular diseases1. Current methods for the analysis of cholesterol (single, esterified, sulphated), as well as other sterols and lipid
classes, include TLC, SPE, GC, HPLC and SFC2, 3. In this study, chromatographic separation of sterols was optimized for their fraction collection and subsequent MALDI MS determination using silver nanoparticles4 as matrix. Appropriate separation of sterols was obtained
in less than 9 minutes on Luna C8 column (150 × 4.6 mm I.D., 3 µm) with MetOH:water (97:3) as eluent. Experiments were performed
on HPLC system 10AVP with DAD and TSP HPLC system; wavelength was set to 206 nm. The fractions were collected and deposited
on MALDI target. The LC effluent was collected as 4-second fractions with 500 nanoliter drops. MALDI MS signal of separated sterols
(~ 15 picomoles injected, splitter 1:100) was two to three orders higher than MS LOD. Silver ions form readily adducts with sterols,
which enables sensitive MS analysis in positive mode. The MALDI MS detection limit of sterols (S/N = 3) were about 10 femtomoles
per spot. The off-line HPLC-MALDI MS was found to be suitable for analysis of nonpolar compounds and can be utilized as alternative
technique to gas chromatography.
We gratefully acknowledge Czech Science Foundation, grant No. 203/09/1025 and Ministry of Education, Youth and Sports of the Czech
Republic, grants No. MSM0221622415 and LC06035.
P098.
1 Kritchevsky, D., Chen, S.C. Nutr. Res. 25 (2005) 413-428
2 Abidi, S.L. J. Chromatogr. A 935 (2001) 173-201
3 Holen, B. J. Am. Oil Chem. Soc. 62 (1985) 1344-1346
4 Sluszny, Ch., Yeung, E.S. J. Am. Soc. Mass Spectrom. 16 (2005) 107-115
P097:
Accurate quantitation of glutathione in cells through surface-assisted laser desorption/ionization mass spectrometry using gold
nanoparticles
Cheng Kang Chiang, Yang Wei Lin, Wen Tsen Chen, Huan Tsung Chang
Department of Chemistry, National Taiwan University, Taipei, Taiwan
We developed a straightforward method for the determination of three aminothiols—cysteine (Cys), glutathione (GSH), and homocysteine (HCys)—using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) The analytes were first captured using the
unmodified 14-nm Au NPs; N-2-mercaptopropionylglycine (MPG) modified Au NPs served as internal standard was sequentially added
and then the sample was analyzed using SALDI-MS. This approach provided good quantitative linearity of the three analytes (R2 = ca.
0.99), with good reproducibility (relative standard deviations: <10 %), in the analyses of GSH in the lysates of human red blood cells and
MCF-7 cancer breast cells in the presence and absence of the anti-inflammatory drug sulfasalazine. Relative to the absorbance approach
for the detection of GSH in MCF-7 cells, our method is simpler (it does not require an expensive enzyme), faster, and more reproducible.
This SALDI-MS technique exhibits good linearity, superior reproducibility, and high accuracy; it holds great potential for use in clinical
diagnoses, such as for the multiple quantitation of small molecules under the influence of a range of drugs. Although herein we have
demonstrated the detection only of Cys, GSH, and Hcys, this simple technique should be applicable to the determination of a range
of analytes—from small solutes to biopolymers—when using different internal standards. Recently, we demonstrated (manuscript in
preparation) that Pt nanomaterials are effective matrices for proteins having molecular weights as high as 25 kDa; therefore, we expect
that our internal standard approach holds great potential for use in proteomics.
P098:
Capillary electrophoresis coupled with time-of-flight mass spectrometry for screening purpose
Isabelle Kohler, Julie Schappler, Jean-Luc Veuthey, Serge Rudaz*
109
Poster Abstracts
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d’Yvoy 20 - 1211 Geneva 4 - Switzerland
Swiss Centre for Applied Human Toxicology (SCAHT), University of Geneva, CMU, Rue Michel-Servet 1 - 1211 Geneva 4 - Switzerl
Capillary electrophoresis (CE) is a powerful separation technique for polar compounds with several advantages such as high efficiency,
low solvent and sample consumption and short analysis time. In addition, mass spectrometry (MS) detection provides high sensitivity
and selectivity, especially with time-of-flight (TOF) mass analyzer, due to its high mass resolution and accuracy. Because of its robustess and versatility, the sheath liquid interface is the most commonly used configuration for coupling CE to TOF with an electrospray
ionization (ESI) source. Conventionally, acidic background electrolyte (BGE) and ESI in the positive mode with an acidic hydro-organic
sheath liquid are used for cations analysis, while basic BGE is used for anions analysis in ESI negative mode with the addition of a basic
hydro-organic sheath liquid. In this study, a generic method was developed to allow simultaneous analysis of cations and anions with
only one capillary, one volatile BGE and one sheath liquid to avoid changes and re-equilibration of analytical conditions at each run.
CE and TOF parameters were optimized to achieve the best electrophoretic performance and ionization efficiency. An ammonium acetate
buffer at pH 5.2 permitted the migration of all compounds in positive polarity mode (30 kV, 25°C) according to their pKa thanks to the presence of electroosmotic flow (EOF), each sample being injected hydrodynamically at 50 mbar for 30 s (corresponding to 2.7 % of the capillary length) with acetonitrile added in the injection plug. A sheath liquid composed of isopropanol and water with ammonium allowed simultaneous
deprotonation of acids and protonation of bases, with fragmentor voltage of 150 V which gave the best compromise for all compounds. This
method was applied to the screening of small polar compounds of toxicological or pharmaceutical interests with a mass-to-charge ratio (m/z)
located between 100 and 1000 (e.g. amino acids, drug metabolites, endogenous compounds, contaminants, vitamins). With these conditions,
limits of detections (LOD) down to 100 ppt were achieved for basic analytes and 500 ppb for acidic compounds. Monday, March 22nd, 2010 P099:
Identification of toxicologically important compounds in a spot of human blood using desorption nano-electrospray ionization mass
spectrometry
Martin Švidrnoch, Václav Ranc, Lucie Hartmanová, Vítězslav Maier, Vladimír Havlíček, Karel Lemr
Deparment of Analytical Chemistry, Faculty of Science, Palacký University
Desorption electrospray1 is a useful tool for direct analysis of surfaces including their imaging by mass spectrometry. It has been applied in
many areas covering analysis of small and large molecules2. In this work, its modification, desorption nano-electrospray (nano-DESI)3,4, was
tested for screening of toxicologically relevant compounds in dry human blood samples. Experiments were performed using home-made
nano-DESI source and an ion trap mass spectrometer (LCQ, Thermo Finnigan, San Jose, USA). Nano-DESI gave longer lasting signal for the
same amount of sample, consumption of chemicals as well as contamination of mass spectrometer caused by sample matrix were smaller
in comparison to DESI. Spiked whole human blood was spot on target plate, let to dry and directly analyzed. Rough glass and glassy microfiber filter as sample target plates and influence of spray liquid composition were evaluated in analysis of nicotine, caffeine, selected opiates,
benzodiazepines and aphetamines. Important improvement of signal intensity was observed for glassy micro-fiber filter. The obtained nanoDESI-MS/MS spectra allowed identification of analytes by comparison with ESI-MS/MS spectra of standards. Application of nano-DESI mass
spectrometry represents a fast (analysis time less than 5 min) and easy (none sample pretreatment) method for screening of toxicologically
relevant compounds in human blood.
P100:
Capillary isotachophoresis with column-coupling technology acting as a powerful sample pretreatment tool in mass spectrometry
Andrea Staňová*, Jozef Marák, Soňa Žákovičová, Dušan Kaniansky
Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská Dolina CH-2, SK-842 15 Bratislava, Slovak Republic
Micro-preparative capillary isotachophoresis (pITP) was developed for several isolation techniques1. One of these, based on a fractionation
microvalve technique, was introduced for an off-line coupling of pITP with MS2. Apparently, pITP was significantly enhanced for using ITP with
the column-coupling technology3. Such an advanced ITP approach [p(ITP-ITP)] is offering a micro-preparative analytical tool, for example, as
applicable for an off-line coupling p(ITP-ITP)/MS. This work was studied potentialities of p(ITP-ITP)/MS and taking busereline as a (model) analyte
110
Poster Abstracts
(nona-peptide busereline, Mw = 1238.66 g/mol, pI = 10.5) while including a urine matrix. Our model analyte, at various concentration levels,
was reflecting for different approaches as regarding urine by using p(ITP-ITP)]. In our detailed experiments were covered: a direct injection in a
MS mode and, subsequently, for LC/MS and p(ITP-ITP)/MS. In addition, for p(ITP-ITP)/MS was used discrete spacers in the p(ITP-ITP) stage of the
combination, especially, to minimize very significantly for the urine matrices and highly reproducing for remainders of the matrix constituents
as accompanied for the analyte. MS and MS/MS spectra, obtained from the reconstructed sample fractions, proved both the p(ITP-ITP) cleanup effect and its ITP concentrating power for very low concentration levels of the analyte as present in very complex urine matrices.
This work was supported by a grant from the Slovak Research and Development Agency (No.VVCE-0070-07) and the grant of Slovak Grant
Agency, No.1/0882/09.
1 T. Hirokawa, Y. Kiso, J. Chromatogr. A, 658(1994) 343
2 E. Kendler, D. Kaniansky, J. Chromatogr., 209(1981) 306
3 D. Kaniansky, V. Madajová, M. Hutta, I. Žilková, J. Chromatogr., 286(1984) 395
P101:
Development of a microfabricated micronebulizer for bioapplications
Jakub Grym, František Foret
Institute of Analytical Chemistry ASCR, v.v.i., Brno, Czech Republic
Nebulizers are commonly used for dispersing liquids in many areas of science and technology. Micronebulizers are also often used
in analytical chemistry, e.g., in atomic emission spectroscopy, ICP mass spectrometry or for preparation of thin films for MALDI mass
spectrometry. Various forms of nebulizers are also used in many electrospray interfaces for mass spectrometry. Typical micronebulizer
utilizes a concentric tube arrangement with the flow rate of the dispersed liquid on the order of tens to thousands of microliters per
minute. Lowering the flow rate of the dispersed liquid is often desirable, for example when dealing with very small available samples.
Additionally, miniaturization typically brings additional system improvements, e.g., the ionization efficiency and sensitivity of the MS
analysis can be achieved by lowering the flow rate of the electrosprayed sample. While capillaries with diameters down to submicrometer range are available making further miniaturization for lower flow rates possible, connecting such micronebulizers to the external
device is less practical. Integration of a microfacricated nebulizer is a viable option for achieving reliable dispersion of low volume flows.
The devices under development were microfabricated in glass and for repeatable use a sandwich design with electrode chambers and
sample inlet ports located on an external plastic manifold was selected. The performance of several experimental micronebulizers was
tested with respect to the sample and air flows for use in electrospray ionization.
PO102:
Preparation of microsamples for elemental analysis of biological samples
Pavla Jungová*1, Ondřej Peš1, Tomáš Vaculovič1, Viktor Kanický1, Jarmila Navrátilová5, Jan Šmarda5, Jan Preisler1
1 Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic
5 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic
New approaches of sample introduction to inductively-coupled plasma mass spectrometry (ICP MS) which are especially suitable for
minute amount of samples are presented. Defined submicrolitre volumes of samples are deposited on substrate (polyethylene terephthalate glycol) which strongly absorbs laser light at 213 nm and desorbed together with the analyte after application of a few laser
pulses; the process was characterized as substrate-assisted laser desorption (SALD)1. Detailed characterization of SALD ICP MS has
shown large signal stability over a wide range of laser power density values, the independence signal on acquisition modes and linear
dynamic range over six orders of magnitude. Complete sample desorption can be achieved using only 10 pulses with spot-to-spot reproducibility below 6 %. SALD ICP MS with low limits of detection (0.1-14 fmol for Cr, Cu, Co, Fe, Ni, Sn and Zn) is suitable for off-line coupling
with capillary electrophoresis (CE) for speciation analysis or selective detection of metals. Off-line coupling of CE and SALD ICP MS was
applied to separate Cr(III) and Cr(VI) species; has been achieved subpicomolar sensitivity and high separation resolution. Applicability to
biological samples analysis was demonstrated on investigation the role of copper ions to regulate cytotoxicity of disulfirma to myeloid
leukemia cells2. Detection limits about 26 fg for copper were sufficient for determination of copper in U937 cells at physiological level.
111
Poster Abstracts
The protocol for biological sample preparation will be discussed. SALD ICP MS allows rapid, sensitive and reproducible method for
quantitative analysis of metals with very low consumption of sample. The advantages of SALD are also sample archiving and possibility
of consequent analysis in different detection modes, such as MALDI MS or fluorescence. The project was financial supported by the Czech Science Foundation (Grant No. 203/09/1025) and the Ministry of Education, Youth
and Sports of the Czech Republic (MSM0021622415).
1 O. Pes , P. Jungova, R. Vyhnanek, T. Vaculovi, V. Kanicky, J. Preisler, Analytical Chemistry, 2008, 80, 8725
2 J. Navrátilová, P. Jungová, P. Vaňhara, J. Preisler, V. Kanický, J. Šmarda, International Journal of Molecular Medicine, 2009, 24, 661
P103:
CE-MS/MS Analysis of 3-Nitrotyrosine using pH junction velocity difference induced focusing and a low dilution ESI interface
Yu Hui, Koen Raedschelders, Elizabeth J. Maxwell, David M. Ansley, David D. Y. Chen
The University of British Columbia
Patients with low cardiac output syndrome face a four-fold increase in overall mortality rates following aortocoronary bypass surgery.
Ischemia-reperfusion injury during this clinical procedure is the main factor contributing to postoperative low cardiac output syndrome.
The first few minutes of post-ischemic reperfusion induce a large-scale generation of reactive oxygen species that quickly react with
endogenous nitric oxide to generate the highly reactive and injurious peroxynitrite. Peroxynitrite is a short-lived mediator of nitrosative
stress, which is challenging to capture and analyze. 3-Nitrotyrosine is a stable end product of peroxynitrite, and is therefore well suited
as a biomarker of nitrosative stress.
Quantitative analysis of nitrotyrosine is complicated by several factors. 1) The basal concentration of 3-nitrotyrosine in proteins from
human plasma ranges from 1 to 5 nM. 2) Nitrotyrosine artifacts are easily generated during sample preparation. 3) Biological samples
often contain isobaric polar molecules co-eluting with 3-nitrotyrosine.
We are currently using CE-MS/MS to overcome these analytical challenges. CE is well suited for the separation of hydrophilic zwitterionic
analytes similar to 3-nitrotyrosine. In order to achieve sufficient selectivity for eventual in vivo 3-nitrotyrosine analysis, we coupled CE
with tandem mass spectrometry using a low dilution ESI interface developed by our lab. The sample was suspended in a 50 % methanol
matrix containing 1 % ammonium hydroxide and loaded onto the capillary under 5psi for 60 seconds. The sample was subjected to online preconcentration using pH junction velocity difference induced focusing with a background electrolyte consisting of 50 % methanol
with 1 % formic acid. Electrophoretic separation was performed with a modifier flow rate of 0.3µL/min at the ESI interface.
Our preliminary results from this CE-MS/MS technique indicate a limit of detection for 3-nitrotyrosine in the nM range with run times
below 10 minutes. Our goal is to further optimize this technique and apply it to the analysis of nitrosative stress in patients undergoing
aortocoronary bypass grafting with cardiopulmonary bypass. Wednesday, March 24th, 2010
P104:
Determination of D,L-lactic acid enantiomers by capillary electrophoresis with electrospray mass spectrometry
Vítězslav Maier, Romana Kupcová, Václav Ranc, Joanna Znaleziona, Radim Knob, Pavlína Ginterová, Magdalena Megov,
Martin Švidrnoch, Juraj Ševčík
Department of Analytical Chemistry, Faculty of Science, Palacký University, Tr. 17. Listopadu 12, Olomouc, CZ-77146
A method for the separation and simultaneous determination of D- and L-lactic acid enantiomers by capillary electrophoresis with
electrospray ionization mass spectrometry (CE-ESI-MS) is presented. The CE separation was performed in a polyacrylamide coated fused
silica capillary when the vancomycin as chiral selector was filled only into a part of the capillary to avoid the contamination of mass
spectrometer by the non-volatile chiral selector. Counter current method was used for direct enantiomeric separation of D,L-lactic acid
enantiomers when positively charged vancomycin and negatively charged D,L-lactic acid enantiomers migrate in opposite direction due
to the appropriate selection of the running electrolyte pH. Mass spectrometry detection of separated D,L-lactic acid enantiomers was
performed utilizing the negative ionization mode when the [M-H]- ions are measured. The effect of several experimental parameters on
the enantioresolution such as vancomycin concentration (0-15 mM), pH of the running electrolyte (pH 4-6) was studied. The effect of
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Poster Abstracts
experimental conditions on mass spectrometry detection such as composition of sheath-liquid, nebulizer gas pressure, gas temperature
and gas flow was studied as well. The advantages of presented method lie in high separation selectivity of D,L-lactic acid enantiomers
in CE separation step and in essential structure information of analytes obtained by mass spectrometry detection. Moreover, no derivatization of analytes or using of UV absorbing compound as running electrolyte component are necessary for mass spectrometry
detection. The potential of developed CE-ESI-MS method is demonstrated on the identification of D,L-lactic acid enantiomers as chiral
metabolites in clinical and food analysis.
This work was supported by the Research Project of the Ministry of Education of the Czech Republic No. MSM6198959216 and by the
Research Project of the Grant Agency of Czech Republic No. 203/07/P233.
P105:
Determination of tyrosine kinase inhibitors in human plasma by UHPLC-MS/MS
Katerina Micova, Adriana Polynkova, David Friedecky, Tomas Adam
Laboratory for inherited metabolic disorders, University Hospital and Palacky University Olomouc, I.P. Pavlova 6, 775 20 Olomouc, Czech Republic
The aim of this study was to develop new rapid and sensitive UHPLC method with tandem mass spectrometry detection for determination and quantification of plasma concentration of tyrosine kinase inhibitiors dasatinib, nilotinib, lapatinib, imatinib and its main
metabolite (CGP74588). Sample preparation is very fast and quick – 20 µl of pacient's plasma was deproteinated by 180 µl of methanol
with addition of internal standard (deuterated imatinib), sample was shaked, centrifugated 5 min. at 14000 RPM and supernatant was
analysed. Chromatographic separation of drugs and internal standard was achieved on a reverse phase analytical column ACQUITY
UPLC®BEH C18 1.7 µm (2.1 × 50 mm; Waters). Mobile phase consisted of acetonitrile (B) and 4 mM ammonium formiate; pH = 3.2 (A).
The total run time of the analysis was 2.2 min at a flow rate 0.5 ml/min. A triple quadrupole detector (API 4000, Applied Biosystems)
with electrospray ionization in positive mode was used for detection. Mass spectrometer was operated in multiple-reaction monitoring (MRM) mode (m/z transitions for IM 494->394, CGP74588 480->394, D8-IM 502->394, NIL 531->290, DAS 489->402 and for LAP
582->366; dwell time 200 ms). We demonstrated the suitability of this assay for TKIs using to quantify their concentrations in plasma
of patients with chronic myeloid leukemia on imatinib and nilotinib therapy and for patients with breast cancer on lapatinib therapy.
This project is supported by grants IGA MZCR NS9627 and MSM 6198959205.
P106:
Application of GCxGC-TOF MS in metabolomic profiling associated with human disorders
Jitka Zrostlíková1, Tomáš Kovalczuk1, Tomáš Adam3, Petr Wojtowič3, Jakub Schůrek1
1 LECO Application laboratory, Sokolovská 219, 190 00 Praha 9, Czech Republic
3 Laboratory of Inherited Metabolic Disorders, Department of Clinical Biochemistry, University Hospital Olomouc, I.P.Pavlova 6, 775 20 Olomouc, Czech Republic
Metabolomics is a rapidly developing technique serving as an important tool in many research areas, such as genetic research,
fingerprinting of species, metabolic pathway studies, metabolic disorders research and laboratory diagnostics. Among the analytical
methods used in metabolomics, both capillary gas and high performance liquid chromatography, coupled to mass spectrometry play
an important role, thanks to their ability of determination and identification of the large number of compounds.
In the area of human metabolomics, research efforts have been focused to improve laboratory diagnostics and also to elucidate
metabolic mechanisms. Basically, two types of tasks stand in front of a human metabolomics analytical chemist: (i) the determination of the given set of metabolites for the purpose of routine diagnostics and (ii) the study of complete collection of metabolites
present in cells or body fluids. Obviously the latter task is much more challenging and lays high demands on the analytical technique
used. Classic scanning GC-MS techniques can struggle with the high number of components present and the occuring coelutions.
Also these techniques are not usually designed as non-target screening methods for the identification of all sample components.
Recently, comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GCxGC-TOF MS) has
become an emerging technique in the field of plant1, 2 animal3 and human4 metabolomics. Using this technique, the enormous separation power of GCxGC is combined with the identification capability provided by full mass spectra of TOF mass spectrometer and
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Poster Abstracts
Automated Peak Find and identification capabilities of the applied software.
In this work, the GCxGC-TOF MS (LECO Pegasus 4D) method has been developed for the analysis of derivatized samples of body fluids
as well as extracts from human fibroblasts. The performance of this method has been compared to a classic quadrupole GC-MS method. Contrary to one-dimensional GC-MS many more compounds were revealed and this technique has been shown as very powerful
for the purpose of comprehensive sample profiling. Initial results from the comparison of human fibroblasts samples from metabolic
disordered vs. healthy persons will be shown.
1 Fiehn et al., Nat Biotechnol., 18 (2000) 1157-1161
2 O´Hagan et al., Anal. Chem, 79 (2005) 464-476
3 Shellie at al., J. Chromatogr. A 1086 (2005) 83-90
4 Konstantinos et al., J. of Chromatogr. A, 1217 (2010) 104-111
P107:
Microbial targeted metabolome analysis of common nucleotides and coenzymes by capillary electrophoresis
Jindra Musilová*, Zdeněk Glatz
Department of Biochemistry, Faculty of Science, Masaryk University
The quantitative determination of nucleotides is important in many fields of biochemical research. Intracellular level of nucleotides
characterizes the energetic state of the cell under a variation of physiological conditions during the cell growth. The main aim of this
work was to optimize conditions for selective and rapid determination of 17 energetically important metabolites (purine and pyrimidine
nucleotides, adenine coenzymes and Acetyl CoA) using capillary electrophoresis (CE). Because of low intra- and extracellular concentration of these metabolites, the capillary zone electrophoresis was combined with the on-line preconcentration technique - field enhanced
sample stacking - to improve the concentration sensitivity. The optimal separation was reached in a bare fused silica capillary (effective
length: 56 cm; internal diameter: 75 micro m) using separation voltage 23 kV (positive polarity), temperature of capillary 21°C and
direct detection at 260, 280 and 340 nm. 50 mM concentration of phosphate buffer (pH 5.8) provided the best resolution of peaks.
Metabolite samples were dissolved in deionised water and injected into the capillary hydrodynamically with a pressure of 50 mbar for
14 s. Resulting method showed RSD in the range from 0.65 to 1.40 % for migration time and from 2.37 to 5.99 % for relative peak area
of metabolites (n=10). Optimized method was used for analyzing of cell-extract of bacterium Paracoccus denitrificans.
This work was supported by research project No. MSM0021622413 and research centre LC06023 both from Czech Ministry of Education.
P108:
Large Volume Injection GC/MS Analysis of Cultured Human Skin Fibroblasts for Metabolomic Studies
Petr Wojtowicz, Lenka Žídková, Vojtěch Bekárek, Tomáš Adam
Laboratory for Inherited Metabolic Disorders, University Hospital and Medical Faculty, Palacký University, I.P. Pavlova 6, 775 20 Olomouc, Czech Republic
Introduction: Gas chromatography-mass spectrometry (GC/MS) is often used technique for metabolomic studies to analyze organic
acids, amino acids and sugars. Human cells in a culture provide an important research and diagnostic tool because influence of major
part of the external factors is minimized in culture where defined extracellular environment takes place. Sample volume is key limiting
factor since 1 mL of total cell mass is usually harvested per cultivation flask. Under standard derivatisation and injection conditions it
is possible to analyze only a limited number of metabolites because of the limited amount of material injected on column. We report
here the method for analysis of cultured human skin fibroblasts by large volume injection using programmable temperature vaporizing
(PTV) injector.
Methods: Fibroblasts (one million cells) were cultured by standard protocol (Dulbeco’s medium supplemented with 10% fetal bovine
serum and antibiotics). Cells were quenched by spraying with cold 60 % aqueous methanol (v/v) and immediately extracted into cold
80 % aqueous methanol (v/v). The cell extract was freeze-dried, derivatizated to trimethylsilylderivates (Koek, MM, Anal. Chem. 2006)
and analyzed. Final amount of derivatized sample in the conical vial insert is 30 mL which makes it possible to use an autosampler.
Conditions of the PTV injector were optimized using Design of Experiment method (model mixture of organic and amino acids). A
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Poster Abstracts
Plackett-Burman design was used to identify the significant factors and a central composite design for obtaining their best values.
Results: In the intracellular content we totally determined 35 metabolites – 13 amino acids, 5 organic acids, cholesterol, 7 peaks of
not fully characterized sugars/polyols and 9 undefined peaks. The efficiency of separation with 5mL-injection in splitless mode (250°C,
splitless time 1.1 min, no backflush and surge) was 200 000 theoretical plates.
Conclusion: Large volume injection technique was used for substantially improvement of GC/MS sensitivity and it allowed analysis of
intracellular metabolites from human skin fibroblasts. The developed method can be robotized and allow analyzing large series of
samples.
Supported by grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism and the Norwegian Financial Mechanism (A/CZ0046/2/0011) and MSM6198959205. Tuesday, March 23rd, 2010
P109:
Comparison of Derivatisation Techniques for GC/MS Metabolomic Analysis of Cultured Human Skin Fibroblasts
Petr Wojtowicz, Vojtěch Bekárek, Tomáš Adam
Introduction: Gas chromatography-mass spectrometry (GC/MS) is often used method for analysis of organic and amino acids. There are
many derivatisation approaches based on formation of compounds with lower polarity and higher volatility. Silylation is mostly used
but it has certain limitations, for example it forms more products from single analyte which are changing in time. We report here the
comparison of three derivatisation techniques: trimethylsilylation by N-methyl-N-trimethylsilyltrifluoroacetamide (TMS) and indirect
alkylations via methylchloroformate (MCF) or ethylchloroformate (ECF; Hušek P. et al., J. Chromatogr. 2003).
Methods: Cultured human skin fibroblasts (hCSF) were cultured by standard protocol, quenched by spraying with cold 60% aqueous
methanol (v/v) and extracted into cold 80 % aqueous methanol (v/v). The cell extracts were freeze-dried, derivatised and analyzed by
GC/MS (Thermo Electron Corporation, USA, ZB-5MS, 30 m ´ ID 0.25 mm, df 0.25 µm, Phenomenex, USA). Results: Obtained chromatograms were evaluated by number of peaks belonging to chemical species that could be quantified (S/N >
10). In silylated sample 34 peaks were determined. In case of MCF- and ECF-alkylation it was determined 60 and 62 peaks, respectively. ECF-amino acids provide higher sensitivity compare to MCF. Alkylated chromatograms are simpler because there are no peaks of
sugars which are the most abundant in silylation method, cannot be positively identified and most probably mask lower concentrated
compounds.
Conclusion: Indirect alkylation using ECF is effective derivatization method for metabolomic analysis of hCSF. This approach allows
analysis of highest number of compounds in the sample of all methods studied.
This work was supported by a grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism and the Norwegian
Financial Mechanism (A/CZ0046/2/0011).
P110:
Principles and applications of CE-MS in metabolomics
Martin Greiner1, Tomoyoshi Soga2
1 Agilent Technologies, Waldbronn, Germany
2 Keio University, Tsuruoka, Japan
For higher sensitivity and better compound identification, the coupled technique of capillary electrophoresis (CE) and mass spectrometry (MS) has significant advantages compared to standard detection methods based on UV absorbance or laser-induced fluorescence
(LIF). Interfacing capillary electrophoresis equipment to commercial mass spectrometers equipped with electrospray ionization (ESI)
sources is possible in various ways, whereby sheath liquid interfaces are most predominant although direct interfaces are also available. We used a sheath liquid interface approach and discuss the advantages and disadvantages of this technique. Data shown are
from Prof. Soga metabolomics research team at Keio University, Japan. Metabolomics applications have to deal with a multitude of
polar compounds in complex matrices and therefore offer an ideal deployment field for CE analysis, especially when combined with
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Poster Abstracts
mass spectrometry detection. Fast scanning and accurate MS tools such as accurate mass time-of-flight (ToF) or Q-ToF instruments
are ideally suited for sensitive and unique identification of such complex samples. It also proved to be suitable to meet requirements
such as generating robust and reproducible data in a routine manner. Basic setup of instrumentation as well as metabolite profiling
and identification in this highly comparative type of analysis is demonstrated with anionic compounds on CE-ToF-MS instrumentation.
P111:
Quenching and extraction of human skin fibroblasts in adherent culture for metabolomic studies
Lenka Žídková, Petr Wojtowicz, David Friedecký, Tomáš Adam
Introduction: Metabolomics aiming to characterize of a phenotype through analysis of metabolome developed to an accepted and valuable
tool in life sciences over the recent years. Human cells in a culture provide an important research and diagnostic tool but analysis of adherent
cell cultures present complex challenges mostly given by limited sample size. Rapid quenching of intracellular metabolism simultaneously with
considerable removal of superabundant growing medium are the main prerequisites. The aim of our work was to develop efficient quenching
and extraction procedure for human skin fibroblasts in culture for metabolomic studies.
Methods: Fibroblasts were cultured in DMEM using standard protocol. We optimized manipulation technique, solvent volume and constitutions. Final quenching technique was performed by spraying out media from cultivation flask using syringe with a needle by 20 ml of 60%
methanol at – 50°C. Standard cell harvesting by trypsinisation was used for comparison. Cells were extracted twice by 1 ml of 80% methanol
at – 50°C. Leakage of intracellular metabolites during quenching procedure and intracellular metabolites were determined by capillary electrophoresis (Friedecky D, 2007) and gas chromatography – mass spectrometry (Koek MM, 2006).
Results: Average leakage of the measured compounds was below 4%. Removal of original cultivation media is very efficient and it contributes
by 1.2% to final analyzed material. The quenching procedure takes approximately 6 s. Influence of harvesting method on metabolome (quenching vs. trypsinisation) were compared as the natural logarithms of ratios of means and variations. Harvesting method substantially affects
concentrations of number of metabolites. Several metabolites (e.g. citrate, lysine) differ approximately order of magnitude. From variations it is
also evident that sample preparation by conventional trypsinisation provides substantially more variable data compared to quenching by spray.
Conclusion: We developed quenching technique for cultured adherent human skin fibroblasts providing stable data sample-to-sample with
minimal leakage. The methods rapidly quenches intracellular metabolism and effectively removes cultivation medium.
Supported by grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism and the Norwegian Financial Mechanism
(A/CZ0046/2/0011) and MSM6198959205. Tuesday, March 23rd, 2010
P112:
Identification of individual cytochrome P450 enzymes involved in the metabolism of ketamine and norketamine in man
Simone Portmann1, Regula Theurillat1, Andrea Schmitz3, Meike Mevissen3, Wolfgang Thormann*1
1 Department of Clinical Pharmacologyy and Visceral Research, University of Bern
3 Division of Veterinary Pharmacology and Toxicology, University of Bern
Ketamine, an injectable anesthetic and analgesic consisting of a racemic mixture of S-and R-ketamine, is routinely used in veterinary
and human medicine. Metabolism and pharmacokinetics of ketamine in man have not been characterized sufficiently. An enantioselective CE assay for ketamine and its metabolites in microsomal preparations has been developed and applied to the identification of
individual human CYP450 enzymes catalizing ketamine to norketamine and norkatemine to further metabolites in vitro. The assay is
based upon analyte liquid/liquid extraction at alkaline pH and the use of a CE buffer composed of tris/phosphate at pH 2.5 containing
10 mg/ml of multiple isomer sulfated beta-cyclodextrin as chiral selector. This approach permits the simultaneous anionic analysis of
the stereoisomers of ketamine, norketamine, dehydronorketamine and hydroxylated norketamine metabolites with hydroxylation at
the cyclohexanone ring1. Commercially available recombinant human CYP450 enzymes (SUPERSOMES) were incubated with racemic
ketamine or racemic norketamine and samples were analyzed after incubation time intervals of 0, 60 and 120 min. Six CYP450 enzymes
(3A4, 2C19, 2B6, 2A6, 2D6 and 2C9) were identified to catalyze ketamine N-demethylation among which three enzymes (3A4, 2B6
and 2A6) generated norketamine stereoselectively. Two enzymes (2B6 and 2A6) were found to be associated with the formation of
norketamine metabolites (hydroxylated norketamine metabolites and dehydronorketamine). With three enzymes (2E1, 1A1 and 1A2),
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Poster Abstracts
no ketamine metabolites were detected. The results suggest that the metabolism of ketamine in man is mainly catalized by CYP3A4,
CYP2B6, CYP2C9 and CYP2C19. The work highlights the value of enantioselective CE in drug metabolism studies.
This work was supported by the Swiss National Science Foundation. 1 A. Schmitz, R. Theurillat, P.-G. Lassahn, M. Mevissen, W. Thormann, Electrophoresis, 30, 2912-2921 (2009)
P113:
Metabolomic study of human cerebrospinal fluid using CE-MS
Clara Ibañez1, Carolina Simo1, Angel Cedazo-Minguez3, Alejandro Cifuentes1
1 Department of Food Analysis, Institute of Industrial Fermentations, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain
3 Karolinska Institutet, NVS Department, 14186 Stockholm, Sweden
In this work, capillary electrophoresis-mass spectrometry (CE-MS) is used to carry out a metabolomic study of human cerebrospinal
fluid (CSF). A CE-MS coupling with an electrospray sheath-liquid interface (ESI) and a time of flight (TOF) MS analyzer were used in
this work. Due to the high scan speed and high mass resolution of TOF MS, this analyzer is well-suited for on-line coupling with fast
separation techniques as CE to analyze complex samples. TOF MS also provides high mass accuracy determination, essential to obtain
as much information as possible from complex biological fluids as CSF. The CSF was analyzed with minimal sample pre-treatment:
deproteinization was carried out by using ultracentrifugation and a membrane with 3000 Da cutt-off. The ultrafiltrate was directly
injected to the CE-MS. For the metabolic profiling of human CSF by CE-ESI-TOF MS an acidic volatile buffer was used together with
a bare fused-silica capillary. Reproducible analyses were obtained with %RSD values within the same day (five consecutive injections)
lower than 0.5 % and 4 % for analysis time and peak area, respectively. A metabolic profile in the positive ionization mode was obtained
in less than 30 min. After discarding the potential interferences from solvent ions, adducts and other contaminants, a total number of
120 peaks were subjected to peak identification. The high mass accuracy provided by TOF MS allowed generating a molecular formula
for each metabolite that was then used for metabolite identification using HDBM, Metlin, Kegg, Chemspider, PubChem, etc. databases.
Tentative identification of more than 80 compounds is achieved in this work, to our knowledge, the highest number of compounds
identified in human CSF by CE-MS so far. The potential of this CE-MS metabolomic approach will be used to detect biomarkers of a
variety of neurological diseases.
P114:
Stable Coating Using Nafion Thin Membrane for Fast Electroosmotic Flow in Poly(dimethylsiloxane) Microchips
Mariko Seno, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka
Kyoto University
In microchip electrophoresis (MCE) on a poly(dimethylsiloxane) (PDMS) microchip, a modification of the inner surface of the channels
is one of the important issues since an untreated PDMS microchip provides the unstable and slow electroosmotic flow (EOF) and the
nonspecific adsorption of biomolecules. To overcome these drawbacks, we have studied on the coating method using Nafion which has
negatively charged side chains and easily forms a durable thin membrane by drying.
A PDMS microchip with a straight-channel was fabricated by the conventional photolithographic technique. In the multi-layer coating
method, a 0.5 % (v/v) polybrene (PB) and 1.5% (v/v) Nafion aqueous solutions were alternately introduced into the microchannel, resulting in the strong adsorption of Nafion onto the surface due to the electrostatic interaction. After the 3-times repetition of the alternate
injections of the two polymer solutions to increase the amount of the adsorbed Nafion polymer, the PDMS chip was baked at 110°C for
30 min to form the Nafion thin membrane.
To evaluate the characteristic of the Nafion-membrene coated microchip prepared with the multi-layer coating method, the EOF measurements using uranine were carried out. In the Nafion coated microchip, the electroosmotic mobility (mEOF) was ranging from 3.0 to
7.5´ 10–4 cm2/V×s during the 30-runs. It should be noted that the observed mEOF became higher upon increasing the number of runs,
suggesting that the cationic polymers desorbed from the channel surface, whereas the Nafion coatings remained due to the formation
of the durable membrane. The averaged value of mEOF was estimated to be 5.1´ 10–4 cm2/V×s which was higher than the PB/dextrane
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Poster Abstracts
sulfate coated PDMS chip (3.6´ 10–4 cm2/V×s)1, fused silica capillary (4.6´ 10–4 cm2/V×s) and poly(methyl methacrylate) microchip (2.3´
10–4 cm2/V×s)2. These results indicated that the durable Nafion membrane was successfully formed onto the inner surface, providing
the fast EOF. In addition, the Nafion coated microchip could suppress the adsorption of derivatized labeled carbohydrates. Therefore,
the Nafion coated PDMS microchip should be promising for rapid MCE analyses of carbohydrates.
1 Liu, Y. et al. Anal. Chem. 2000, 72, 5939-5944
2 Soper, S. A. et al. Anal. Chim. Acta 2002, 470, 87-99
P115:
Modeling and simulation, design and fabrication, instrumentation and control for dielectrophoresis
Jiri Zemanek1, Zdenek Hurak1, Yves Bellouard3
1 Faculty of Electrical Engineering, Czech Technical University in Prague
3 Technische Universiteit Eindhoven
The poster gives an overview of the authors' recent research activities and achievements in the domain of dielectrophoretic manipulation with meso- and microscale particles. It recalls the mathematical models for the dielectrophoretic phenomenon, describes a
few classical and introduces some new designs of prototyping dielectrophoresis-ready microfluidic chambers, visualizes the results of
numerical simulations for these electrode layouts, documents and comments on a few selected fabrication procedures and shares a
few practical fabrication tricks that can only be earned by spending dozens of hours in a lab (such as how to connect electrical cables
to the glass made dielectrophoretic chips). Finally, it elaborates in some detail on both commecial and home-made instrumentation and
computational algorithms related to computer-vision based detection of particle positions. The motivation for this practical and theoretical investigation of dielectrophoresis was given by its intended use as the key driving force for self-assembly experiments wherein
particles such as glass or polystyrene beads, silicon dust, LED chips or thick-film resistors are immersed in a liquid and steered around
"somewhat randomly" over a planar substrate. Visit http://www.golem-project.eu/ for the overall self-assebly motivation since it will
not be covered by the presentation. The goal of self-assembly induces a few control strategies such as random perturbation of phase
delays of applied harmonic voltages or random switching among a few values of phase delays. The presented findings are believed to
be applicable also in other more conventional applications of dielectrophoresis such as planar transportation, separation and mixing.
P116:
Using Micro Electrode Chip for Cell Migration via Moving Electric Field
Chia-Hsien Yeh, Po-Yu Tu, Yu-Cheng Lin
Department of Engineering Science, National Cheng Kung University
Abstract: This study describes using the technology of Micro Electro Mechanical Systems (MEMS) to fabricate the chip with different
gaps of microarray electrode, and cell migration was observed when the moving electric fields by employing high frequency AC signal.
Our strategy is to simulate the four asymmetric waveform signals (sine, square, triangle, and ramp) in the chip with various gaps (10
μm, 20 μm, and 30 μm) to generate the moving electric field by the ANSYS software. The ramp asymmetric waveform signal could be
generated the better effect of the moving electric field than other asymmetric waveform signals. When fixing the electric filed intensity
in the ramp asymmetric waveform signal, the 10 μm gap micro-assay electrode chip had a better obvious effect. The migration velocity
of the cells was 21.25 μm/hr in 10 μm micro-assay electrode chip.
Introduction: In the past years, a lot of researches demonstrated the enhancing effects of DC electric field on cells in vitro. Moving
electric fields generated by AC voltage have been applied in separating particles and cells. The analysis of cell shape changes and cell
migration by DC electric field were reported. In this study, a simulation and comparison of the electric field distribution of different
waveform types used for migrating cells is reported.
Materials and methods: The chip was designed by using the AutoCAD® 2009 software, and the electrodes were the arrayed-finger type.
The size of the electrode width was 10 μm and the various gap sizes were 10 μm, 20 μm, and 30 μm. In this study, we have simulated
the four asymmetric waveforms, including the sine waveform, square waveform, triangle waveform, and ramp waveform. After simulation, the effect of gap size of various electrodes and the most suitable waveform were discussed.
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Poster Abstracts
Results and Discussion: We have found that the ramp waveform has the smaller time ratio of positive electric field than other waveforms. The electric field distribution of the ramp waveform was obviously focused on same direction. Moreover, the ramp waveform
has a larger difference between peaks of the positive and negative electric field. It shows that the ramp waveform provides the uniform
electric field in high frequency, so the ramp waveform would be used for the micro arrayed-electrode chip to drive the cells migration.
The cell migration was measured in the chips of various gap sizes under the same condition: 100 KHz and 0.6 V/mm. We have found
that the chips with 10 µm have a larger migration effect than that of other gap sizes during 4 hr. The average migration distance was
21.25 µm in 10 µm gap size chip.
Conclusions: This study has successfully demonstrated the ramp waveform provided the better moving electric field to drive the cells
migration than other waveforms. The smaller gap size (10 µm) had the better effect on cell migration under 100 KHz and 0.6 V/mm.
This design could be applied to cell migration, DNA separation and other biology applications.
P117:
Specific detection of proteins by immunoprecipitation combined with high sensitivity protein sizing on microchips
Ela Herwig1, Christian Wenz2, Martina Marchetti-Deschmann1, Günter Allmaier1, Andreas Rüfer2
1 Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria
2 Agilent Technologies R&D and Marketing GmbH & Co. KG, Waldbronn, Germany
Today, immunoaffinity is a crucial tool for the targeted and highly specific analysis of proteins in complex samples. Techniques like
Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting are widely used for a wide range of applications such as biomarker candidate verification in body fluids or clone selection for recombinant protein expression. Here we present a new method that
combines the specificity of an immunoprecipitation approach out of e.g. a total cell lysate with the high sensitivity of protein detection
on microchips. Initially, sample proteins are derivatized to a low degree with a fluorescent dye (optimal one dye molecule per analyte
molecule). After incubation with a specific target antibody, immunocomplexes are captured with Protein A/G coated magnetic beads,
washed and eluted by heat denaturation in the presence of SDS. Samples are then directly loaded onto microfluidic chips and analyzed
automatically for protein size and quantity. The final on-chip analysis takes about 30 min for 10 samples and yields digital data. Together
with the sample preparation steps, the total assay time is about 3 hours. Results are compared to Western Blotting and ‘pros and cons’
of both methods are discussed.
P118:
Carbohydrate profiling using CE-on-a-chip implemented on an Agilent Bioanalyzer 2100
Petr Smejkal1, Markéta Ryvolová2, Andras Guttman3, František Foret4, Fritz Bek5, Mirek Macka2
1 National Centre for Sensor Research and School of Chemical Sciences, Dublin City University, Dublin 9, Ireland;Institute of Analytical Chemistry of the ASCR, v.v.i.,
Veveří 97, 60200 Brno, Czech Republic; Faculty of Chemical Technology
3 Horváth Laboratory of Bioseparation Sciences, Institute of Analytical Chemistry, University of Innsbruck, A-6020 Innsbruck, Innrain 66, Austria
5 Agilent Technologies, P.O. Box 1280, 76337 Waldbronn
Carbohydrate analysis and profiling is important in numerous areas form food industry and dietetics to glycoproteomics and disease
detection. Post-translational modifications such as glycosylation play a key role in the function of a variety of proteins in living organisms. Glycoproteins mediate essential processes such as signal transduction and/or intercellular interactions. Carbohydrate profiling can
be used as a screening method for biomarkers of various diseases, is based on capillary electrophoresis (CE) analysis of oligosaccharides
released from glycoproteins.
Due to the lack of a chromophore or fluorophore in carbohydrates fluorescent tags such as 8-amino-1,3,6-pyrenetrisulfonic acid (APTS)
have to be incorporated providing both the means of very sensitive LIF detection and a charge to allow for CE separation.
Agilent Bioanalyzer 2100 is a separation CE-on-a-chip analyzer using glass chips with a 14 mm separation channel and laser-induced fluorescence detection with current methods specifically tailored for analysis of DNA, RNA and proteins. In contrast here the Bioanalyzer
is used in a flexible research-mode as a CE-on-a-chip instrument for separations and analysis of carbohydrates.
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Poster Abstracts
In this work the potential of Bioanalyzer for carbohydrate screening was investigated. Blue LED-IF detection (lex= 470/25 nm, lem= 525/30
nm) provided by the Bioanalyzer was initially tested using Fluorescein and Fluorescein isothiocyanate (FITC) with limits of detection 15
nM and 1.8 mM, respectively. APTS-labelled maltooligosaccharide ladder separated in an electrolyte system of 50 mM acetate buffer
pH 4.75 and Agilent DNA gel in a ration of 2:1 (v/v) showed well separated peaks for up to twenty maltooligosaccharide units documenting the separation power of the short separation channel. Applications were explored in the areas of food analysis and medical
diagnostics. Wednesday, March 24th, 2010 P119:
Development of a Rapid Two-Step Micro Southern Hybridization Analysis System
Kiichi Sato1, Keisuke Aono2, Etsuro Yoshimura2
1 The University of Tokyo, Japan Science and Technology Agency
2 The University of Tokyo
A Southern analysis is a method routinely used in molecular biology to confirm the presence or absence of a target DNA in biological
samples. The analysis combines gel electrophoresis for size separation of DNA fragments with transfer of the separated DNA to a
membrane for probe hybridization. Because the method requires troublesome manual operations and long analysis time, reduction of
the analysis time is welcomed.
In this paper, we propose a microchip-based Southern hybridization analysis system, which combines DNA separation by microchip
electrophoresis with hybridization in a micro square space. Instead of blotting to a transfer membrane and successive hybridization
on the membrane required in the conventional method, the separated DNA is electrophoretically transferred to hybridize with a probe
DNA fixed in a soluble polymer in the microchip.
Hydroxyethyl cellulose was used as a sieving matrix. Probe DNA was copolymerized with dimethylacrylamide and the resulting conjugate was used as a hybridization polymer. The PDMS microchip had a narrow microchannel and a square space. The narrow channel
and a part of the square space was filled with the sieving matrix solution and the other part of the square space was filled with the
sieving matrix solution.
HaeIII digest of phi X174 DNA was analyzed with the chip. After electrophoretic separation, only the target DNA was trapped at the
beginning of the hybridization zone, while the other DNA passed through it. The target DNA was clearly shown as a trapped band and
its position indicated its size. Therefore, the system could realize both the size separation and sequence-dependent detection within 5
min instead of 2 days in the conventional methods. The method will be useful to detect a trace amount of target DNA in crude samples.
P120:
Development of an Intergrated Microscale Ceramic Separation Device to Address Limited Sample Volumes in Bioanalysis
Paul D. Rainville1, Mike Tomany2, James Murphy2, Norman W. Smith4, Robert S. Plumb5
1 Kings College London, Waters Corporation
2 Waters Corporation
4 Kings College London
5 Imperial Collge London
Introduction: Small sample volumes from tail-bled rodents and dried blood spot (DBS) cards present an extra challenge for the
bioanalytical scientist. Microscale separations have shown potential for the high-sensitivity analysis of limited-volume samples
in proteomics. However, this traditionally requires experienced analysts and specialized instrument configurations. In this paper,
we will discuss the use of a capillary-scale ceramic separation device to achieve pg/mL levels of sensitivity for candidate drugs
in a few microliters of biofluid. The sensitivity of the capillary system with a dedicated nano-spray source was compared to a
standard tandem quadrupole LC/MS system and found to 25 times more sensitive.
Methods: Various drug compounds and associated metabolites were spiked into rat plasma and whole blood. Plasma samples
were prepared by protein precipitation utilizing 2:1 (acetonitrile: plasma), centrifuged and diluted 1:1 with water. Whole blood
samples were prepared by spotting 15 µL of blood onto Whatman 903 ® Specimen collection paper, drying for two hours and
extracting the blood spot with methanol. Samples were then injected onto a 0.300 × 100 mm channel packed with 1.7 µm BEH
C18 and eluted using a linear gradient housed on a ceramic nanotile at 12µL/min. Mass spectrometry was performed on a tan120
Poster Abstracts
dem quadrupole MS operating in positive electrospray mode. The capillary and cone voltages were optimized for each compound.
Results: Utilizing samples derived from the protein precipitation of rat plasma and whole blood from blood spot cards, the
reproducibility of the system was evaluated. The separation device showed excellent robustness with greater than 1,000 injections obtained from protein precipitated plasma, maintaining peak widths similar or superior to standard microscale LC/MS. The
average peak width for a small molecule was 2.3 seconds at base, giving a peak capacity of 130 for a 5 minute run. Retention
time variation of test analytes between different lots of ceramic devices was less than 0.6 % CV and resolution between critical
pairs was between 1.2 and 1.5 for a test mix. The maximum injection volume was determined to be 1.2µL with a 66% acetonitrile aqueous mixture. The assay sensitivity of the micro LC system was determined to be 25 times that of the standard 2.1mm
system, for a 1µL injection of sample; making this system ideal for low sample volume assays. Bioassays for various compounds
were successfully developed from 15 µL DBS with LLOQs as low as 100 pg/mL utilizing only 1 µL of injected sample.
P121:
Photolithographically Defined Monolithic Materials in Microfluidic Devices
Seth M. Madren, Stephen C. Jacobson
Indiana University
Monolithic materials are a convenient scaffold in capillaries and microfluidic devices for incorporating stationary phases for
separations and enzymes for biochemical reactions. To enhance device functionality, confining the monolith to specific regions
in the microchannels is preferred. To accomplish this, we are developing photolithographic methods to form these monolithic
materials. With photolithography the boundary of the monolithic material is established without a need for physical barriers.
However, one complication in developing this technique has been formation of monolithic material in regions that are not exposed UV radiation. We believe that this problem is exasperated by the high surface-to-volume ratio of the microchannels (10
µm deep by 50 µm wide). Inclusion of trace amounts of a radical scavenger to the polymer solution eliminates this unwanted
polymerization without inhibiting the monolith formation in regions exposed to UV. We have used this procedure to form a
methacrylate monolith suitable for gradient CEC separations for one and two dimensional separations. In addition to creating
stationary phases, forming multiple monoliths with various functionalities in the same device is possible. For example, enzymes
have been attached to monoliths that contain epoxy groups to create bioreactors for digesting proteins and cleaving glycans. The
long term goal is to develop devices that contain bioreactors and affinity columns that can perform sample preparation prior to
one or two dimensional separations. Wednesday, March 24th, 2010 P122:
Highly Effective Sample Preconcentration and Separation Based on Transient-Trapping in Microchip Electrophoresis
Kenji Sueyoshi, Kota Hashiba, Ryuta Tanaka, Fumihiko Kitagawa, Koji Otsuka
Recently, several on-line sample preconcentration techniques have been applied to improve the insufficient detectability of
microchip electrophoresis (MCE). However, the effective separation length is often reduced since a sample solution is injected as
a long plug, resulting in the unexpected decrease of resolution. To overcome this drawback, several preconcentration techniques
using the micro/nano structures were also combined with the MCE devices, so that the sensitivity could be improved without the
decrease in resolution. In this paper, a novel on-line sample preconcentration technique named transient-trapping (tr-trapping)1
is investigated for improvements of both resolution and sensitivity in MCE.
In tr-trapping, the highly effective preconcentration and separation can be achieved by the trap-and-release mechanism without
the complicated fabrications1. Briefly, a micellar solution (M) plug is partially injected in front of a sample solution (S) plug, and
then the separation voltage was applied. When the samples reach the boundary between the S and M plugs (S/M boundary) by
the EOF, strongly hydrophobic samples are “trapped” and concentrated by the micelle nearby the S/M boundary. At the same
instance, the concentration of the micelle is gradually decreased due to the molecular diffusion, so that the sample retention by
the micelle is also reduced. Thus, the trapped analytes are “released” from the S/M boundary and penetrate into the M plug.
Due to the difference in the releasing time, the samples are effectively separated. After releasing, the samples migrate in the M
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Poster Abstracts
on the basis of microchip micellar electrokinetic chromatography (MCMEKC) and then move in the BGS zone based on microchip
zone electrophoresis (MCZE).
To observe the detailed process of tr-trapping–MCMEKC, quartz microchips with a 5 way-cross channel geometry were fabricated
by the photolithographic technique for the fluorescence imaging. The injection volumes of the S and M plugs were adjusted by
the voltage control. Detection was carried out with the laser-induced fluorescence scheme. The background (BGS) and M solutions were 30 mM of phosphate buffer and 25 mM of sodium dodecyl sulphate / 22 mM phosphate buffer both containing 10% (v/v)
methanol, respectively. Sulforhodamine B (SRB) and 101 (SR101) were used as the test analytes. As a result, the estimation of the
effective diffusion constant clarified that the tr-trapping–MCMEKC analysis consists of the four successive steps: (i) preconcentration due to the trapping, (ii) MCMEKC in the M zone (iii) band broadening due to the difference in the velocity of the samples
with/without micelles and (iv) MCZE in the BGS zone. This result supported the proposed mechanism in tr-trapping–MCMEKC.
The fluorescence imaging also suggested that the tr-trapping analysis could be demonstrated on the shorter effective separation
length. To realize more rapid and sensitive MCE, the analysis of rhodamine dyes were demonstrated at the effective length of 5.0
mm. As a result, the released SRB was well separated from the trapped SR101 within 5.0 s under the tr-trapping condition, whereas these dyes could not be resolved in conventional pinched injection–MCMEKC (PI–MCMEKC). Furthermore, the detectability
of SR101 in tr-trapping–MCMEKC was 580-fold improved as comparing to the conventional PI–MCMEKC analysis.
1 Sueyoshi, K.; Kitagawa, F.; Otsuka, K. Anal. Chem. 2008, 80, 1255–1262
P123:
Microchip Electrophoresis of Proteins Using Microheater Integrated PDMS Chip. 3
Kana Tanigawa, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka
Protein analyses in microchip electrophoresis (MCE) have great advantages such as the rapid separation, high resolution and low consumption of reagents, while the low-concentration sensitivity is still one of major problems. To overcome this drawback, we developed
a new on-line sample preconcentration technique based on the sodium dodecyl sulfate (SDS) denaturation of proteins enhanced by
heating on a microheater integrated poly(dimetylsiloxane) chip.
In this technique, a solution of native proteins without SDS is injected as a long plug into the separation channel filled with a background solution (BGS) containing SDS. The proteins move fast by the electroosmosis flow (EOF) in the sample solution. After the proteins
penetrate into the BGS zone, they are slightly denaturated by SDS. The proteins then reach the microheater, and the SDS denaturation
of the proteins is drastically accelerated by heating. Since the electrophoretic mobility of the proteins should be increased in the opposite direction of the EOF by the binding of the anionic SDS, the long sample zone is swept around the microheater by the difference in
the effective velocities. Finally, the concentrated samples are separated on the basis of gel electrophoresis.
To realize the proposed method, a copper film, which worked as a heater, was installed under the separation channel on the microchip
with a cross-type channel. To verify the performance of the integrated microheater, the temperature of the buffer solution inside the
microchannel was estimated by the observing the fluorescence quenching of rhodamine B. When the current of 3.5 A was passed
through the copper film, the temperature around the film was increased to ca. 80 °C. The effect of the heating on the preconcentration
of proteins was estimated: bovine serum albumin was detected as a sharp peak with heating, whereas a broad peak was observed
without heating. These results demonstrated that the preconcentration effect based on the denaturation of proteins with SDS was
enhanced by the heating on the integrated microheater. In addition, the size separation of the proteins by gel electrophoresis was
achieved with higher sensitivity.
P124:
Column-coupling chip technology for zone electrophoresis in the determinations of inorganic cations and anions in drinking water
Marián Masár*1, Róbert Bodor1, Benjamin Bomastyk3, Milan Luc1, Heinz-Martin Kuss3, Dušan Kaniansky1
1 Department of Analytical Chemistry, Faculty of Science, Comenius University, Mlynska Dolina CH-2, SK-84215 Bratislava, Slovakia
3 Department of Instrumental Analytical Chemistry, Gerhard-Mercator University, Lotharstrasse 1, D-47048 Duisburg, Germany
122
Poster Abstracts
Ion chromatography (IC), for the determinations of some anionic and cationic constituents in drinking water, belongs for relevant analytical methods as included for drinking water regulations1. Clearly, capillary zone electrophoresis (CZE) is offering significant separation
potentialities for these ions. However, a common CZE approach2 is analytically limiting for the separation capacity as linked with the
opened separation system. Our work was focused to assemble an electrophoresis chip technology for the column-coupling technique
while favoring a closed separation system3. Further, the chip itself was joined tightly with a platform as connected for a set of peristaltic
micropumps3. The micropumps operate: (1) to fill-up the solutions with the separation channels on the microchip and (2) to close the
microchip channels by stopping micropumps3. In addition, our task was to elaborate an analytical procedure as applicable for sequential determinations of cationic and anionic constituents in drinking water. In CZE was determined for the anionic macro-constituents
(sulfate, chloride and nitrate) as diluted for a particular drinking water (not included for bicarbonate and carbonate). For the cationic
macro-constituents (calcium, magnesium, sodium and potassium along with ammonium for a lower concentration), in an identical water sample, required very significant dilution of the sample (consider electroneutrality) in the CZE determination. Such a situation in the
dilutions of samples is considered for the time steps in our CZE runs while operating for sequential determinations of the cationic and
anionic constituents. Our experiments are introducing chip technology to join the CZE determinations of inorganic cationic and anionic
constituents with a focus for drinking waters. For example, concentration limits of detections were reached (anions at 100 μg/l concentrations and cations at 10 μg/l concentrations). In addition, highly reproducible determinations (about 0.5% RSD of the peak areas for
anions and 0.6-5% RSD for cations) and still a rapid procedure (sample pretreatments were not demanding) should be considered as a
very potent analytical tool as applicable for ions in (drinking) water analyses.
This work was supported by the Slovak Grant Agency for Science (VEGA 1/0672/09 and 1/0882/09), the Slovak Research and Development Agency (VVCE-0070-07), Comenius University (UK/307/2009) and, in part, supported by Merck (Darmstadt, Germany).
1 P.Hecq, A.Hulsmann, F.S.Hauchman, J.L.McLain, F.Schmitz, Drinking Water Regulations, in P.Quevauviller and K.C.Thompson (Editors)
Analytical Methods for Drinking Water. Advances in Sampling and Analysis, Wiley, Chichester, 2006, Ch. 1, p. 1-37
2 J.W.Jorgenson, K.D.Lukacs, Anal.Chem., 53 (1981) 1298
3 D.Kaniansky, M.Masar, R.Bodor, M.Zuborova, E.Olvecka, M.Johnck, B.Stanislawski, Electrophoresis, 24 (2003) 2208
P125:
Thermooptical detection in microfluidics: Improvement of DT-TLM
Adelina Smirnova, Kazuma Mawatari, Takehiko Kitamori
The University of Tokyo, Tokyo, Japan
Thermal lens microscopy is a sensitive detection method for nonfluorescent molecules and widely applied to detection in a capillary
or on a microchip. These miniaturized systems require sophisticated microfabrication, microfluidics, and detection of low concentrations of target substances in a small volume and short optical length. Therefore, a highly sensitive and versatile analytical technique
is crucial. We have developed a thermal lens microscope (TLM) for sensitive detection of nonfluorescent molecules in microspace1. TL
detections with sensitivity of a single molecule concentration under optimized conditions was demonstrated. Since then, TLM has been
successfully applied to various integrated analytical systems on microchips for environmental analysis, clinical diagnosis, food analysis,
and single cell analysis. TLM has also been coupled with separation methods like electrophoresis to realize pretreatment, separation
and detection of carabamate pesticides in water2.
In fact this optimized TLM instrument was large and had very complicated optics for controlling the chromatic aberration; a more
compact desktop instrument was later developed and is now commercially available. However present DT-TLM has also some drawbacks which limit its application. At first there are still many optical elements with complicated adjustment procedure and the second
problem is insufficient signal stability, which caused by long way from sample to detector and optical elements on this way. To improve
performance characteristics and simplify the design of DT-TLM, we reduced the number of optical elements, change the lasers and
introduce new detection system. As the result, signal fluctuations were lowered, LODs for new DT-TLM were reduced almost in one
order (1´10-7 M of Sunset Yellow in water) and adjustment procedure became more easy and faster. Besides, the microscope with new
optics became more compact, light and portable. New DT-TLM is planning to be applied as a detection method in nanotitration system
on a microchip.
1 Kitamori, T., Tokeshi, M., Hibara, A., Sato, K., Analytical Chemistry 2004, 76, 52a-60a
2 Smirnova, A., Shimura, K., Hibara, A., Proskurnin, M. A., Kitamori, T., Journal of Separation Science 2008, 31, 904-908
123
Poster Abstracts
P126:
A microfluidic hydrogel for long-term cell preservation under cell-based assay conditions
Yan Xu1, Kihoon Jang1, Tomohiro Konno3, Kazuhiko Ishihara3, Kazuma Mawatari1, Takehiko Kitamori1
1 Department of Applied Chemistry, School of Engineering ,The University of Tokyo
3 Department of Materials Engineering, School of Engineering ,The University of Tokyo
The study addressed a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer hydrogel, as having the capability of spontaneous
encapsulation and long-term preservation of cells on chip without perfusion culture under cell-based assay conditions. Model cells
including mouse fibroblast cells (L929) and human arterial endothelial cells (HAECs) were preserved in the hydrogel in microfluidic
chips to demonstrate cell viability and proliferation. The results showed that in microfluidic chips the hydrogel could maintain high cell
viability and restrain cell proliferation for a long time (for example 18 days). As a model application, toxin screenings were performed to
demonstrate cell function, and as a result the preserved cells had same resolution to various toxins as the cells conventionally cultured
in medium. Therefore, the use of the hydrogel would bring a new strategy for cell-based applications to be performed on microfluidic
chips in a more flexible, simpler, and cheaper way.
P127:
Microfabricated pillar array used as a photonic crystal sensor for on-column refractive index detection
Pedro S. Nunes1, Jörg P. Kutter1, Niels A. Mortensen3, Klaus B. Mogensen1
1 Department of Micro- and Nanotechnology, Technical University of Denmark, 2800 Kgs Lyngby, Denmark
3 Department of Photonics engineering, Technical University of Denmark, 2800 Kgs Lyngby, Denmark
A waveguide based refractive index sensor has been fabricated in silicon oxynitride glass by standard UV lithography and dry etching
processes. The refractive index sensor consists of a pillar array with similar dimensions as microfabricated pillars for support of the
stationary phase in chip electrochromatography. This makes on-column detection possible without addition of a separate detection
cell, thereby minimizing the influence of the detection on the total plate height.
In this presentation improvements in the optics and fluidics are reported when compared to previously published devices [P. S. Nunes
et al. 2008], resulting in two orders of magnitude better detection limit.
The pillars in the array have a period of 5.0 µm, a width of 2.0 µm and a length of 20 µm. They act as a 1-dimensional photonic crystal
embedded in a microfluidic channel and are addressed by fiber-terminated planar waveguides perpendicular to the channel. Infra-red
light transmitted through the liquid infiltrated photonic crystal experiences multiple reflections depending on the refractive index of
the surrounding liquid. An increase of the liquid’s refractive index leads to a red shifted spectrum. Here, we report a significant improvement on both the fabrication process, as well as on the limit of detection of earlier 1D photonic crystal based sensors. Experiences
performed with several ethanol solutions ranging from a purity of 96.00 % (n = 1.36356) to 94.08 % (n = 1.36397) yielded a sensitivity
Δλ/Δn = 532 nm per refractive index unit (RIU) and a limit of detection (LOD) of 5 × 10 -5 RIU for a resonator with 18 pillars. This in
the same range as the measured LOD of waveguide based evanescent wave sensors. The sensitivity of 532 nm/RIU is, however, much
higher than for evanescent based sensors, because in our case all the measured light has passed through the liquid, thus increasing
the overlap between the liquid and the optical field, compared to evanescent based sensors, where only a small fraction of the light
interacts with the liquid.
The theoretical LOD based on the noise of the tunable laser source was around 2 × 10-6 RIU. We expect to get closer to this value by
increasing the number of pillars in the array and by improving their uniformity.
It is furthermore possible to fabricate the device in cyclic olefin copolymer (COC) by means of nanoimprint lithography, which significantly decreases the fabrication time and cost. The performance of polymer resonators might even be better than glass based devices,
because heating of the polymer leads to a smoothened pillar sidewall, resulting in more uniform reflections and thereby in a higher
finesse of the optical resonator. Initial results on this work will also be presented.
P. S. Nunes, N. A. Mortensen, et al. (2008). "Photonic crystal resonator integrated in a microfluidic system." Optics Letters 33(14), 1623-1625
124
Poster Abstracts
P128:
Reverse of Mixing Process with a Two-Dimensional Electro-Fluid-Dynamic Device
Chang Liu*1, Yong Luo2, E. Jane Maxwell1, Ning Fang2, David D. Y. Chen1
1 Department of Chemistry, University of British Columbia
2 Ames Laboratory-USDOE and Department of Chemistry, Iowa State University
Mixing of two solutions into one is a spontaneous process with a net increase in entropy. However, the reverse of the mixing process is thermodynamically un-favored and can only be achieved when certain conditions are met. The equipment available today for obtaining pure
compounds from complex mixtures is limited to the use of one-dimensional column techniques such as chromatography or electrophoresis, and is often inadequate in meeting the need for extracting pure compounds from small amounts of biological samples. A continuous
solution stream containing a mixture of two compounds can be separated into two channels, each containing a pure compound, thus
reversing the mixing process, using a two-dimensional microfluidic electro-fluid-dynamic (EFD) device. When electric field and pressure are
strategically applied in the interconnecting channels of an EFD device, the conditions for reversing the mixing processes can be calculated
by combining electric field and fluid dynamic calculations with the mass balance equation. If the pressure and electric potential at various
inlets and outlets satisfy these predetermined conditions, the reverse of a mixing process is observed. Conventional microfluidic devices
have been used to introduce samples from interconnecting channels or efficiently mix different solutions into a single channel. The EFD
devices expand the spatial separation of analytes from one dimension to two using both differential migration behaviour of analytes and
the velocity field distribution in different channel geometries. The devices designed according to these basic physicochemical principles can
be used for complete processing of minute samples and to obtain pure chemical species from complex mixtures. The predicable nature and
ease of operation could lead to a new generation of purification devices to serve the needs of biomedical research and other commercial
and academic activities.
P129:
Deformability based cell margination - A simple microfluidic design for malarial infected red blood cell filtration
Han Wei Hou1, Ali Asgar S. Bhagat2, Jongyoon Han3, Chwee Teck Lim1
1 Division of Bioengineering, National University of Singapore, BioSyM IRG; Singapore-MIT Alliance for Research and Technology Centre (SMART)
2 BioSyM IRG, Singapore-MIT Alliance for Research and Technology Centre (SMART)
3 Massachusetts Institute of Technology; BioSyM IRG, Singapore-MIT Alliance for Research and Technology Centre (SMART)
The flow of blood through the microvascular system has been extensively studied over the past century. In blood vessels with luminal
diameter less than 300µm, red blood cells (RBCs) which are smaller in size and more deformable than leukocytes, tend to migrate to the
axial centre of the vessel due to the Poiseuille nature of flow within these small capillaries1, thus displacing the larger (and less deformable)
leukocytes to the vessel wall; a phenomenon aptly termed as margination. In this work, this physiological event is mimicked in microfluidic
systems for the biological separation of malarial infected RBCs (iRBCs) from whole blood. Change in cell stiffness is a characteristic of iRBCs
which can act as an intrinsic biomarker for separation2.The malarial infected blood sample was tested using a 3 cm long 15 × 10 µm (W × H)
microchannel, fabricated in polydimethylsiloxane using standard microfabrication soft-lithographic techniques. By designing low aspect
ratio microchannels, thus allowing cell margination along the channel width, the iRBCs align near each sidewall and are removed using an
asymmetrical 3-outlet system, thus achieving filtration. Before testing iRBCs infected blood, the filtration principle was corroborated using
hard polystyrene 3µm beads suspended in blood. Tests were also conducted using early ring stage and late trophozoite/schizont stage
iRBCs which vary significantly in their deformability. Filtration efficiency was quantified by analyzing the dispersion of these fluorescently
labeled microbeads and iRBCs across the microchannel width at the outlet. Flow cytometry analysis was also conducted on the outlet samples to confirm filtration results. Our results indicate filtration efficiency of ~75% for early ring stage iRBCs and >90% for late stage iRBCs.
This is the first demonstration applying this unique biomimetic separation technique to iRBCs filtration for disease diagnostic application.
The simple and passive operation of the system makes it ideal for on-site testing in resource poor settings and can be readily applied to
other blood cell diseases such as sickle cell anemia and leukemia which are also characterized by change in cell stiffness.
1 Pries, A.R., T.W. Secomb, and P. Gaehtgens, Biophysical aspects of blood flow in the microvasculature. Cardiovascular Research, 1996. 32(4): p. 654-667
2 Suresh, S., J. Spatz, J.P. Mills, A. Micoulet, M. Dao, C.T. Lim, M. Beil, and T. Seufferlein, Connections between single-cell biomechanics and human disease states:
gastrointestinal cancer and malaria. Acta Biomaterialia, 2005. 1(1): p. 15-30
125
Poster Abstracts
P130:
HepaChip – microfluidic 3D coculture of in vivo like artificial liver sinusoids for use in substance screening
Julia Schuette*1, Karin Benz1, Christian Freudigmann1, Britta Hagmeyer1, Felix Holzner1, Massimo Kubon Simon Werner1, Jan Böttger7,
Rolf Gebhardt7, Patrick Emmerlich9, Peter Röhnert9, Martin Stelzle1
1 NMI - Natural and Medical Science Institute at the University of Tuebingen, Markwiesenstr. 55, 72770 Reutlingen, Germany
7 Institut für Biochemie der Medizinischen Fakultät der Universität Leipzig, Germany
9 KeyNeurotek Pharmaceuticals AG, Magdeburg, Germany9 KeyNeurotek Pharmaceuticals AG, Magdeburg, Germany
Reliable tools for screening substances for possible toxic side effects are a critical prerequisite in drug development. Especially predicting liver toxicity still presents a major challenge as animal models lack validity when compared to the human metabolism. Furthermore, state of the art in vitro liver cell cultures typically suffer from a rapid loss of liver specific functions.
To this end, we are developing a novel microfluidic test system capable of assembling primary human liver cells into cell aggregates
resembling the structure of liver sinusoids. If successful, we expect to maintain a liver specific metabolism in a long-term culture for
several weeks.
Hepatocytes and endothelial cells are assembled in the HepaChip into an organ like coculture by means of dielectrophoresis. As a particular advantage of this technology, dielectrophoresis enables separation and selective assembly of viable and plateable hepatocytes
from a suspension of cryopreserved cells. Therefore only the healthiest cells will be used for long term culture. In addition, an extra
cellular matrix (ECM) protein coating and an organ like perfusion of the artificial liver sinusoid support a stable phenotype of the hepatocytes, thus allowing for a long term culture in the microfluidic system.
We will present the design of the HepaChip which was optimized with respect to cell trapping rate using numerical simulation. On-chip
coassembly of hepatocytes and endothelial cells in a sinusoid like fashion will be demonstrated. We observed cell adhesion onto the
ECM coated surfaces and formation of characteristic hepatocyte morphology after several hours of cultivation. Results obtained from
cell cultures in the HepaChip over a period of several days will also be presented.
P131:
A Clinical Trial for Therapeutic Drug Monitoring Using Microfludic Devices
Manabu Tokeshi, Tomoya Tachi, Tetsunari Hase, Yukihiro Okamoto, Noritada Kaji, Takeshi Arima, Hiroyuki Matsumoto,
Masashi Kondo, Yoshinori Hasegawa, Yoshinobu Baba
Nagoya University
Background: Microchip analysis is a promising method for therapeutic drug monitoring (TDM). We evaluated a microchip-based fluorescence polarization immunoassay (FPIA) system for point-of-care testing on patients treated by theophylline.
Methods: We constructed a microfluidic FPIA system composed of a newly designed microchip, a laser, a CCD camera and an optical
microscope which had two rotatable polarizers. Theophylline-containing serum and fluorescence-labeled theophylline solution were
introduced through different inlets and combined in a 100 μm-wide microchannel where anti-theophylline antibody was added. The
sera were collected from 20 patients treated by theophylline. Fluorescence polarization was measured on the microchip and theophylline concentrations in serum were obtained. And regression analysis of the correlations was done between the results given by
the microchip-based FPIA and the conventional cloned enzyme donor immunoassay (CEDIA), and between the results given by the
microchip-based FPIA and the conventional particle-enhanced turbidimetric inhibition immunoassay (PETINIA).
Results: We successfully carried out a quantitative analysis of theophylline in serum near the therapeutic range in 65 s. The results
obtained by the microchip-based FPIA correlated well with the CEDIA and the PETINIA; the correlation coefficients (R2) were 0.986 and
0.989, respectively.
Conclusions: The microchip-based FPIA system is a simple and rapid method for point-of-care testing of drugs in serum, and this system
is precise the same as the conventional CEDIA and PETINIA.
126
Poster Abstracts
P132:
Development of a rapid microchip-based electrophoretic immunoassay for the determination of secretory immunoglobulin A in
human saliva
Yoshihide Tanaka, Nahoko Naruishi, Hidenori Nagai, Shin-ichi Wakida
Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST)
The annual number of suicides exceeded thirty thousand for the last twelve consecutive years in Japan. An objective and quantitative
method for stress measurement will be contributed to the prevention of some suicides. Hence, we have developed a rapid point-of-care
testing platform for stress measurement based on microfluidic chip and electrophoretic separation technique. Microchip-based capillary
electrophoresis (MCE) is a promising technique for on-site measurements and has advantages over conventional immunoassay methods
in fast analysis times, high separation efficiency, reduced cost, disposability and downsizing of analytical system. As a diagnostic fluid,
saliva offers distinctive advantages over blood because the collection is easy, stress-free and non-invasive. Secretory immunoglobulin
A (sIgA) in saliva has been suggested to be a potential marker of chronic, long-term stress due to suppression of the immune system.
This presentation includes applications of sIgA measurements in human saliva using a poly(methyl methacrylate) (PMMA) chip and our
developed MCE system. Blocking proteins designed for enzyme-linked immunosorbent assay applications (e.g., Block Ace and ImmunoBlock) were used as dynamic coating agents for both the manipulation of electroosmotic flow (EOF) and the minimization of sample
adsorption to PMMA surface1. The proposed microchip design has a round-bottom reservoir to reduce an on-chip immunoreaction time
within 3 min. After equibrium immunoreaction of FITC-conjugated antibody and sIgA (antigen), CE separation of immune complex from
unbound antibody was performed. The analytical method for sIgA determination is rapid compared with a conventional immunoassay
method, and provides an acceptable degree of accuracy and precision.
1 N. Naruishi, Y. Tanaka, T. Higashi, S. Wakida, J. Chromatogr. A, 1130 (2006) 169-174
P133:
Development of an enzyme immunoassay system for measuring stress biomarkers using disk-shaped microfluidic device
Nahoko Naruishi1, Yoshihide Tanaka1, Hidenori Nagai1, Wakana Iguchi1, Kazuhiro Oguchi5, Daisuke Niwa5
1 Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST)
5 Nano Bionics R&D Center, ROHM Co., Ltd.
Psychological stress is of major importance for our health care in recent years, and may lead to mental disorder, various diseases and
loss of quality of life. Because early detection and management of mental disorders are effective for prevention of depression and
suicide, development of portable analyzer for objectively assessing stress is highly desired . Cathecholamines, cortisol, amylase and
chromogranin A are well-known for acute psycological stress biomarkers, however the relationship between these biomarkers and human chronic stresses/daily mental states is not fully established. Among salivary stress-related biomakers, secretory immunoglobulin A
(sIgA) may be a potential one for the assessment of chronic psychological stress. Hence, we are trying to develop a rapid and easy-to-use
system for sIgA determination using an integrated microfluidics platform.
Enzyme-linked immunosorbent assay (ELISA) has been a major method used for the determination of clinical biomarkers. However,
ELISA requires long incubation times, complicated operations, and is a costly and time-consuming method. In our previous research,
a disk-shaped microfluidic device (Lab-on-a-Disk) was developed to allow a rapid immunoassay for the determination of sIgA1. A single
glass bead with immobilized sIgA on its surface was put in each immunoreaction reservoir, then centrifugal force was used to drive the
sample fluids in the reservoirs through reservoir-connected microchannels. After the competitive immunoassay, it utilized enzymatic
oxidation of 10-acetyl-3,7-dihydroxyphenoxazine by horseradish peroxidase to the fluorescent resorufin. In this study, we fabricated
a Lab-on-a-Disk integrated both accurate dispensing of small volume liquid samples and some immunoassay processes (e.g., antigenantibody reaction, bound/free separation, detection). Besides the microchannel dimensions, the hydrophobility or hydrophilicity of
the microchannel surfaces with dynamic coating was investidated on the control of microchannel flow driven by the centrifugal force.
Lab-on-a-Disk has a potential to replace for the conventional ELISA methods, and we will continue to develop a portable analyzer for
point-of-care testing.
1 H. Nagai, Y. Narita, M. Ohtaki, K. Saito, S. Wakida, Anal. Sci.,23, 975-979 (2007)
127
Poster Abstracts
P134:
Total serum IgE quantitation by microfluidic-ELISA using magnetic beads
Gaëlle Proczek*1, Jean-Marc Busnel1, Joël S. Rossier3, Niels Lion4, Hélène Sénéchal5, Hubert H. Girault1
1 Laboratoire d‘Electrochimie Physique Analytique (LEPA), EPFL, Lausanne, Switzerland
3 DiagnoSwiss S.A., Monthey, Switzerland
4 Laboratoire d‘Electrochimie Physique Analytique (LEPA), EPFL, Lausanne, Switzerland; Service Régional Vaudois de Transfusion Sanguine, Lausanne, Switzerland
5 Laboratoire Environnement et Chimie Analytique (LECA), ESPCI, Paris, France
Nowadays, allergy represents a major health issue, affecting 10-20% of the population worldwide. Therefore, fast and simple in vivo and in
vitro methods for allergy diagnosis are required. Allergy diagnosis is generally based on immunoglobulin E (IgE) quantitation. Typical total
IgE concentration in a healthy adult is about 90 kIU/mL (1 IU = 2.4 ng) whereas higher levels usually reflect the atopy of the patients. Total
serum IgE quantitation is generally achieved by enzyme-linked immunosorbent assay (ELISA) related techniques. Classical ELISA methodologies in microtiterplate and the commercially available immunoCAP system (Phadia) are mostly used. Although these in vitro assays
provide suitable sensitivities with low detection limits, they are time and sample/reagent consuming. In this context, we have developed
a miniaturized sandwich-type ELISA using the GRAVI-Cell platform (DiagnoSwiss, Monthey, Switzerland) for total IgE quantitation. This
technology combines the advantages of microfluidic, gravity force-driven fluidic, immunocapture on magnetic beads and electrochemical
detection. The platform consists of a polyimide microchip, which contains eight parallel and independent microchannels incorporating
microelectrodes for electrochemical detection, and an instrument allowing to maintain the microchip in a tilted position for the gravitydriven fluidics. The device also includes an array of eight magnets to trap the beads in the channels. In our sandwich-type immunoassay,
sample, labeled antibody and magnetic beads functionalized with the capture antibody were preincubated off-line. Then, the mixture was
percolated through the channels where the magnetic beads were trapped. Finally, after a washing step, the substrate for enzyme-mediated
generation of electrochemical substance was introduced and the electrochemical detection was performed in parallel within the eight
microchannels.
In this work, the most appropriate conditions for the IgE quantitation assay have been determined and a highly linear response was achieved (IgE concentration ranging from 47 to 3000 ng/mL). A special attention has also been given to the decrease of non-specific interactions
by the development of a proper blocking solution. After validation with a reference material, the assay has been successfully applied to
the quantitation of total IgE in a patient serum sample. The result was in accordance with those obtained by the standard method (ImmunoCAP). Less than 1.5 µL of serum was required and the whole assay was performed in less than 1 hour, the calibration curve and total IgE
quantitation in serum sample being carried out simultaneously thanks to the eight parallel microchannels on the same chip.
P135:
Improvement in Preparation of Magnetic Immunosorbent for Amyloid beta, Alzheimer’s Disease Marker
Zuzana Svobodova*1, Barbora Jankovicova1, Zdenek Plichta3, Daniel Horak3, Zuzana Bilkova1
1 Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
3 Institute of Macromolecular Chemistry, AS CR, Prague, Czech Republic
In this preliminary work, method for improved oriented immobilization of antibodies (Ab) on magnetic microparticles is presented. Alzheimer's disease (AD) markers, 5 Amyloid beta peptide isoforms, occur in CSF of AD patients in low concentration, approximately ng/mL1. The
aim of the study was to enhance efficiency of antigen capturing, preparation of immunosorbent with higher binding capacity. First goal
was to increase number of site-groups on commercially available particles with hydrazide groups. Method for implementation of hydrazide
groups on beads by introduction of adipic acid dihydrazide to carboxy-functionalized beads was performed. Conventional method TNBS
test2 for determination of hydrazide groups was optimized for lower amount of beads (1 mg) which was connected with lower consumption
of toxic component as TNBS and methanol. Also alternative method, BCA test, was optimized and used for the determination of hydrazide
groups3. On such prepared beads human immunoglobulin G (Hu IgG) was immobilized at first. This process is one way how to increase the
number of captured Amyloid beta peptides in microfluidic device where the amount of implemented magnetic beads is limited.
This work was supported by the E.C. project NeuroTAS No. 037953, the Ministry of Education of Czech Republic (MSMT 0021627502) and
by Czech Science Foundation (GA203/08/1536).
128
Poster Abstracts
1 J. Wiltfang, H. Esselmann, M. Bibl et al. Highly conserved and disease-specific patterns of carboxyterminally truncated Aß peptides 1-37/38/39 in addition to 1-40/42
in Alzheimer's disease and in patients with chronic neuroinflammation. Journal of Neurochemistry. 81 (3) (2002) 481-496.
2 G. Antoni, R. Presentini and P. Neri. A Simple method for the Estimation of Amino Groups on Insoluble Matrix Beads. Analytical Biochemistry. 129 (1993) 60-63.
3 P. E. Tylianakis, S. E. Kakabakos, G. P. Evangelatos and D.S. Ithakissios. Direct Colometric Determination of Solid-Supported Functional Groups and Ligands Using
Bicinchoninic Acid. Analytical Chemistry 219 (1994) 335-340. Tuesday, March 23rd, 2010
P136:
Microfluidic-Chip Capillary Electrophoresis for Analysis of Clinical Urinary Proteins
Ruige Wu1, Ying Sing Fung1, W. S. B. Yeung3
1 Department of Chemistry, The University of Hong Kong
3 Department of Obstetrics and Gynaecology, The University of Hong Kong
Microfluidic-Chip Capillary Electrophoresis for Analysis of Clinical Urinary Proteins Excretion of urinary proteins is one of the most
common abnomalities which reflects pathologic conditions affecting the kidney and urinary tract. But high concentration of salts and
metabolic wastes and low concentration of proteins in urine have made it essential to pretreat urine samples with some methods like
ultrafiltration, organic solvent precipitation and dialysis, which usually cause new problems like protein loss and a time-consuming
process. In order to solve such problems, a device coupling the poly (methyl methacrylate) (PMMA) microfluidic chip to capillary electrophoresis is developed using on-chip isotachophoresis (ITP) to realize sample preconcentration and sample clean-up. Standard protein
mixtures including IgG, BSA, α1-Antitrypsin and transferrin in deionized water and 100mM NaCl were injected into the chip-capillary
system to test the performance of preconcentrating and desalting respectively. After removing non-protein contaminants into waste
vial, stacked proteins were then transferred into an embedded capillary for CZE separation with UV-Visible detection at 214 nm. The
enrichment factor obtained in this system developed could reach more than 40, and the whole analysis could be finished in less than 8
minutes. Results on the applicability of the microfluidic-chip capillary electrophoresis device for the analysis of proteins in urine sample
will be presented and discussed at the meeting.
P137:
Highly Sensitive Electrophoretic Analyses of Sugar Chains on Straight Channel Microchip. 2
Takayuki Kawai, Kenji Sueyoshi, Fumihiko Kitagawa, Koji Otsuka
Department of Material Chemistry, Graduate Schoold of Engineering, Kyoto University
Recently, the rapid analysis of sugar chains using microchip electrophoresis (MCE) has attracted much attention as researches in glycomics have been intensively in progress. However, the conventional MCE analysis with the pinched injection (PI) has disadvantages of
the low sensitivity and the complicated voltage control for the sample injection. To overcome these drawbacks, we applied an on-line
preconcentration method, large volume sample stacking with electroosmotic flow pump (LVSEP)1, to MCE. In the LVSEP–MCE analysis,
a straight microchannel is filled with a sample solution, and then just a constant voltage is applied to reservoirs, resulting in both the
sensitivity enhancement and the simplification of sample injection. In this study, the simple, rapid, and highly sensitive LVSEP–MCE
analysis of sugar chains was investigated.
A poly(dimethyl siloxane) microchip was used for the MCE experiment, where the channel length, depth, and width were 80 mm, 50
μm, and 50 μm, respectively. To suppress the electroosmotic flow (EOF) and sample adsorption, the surface of the channel was coated
with poly(vinyl alcohol). A 25 mM HEPES buffer solution (pH 8.0) was employed as the background solution (BGS). The channel was
filled with water-diluted sugar chain sample obtained from bovine ribonuclease B and the constant voltage of 4.0 kV was then applied
to enrich and separate the sample. Laser-induced fluorescence detection was performed at the excitation/emission wavelengths of 488
/520 nm, respectively.
In the LVSEP analysis, the EOF-suppressed channel is filled with a low ionic strength solution containing anionic analytes. The sample is
concentrated around the anode-side sample/BGS boundary according to the difference in the electric field strength between the two
zones. The focused sample in the BGS and the sample matrix without analytes move toward the cathode by the temporally enhanced
EOF due to the low ionic strength of the solution. After the sample matrix is removed out of the channel, the EOF is suppressed again
and the electric field in the BGS zone is drastically increased. The analytes then start moving toward the anode and are separated by
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Poster Abstracts
microchip zone electrophoresis (MCZE). The LVSEP–MCZE analysis of the real sugar chains was performed on the PDMS straight channel
microchip. As a result, the sample injection procedure could be simplified, and the analytes were well enriched and separated with a
2200-fold sensitivity increase compared to the conventional PI–MCZE analysis. Relative standard deviations of the peak height and the
detection time calibrated with an internal standard were 7.2% and 0.6% (n = 3), respectively.
1 He, Y.; Lee, H. K. Anal. Chem. 1999, 71, 995-1001.
P138:
Development of a Microchip-based Fully Automated Immunoassay System ‘μELISA’
Tomohiko Ebata*1, Toshinori Oohashi1, Osamu Fukahori1, Hidekatsu Tazawa1, Aya Harano1, Kazuma Mawatari6, Takehiko Kitamori7
1 Institute of Microchemical Technology Co. Ltd
6 University of Tokyo
Enzyme-linked immunosorbent assay (ELISA) is widely used for quantitative assessments of cytokines, clinical diagnostic markers, food
allergens, and hormones in various biological samples. ELISA is a highly selective and sensitive method that uses antigen-antibody and
enzyme reactions for quantification. However, this method is usually expensive and time-consuming (analysis time: approximately 3–4
h) and requires large volumes of reagents and samples as well as complex liquid-handling procedures on microtiter plates.
In our previous studies, we had developed a microchip-based ELISA technique. In this technique, the antigen-antibody and enzyme
reactions were performed on microbeads trapped in a microchannel with a dam structure, and the dye molecules of the enzymatic
products were detected downstream of the dam by using a thermal lens detector (TLD) probe. In comparison with conventional ELISA,
the microchip ELISA was faster (analysis time: 10–20 min) and required only a few microliters of the sample. However, in this method,
the samples and reagents had to be manually introduced into the microchip.
To facilitate microchip-based ELISA, we developed a fully automated immunoassay system called ‘μELISA’, which is based on the principles of microfluidics and employs a highly sensitive TLD probe.
This system contains a micro-autosampler, a syringe pump, microvalves, a microchip with a dam structured microchannel, a TLD probe,
and a controller board. The original PC software controls these devices and analyse the TLD signals. All the microchip-level procedures,
from bead introduction to TLD detection and bead removal, are automated and can be performed repeatedly. All the components were
integrated into a portable unit (W × D × H: 253 mm × 200 mm × 222 mm).
We used the μELISA approach to measure the levels of human C-reactive protein (hCRP), which is a useful diagnostic marker for both
inflammatory reactions and cardiac infarction. Using this approach, we could reproducibly detect CRP within 10 min by using a sample
volume of only 2 μL.
The quantitative limit for CRP detection with this system was 20 ng/mL, which was lower than the cutoff value for high-sensitivity
detection of CRP (1000 ng/mL). These results suggest that μELISA can be used for high-sensitivity CRP measurement in small amounts
of samples.
Furthermore, this system can be used for various other assays by obtaining the relevant antibodies; the method requires minimal
amounts of reagents and has very low running costs. μELISA can be used for the detection of various biomolecules in small amounts
of samples; therefore, this approach can be used for point-of-care testing, environmental analyses, food evaluation, and drug screening.
In addition, the fluidics-based control system used in μELISA can be employed for other microfluidics systems, e.g. in reactors; in systems for elution, separation, and analysis; and in integrated systems.
P139:
Single Cell Patterning in Semi-closed Microchannel Using Photoactivation
Kihoon Jang, Yan Xu, Yo Tanaka, Takehiko Kitamori
The University of Tokyo
Recently, interest in single cell analysis has been increased, because conventional method usually averages the property of cells, which
cannot represent the individual cell completely. Thereby, single cell analysis became the focus of the frontier in cell biological research.
Microfluidic techniques can demonstrate the feasibility of integration of various process steps involved in single cell analysis, such as
separating, positioning and detecting. To realize these steps in microfliudic system, the positioning of single cell is indispensible before
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Poster Abstracts
analysis and detection. In here, we have developed single cell patterning method directly inside the semi-closed microchannel using
photochemical reaction, which was very difficult in microchannel.
We developed a UV-sensitive surface modification method, in which an exposure to UV changes from hydrophilic to moderately hydrophobic cell adhering surface. The technique is based on a combining 2-methacryloyloxyethyl phosphorylcholine (MPC) containing
polymer which is known as a resistant barrier for cell adherence and nitrobenzyl group photolabile linker for selective cell patterning.
Experimental procedures were as follows: the cleaned microchannel (width: 400 mm, depth: 100 mm) was treated with 3-aminopropyl–triethoxysilane (APTS), and then nitrobenzyl group containing photolabile linker was introduced and reacted for 2 h. Next, MPC was
grafted by EDC coupling for 12 h, which made non-fouling surface. UV (365 nm) irradiation induced cleaving reaction of MPC which
made the surface moderately hydrophobic, cells attached to the UV illuminated area.
For cell immobilization, round-shaped photo-masks with diameter 10 mm, 20 mm, 30 mm, 50 mm were used. UV was illuminated
through the photo-mask respectively, and the endothelial cell suspension was introduced. After 2 h of incubation, the ratio of attached
cells were 3.3 %, 80.0 %, 50.0 % and 86.7 % in each condition respectively. After 10 h of culture in flow condition, the percentage of
immobilized cells was 0.0 %, 46.7 %, 36.7 % and 66.7 % respectively. To confirm the adhered cells were single cell or not, nuclear was
stained using DAPI for 2 min followed by rinsing with PBS. The stained cells were observed under a fluorescence microscope. Only in
the case of photo-mask with 20 mm and 30 mm, the single cells were observed, the ratio of the single cell in each patterned-cells were
36 %, 8 %, respectively. The single cells were modified inside the semi-closed microchannel directly using photochemical activation, even
in the microfluidic system. This technique can be applied to single cell operation and techniques for fundamental cell-based studies that analyze cell signaling and
detect two different types of individual cell pairs.
P140:
AC Electrokinetic Transport and Selective Poration of Bacteria on Microfluidic Devices
Graham S. Erwin, Michelle L. Kovarik, Stephen C. Jacobson
Indiana University Bloomington
The manipulation and characterization of cells on microfluidic devices have multiple benefits including precise control of cell samples
and reagents, increased assay sensitivity, and the ability to track movement of individual cells. Often, electrokinetic transport is preferred over pressure-driven transport on microfluidic devices because electrokinetic transport is easily miniaturized, uses no moving parts,
and generates a flat flow profile. However, one concern with the electrokinetic transport of cells is that cell electroporation or lysis may
occur. Here, we demonstrate that AC waveforms with a DC offset can be used to transport Escherichia coli efficiently in microfluidic
devices without inducing cell electroporation. First, we evaluated the electrokinetic mobility in the device and found that mobility is
independent of frequency. Next, cell electroporation data were gathered for peak voltages from 0.1 to 1.5 kV/cm and frequencies from
1 Hz to 80 kHz. Membrane-permeable SYTO 9 and membrane-impermeable propidium iodide were used to assess cell viability. Cells
fluoresced green when viable and both green and red when the membrane was compromised. The ratio of electroporated to nonelectroporated cells increased with increasing voltage and decreasing frequency. Further, we determined the maximum voltage and
minimum frequency that can be applied to E. coli before electroporation occurs.
P141:
Electrophoretic mobility measurements of fluorescent dyes via on-chip capillary electrophoresis
Denitsa Milanova*, Robert Chambers, Juan Santiago
Stanford University
We present an experimental study of electrophoretic mobility measurements for fluorescein (FL), rhodamine 6G (R6G), and Alexa Fluor
488 (AF 488) dyes via on-chip capillary electrophoresis. Accurate mobilities are crucial to designing electrokinetic experiments and to
quantifying the mobilities of other analytes. We review analytical expressions for effective mobility as a function of dissociation constant, ionic concentration, and electroosmotic flow (EOF). EOF suppression is important for achieving resolution and repeatability. We
describe the electromigration of these fluorophores as a function of ionic strength, for various pH, and for various concentrations of
PVP (polyvinylpyrrolidone), a polymer commonly used in EOF suppression. The latter influences the EOF velocity and is a crucial factor
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Poster Abstracts
in electrophoretic migration behavior of cationic R6G. We report fully ionized mobility values and analytical expressions for the effective
mobility of FL, R6G, and AF 488.
Analyte mobility increases as the degree of ionization increases, and mobility decreases with ionic strength, as expected. Furthermore,
the effect of ionic strength is more pronounced for the divalent FL than for univalent R6G, consistent with electrolyte mobility theory. Based on experimental data, we recommend that ionic strength should be at least 20 mM to prevent aggregation of the neutral marker
(rhodamine B dye). We point out that reduced wall adsorption is critical for clean and accurate separations. We further suppress EOF
to study adsorption-desorption behavior of cationic R6G dye with negatively charged channel walls. With the addition of 2% PVP at low
pH (5.2), EOF is reduced by more than 100´. For the same case, R6G is well behaved, and produces symmetric analyte peaks. We extrapolate data using electrolyte theory to obtain the fully ionized mobilities for R6G and AF 488 to be 13.73´10 ‑9 m2V-1s-1 and 35.98´0 ‑9
m2V-1s-1, corresponding to -1 and +1 valences, respectively. FL shows two limiting mobilities: 18.5´10 ‑9 m2V-1s-1 and 35.77´10 ‑9 m2V-1s-1,
corresponding to the -1 and -2 valence states.
P142:
Ion spray microchip
Jaroslav Pól1, Tiina Kauppila2, Sami Franssila3, Tapio Kotiaho2, Risto Kostiainen2
1 Faculty of Pharmacy, University of Helsinki, Finland and Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
2 Faculty of Pharmacy, University of Helsinki, Finland
3 Micro and Nanosciences Laboratory, Helsinki University of Technology, Espoo, Finland
We have previously reported nebulizer microchip that has been used as an ionization source for mass spectrometry in APCI1, APPI2, SSI 3
and APTSI4 modes and enabled coupling of capillary LC5 and GC6 to the MS. In this study, we have extended the application range of the
nebulizer microchip to ionic, polar drug molecules, and large biomolecules such as peptides and proteins by operating the microchip
in ion spray (IS) ionization mode.
The microchip nebulizer (10 mm wide and 18 mm long) consists of two plates. The lower silicon plate includes etched channel for
mixing liquid sample with nebulizer gas and a nozzle at the end for spraying the vaporized mixture. The upper plate is made of glass
and serves as a cover, which is bonded to the silicon wafer. Nebulizer gas is delivered through etched whole in the silicon wafer. Liquid
sample is introduced via fused silica capillary, which is glued to the rear end of the microchip. High voltage is directly applied to the
silicon wafer. Sample was delivered with a syringe pump in direct infusion experiments or with a capillary LC (Agilent 1100) with a C18
column. Flow rates employed were 0.5 – 10.0 mL/min. The MS used was API-300 PE Sciex.
The performance of the ion spray microchip was compared to a standard ESI in terms of flow rate and concentration characteristic
using direct infusion of verapamil 10 mM methanol/water solution. The optimum high voltage and nebulizer gas pressure were 4 kV
and 6 bar respectively. At flow rates of 5-10 mL/min the ion spray microchip showed comparable signal intensity to ESI. LC-MS analyses
of standard mixture of neurosteroids and triptic digest of BSA show comparable results with standard ESI.
1 Ostman, P., S. J. Marttila, T. Kotiaho, S. Franssila, and R. Kostiainen. 2004. Microchip atmospheric pressure chemical ionization source for mass spectrometry.
Analytical Chemistry 76: 6659-6664.
2 Kauppila, T. J., P. Ostman, S. Marttila, R. A. Ketola, T. Kotiaho, S. Franssila, and R. Kostiainen. 2004. Atmospheric pressure photoionization-mass spectrometry with
a microchip heated nebulizer. Analytical Chemistry 76: 6797-6801.
3 Pol, J., T. J. Kauppila, M. Haapala, V. Saarela, S. Franssila, R. A. Ketola, T. Kotiaho, and R. Kostiainen. 2007. Microchip sonic spray ionization. Analytical Chemistry 79:
3519-3523.
4 Keski-Rahkonen, P., M. Haapala, V. Saarela, S. Franssila, T. Kotiaho, R. Kostiainen, and S. Auriola. 2009. Atmospheric pressure thermospray ionization using a heated
microchip nebulizer. Rapid Communications in Mass Spectrometry 23: 3313-3322.
5 Ostman, P., S. Jantti, K. Grigoras, V. Saarela, R. A. Ketola, S. Franssila, T. Kotiaho, and R. Kostiainen. 2006. Capillary liquid chromatography-microchip atmospheric
pressure chemical ionization-mass spectrometry. Lab on a Chip 6: 948-953.
6 Ostman, P., L. Luosujarvi, M. Haapala, K. Grigoras, R. A. Ketola, T. Kotiaho, S. Franssila, and R. Kostiainen. 2006. Gas chromatography-microchip atmospheric pressure
chemical ionization-mass spectrometry. Analytical Chemistry 78: 3027-3031.
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Poster Abstracts
P143:
Magnet chain sleeve on a capillary for multiplug magnetic beads trapping: numerical simulations and visualizations
Anne-Laure Gassner1, Jacques Morandini2, Jacques Josserand1, Hubert H. Girault1
1 Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland
2 Astek Rhône-Alpes, 1 place du Verseau, 38130 Echirolles, France
Magnetic beads (MBs) have now proven to be a powerful tool in both research and biomedical applications. They are available in a wide
range of sizes and their surface can be modified with molecules having biological specificities and functions. In microfluidics, where the
goals are faster reaction/analysis time and reduced sample consumption, MBs offer many advantages. First of all, compared to an open
microchannel, a packed bed of beads increases the specific surface available for molecule adsorption/binding. The diffusion pathway is significantly reduced, improving interactions between molecules. Moreover, in comparison to classical beads, they can be easily manipulated
by magnets.
The present investigation presents a multiplug trapping system for MBs in a capillary. The capillary is inserted through a chain of cylindrical permanent magnets alternating with cylindrical non-magnetic spacers. The magnets and spacers are simply placed on the capillary
like pearls on a string. The magnets are indeed drilled along their magnetization axis, parallel to the capillary and can be placed either in
attraction or in repulsion. This system has the advantage of a very simple assembly. Moreover the axial symmetry and the proximity of
the magnets and capillary produce high magnetic forces, giving the opportunity to work at higher flow velocities than with other setups
classically made up by two magnets spaced by 1 mm with their magnetization perpendicular to the capillary. Finally only the length of the
capillary theoretically limits the length of the magnets chain, and it is possible to adapt the number of magnets to the desired number of
plugs, increasing in a controllable manner the surface available for protein adsorption in the case of immunoassays or immunoextraction.
Using numerical simulation we have mapped the magnetic force for different geometries. This force was then introduced into a convection-diffusion model to understand the formation of the multiplugs and the influence of the flow velocity on their size and position. Finally
as a proof of concept, the accumulation of MBs was visualized by microscopy in a capillary placed between rectangular magnets having
a magnetization parallel to the capillary.
P144:
Fluorescent Detection of Adenosine Using Gold Nanoparticles and Two Aptamers
Shih-Ju Chen1, Chih-Ching Huang2, Huan-Tsung Chang1
1 National Taiwan University
2 Institute of Bioscience and Biotechnology, National Taiwan Ocean University
In this study, we have developed a simple, cost-effective, label-free fluorescence analytical assay—comprising an adenosine-binding aptamer
(AptAdo), platelet-derived growth factor (PDGF)-binding aptamer (AptPDGF), gold nanoparticles (Au NPs), and the DNA-binding dye Oligreen
(OG)—for the determination of adenosine. AptAdo and AptPDGF are for the recognition of adenosine and for the amplification of fluorescence
signal, respectively. The presence of adenosine induces the conformational switch of the Apt Ado from coiled to a G-quadruplex structure,
leading to the less binding of AptAdo onto the surface of Au NPs. The more the adenosine is present, the less the amount of Apt Ado is adsorbed, resulting in the fluorescence change of the aptamer–OG complexes. When using a mixture of Apt Ado (15.0 nM), Au NPs (0.1 nM), and
OG (0.05 X) in 5.0 mM phosphate (pH 7.4), this sensor provides the limit of detection of 70.0 nM for adenosine at a signal-to-noise ratio of 3.
The LOD for adenosine is down to 5.5 nM when using AptAdo modified Au NPs (AptAdo-Au NPs) and AptPDGF for the enrichment of adenosine
and amplification of fluorescence signal of OG, respectively. The practicality of the present sensor has been validated by the determination
of adenosine in diluted urine samples, showing its advantages of simplicity, selectivity, sensitivity, and minimal matrix interference.
P145:
Robotic microseparation of metallothionein and alpha-methylacyl CoA racemase by use of paramagnetic micro- and nanoparticles
Michal Masarik*1, Jaromir Gumulec1, Jana Chomoutska3, Sarka Kuchtickova1, Roman Hrabec5, Arne Rovny5, Petr Babula7,
Jaromir Hubalek3, Rene Kizek9
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Poster Abstracts
1 Department of pathological physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech republic
3 Department of Microelectronics, Brno University of Technology, Udolni 53, CZ-602 00 Brno, Czech Republic
5 Department of Urology, St. Anne´s University Hospital, Pekarska 53, CZ-65691, Brno
7 Department of Natural Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3, CZ-612 42 Brno, Czech Republic
9 Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic
In many developed countries, prostate cancer (CaP) is the second leading cause of cancer-related deaths among men. Therefore, having
reliable and easy-to-detect marker is needed to treat this disease successfully. Metallothioneins (MT) are a group of proteins rich in
cysteine with molecular weights ranging from 6 to 10 kDa. Their main physiological functions are homeostatic control and detoxification of the metals. The role of MT in tumour tissue remains still unclear, but MT can be considered as new promising tumour marker.
Besides MT, alpha-methylacyl CoA racemase (AMACR) has been proven to be one of the few biomarkers that can help distinguish malignant from benign tumours with high sensitivity and specificity for prostate carcinoma. Four human prostatic cell lines (PNT1A, PC-3,
22RVL, and LNCaP) were used in this study. Total cellular proteins from the experimental samples were extracted by RIPA buffer with
protease inhibitor or by homogenization and heat denaturation. This study provides important new information about expression of MT
and AMACR in prostate tumor cell lines and about amount of MT in human serum samples of patients with CaP and comparison and
selection of suitable separation methods. For this purpose, magnetisable microparticles with modified surface binding antibody to MT
(monoclonal mouse antibody against MT 1 and MT 2) or AMACR (polyclonal rabbit antibody against AMACR) were used. In this study,
also magnetisable nanoparticles modified by antibodies to MT and AMACR were characterized. Microparticles modified by antibody
were incubated for strictly determined time period (10, 20, 30, 60, and 120 min) with MT or AMACR in volume of 5 µl, eventually 10 µl.
Subsequently, microparticles were rinsed by the help of stationary magnet and bounded analyte was released by addition of 100 mM
acetate buffer, pH 3.5. MT and AMACR were subsequently electrochemically analyzed. Recovery factor of above described microseparation process was about 25 % for MT and 12 % for AMACR. Manual separation procedure was modified for fully robotized preparation of
modification of microparticles and nanoparticles by antibodies and isolation of analytes by the use of robotic system EP Motion Blue.
For verification of isolated proteins, 12.5 % SDS-polyacrylamide gels for protein separation and capillary electrophoresis on chip were
used. Subsequent detection by western blotting was performed by antibody against MT and/or AMACR. We found out that expression
of MT is significantly down regulated in 22RVL and PC-3 cells due to significant lower zinc concentration in consequence with prostate
carcinoma cells. The results suggest MT and AMACR as candidate tumor markers for diagnosis of prostate cancer and indicate possible
application of electrochemical methods.
Financial support from GACR 301/09/P436 and NS10200-3 is greatly acknowledged.
P146:
High Efficient Affinity Enrichment of Epitestosterone from Complex Matrix Samples Based on Gold-Coated Magnetic Nanoparticles
Shuang Qui, Li Xu, Yi-Ran Cui, Qin-Pei Deng, Wei Wang, Hong-Xu Hong, Xin-Xiang Zhang
College of Chemistry,Peking University
A solid-phase immunoextraction method based on gold-coated magnetic nanoparticles (MNPs) has been developed for the quantitative
analysis of epitestosterone (ET) in urine by high-performance liquid chromatography (HPLC). Half-IgG of anti-ET monoclonal antibodies
were covalently immobilized onto (Fe3O4)core-Aushell (Fe3O4@Au) MNPs. The MNPs were used in the pseudo-homogeneous extraction
of ET in spiked human urine samples. An external magnetic field was applied to collect the MNPs which were then rinsed with distilled
water followed by elution with absolute methanol to obtain ET as the analyte. The obtained extraction solution was analyzed by HPLC
with UV detection (244 nm) within 12 min. The standard calibration curve for ET showed good linearity in the range of 20-200 ng/mL
in phosphate-buffered saline (PBS) solutions with acceptable accuracy and precision. An enrichment factor of 100-fold was achieved to
fulfill the need for measuring analyte at low concentration levels. The results obtained by the present method for spiked urine samples
were in agreement with those from indirect competitive enzyme-linked immunoadsorbent assays (ELISAs). The approach described
here was highly specific, fast, easy to operate, and is likely to have the advantage of conventional immunoaffinity column technique.
This work was supported by National Natural Science Foundation of China (No. 90713013) and the National Scientific Support Project
2006BAF07B03 and 2009CB320305 (MOST, China).
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Poster Abstracts
P147:
Investigation into the usefulness of carbon nanotubes in on-line enzymatic incubations by capillary electrophoresis
Jochen Pauwels, Jos Hoogmartens, Ann Van Schepdael
K.U.Leuven, Belgium
During the development of new drugs, it is essential to investigate the extent to which these new molecules are metabolized by enzymes such as the cytochrome P450 (CYP) oxygenases. Since a tendency exists to perform this screening in the early stages of drug
discovery, the number of molecules to be investigated is high. In view of this substantial number of candidate drugs, it is advantageous to develop automated analytical methods, which are also miniaturized so that a limited amount of compound is needed. There
is interest in the development of in-capillary incubations of substrates with CYP enzymes by the technique called Electrophoretically
Mediated MicroAnalysis, introduced by Bao and Regnier. However, during these incubations, the capillary wall constantly changes due
to adsorption of protein material from the enzyme, which causes the migration times to gradually increase. Because of the ability of
carbon nanotubes to interact with proteinaceous material, the aim was to investigate whether the repeatability of migration times
could be improved with the use of carbon nanotubes. As a model system the combination of the isoenzyme CYP3A4 with verapamil
substrate was chosen. The enzymatic reaction product of N-demethylation is norverapamil. Different surfactants were first studied
for their potential to disperse carbon nanotubes in an aqueous environment. The possible negative effect of these surfactants on the
enzymatic yield was also verified. The most suitable surfactant was selected in order to perform in-capillary incubations. Here, several
parameters can be varied to obtain the highest yield of norverapamil, such as the plug length, the incubation temperature, the duration of in-capillary incubation, and the composition of the incubation environment inside the capillary. On the other hand, it has been
described that carbon nanotubes are able to enhance the enzymatic turnover of, for example, glucose oxidase. Therefore, it is useful to
comment on this characteristic in the frame of CYP monooxygenases. J. Bao, F. E. Regnier: Ultramicro enzyme assays in a capillary electrophoretic system. J. Chromatogr. 1992, 608: 217-224
P148:
The Application of Cyclodextrin Modified Gold Nanoparticles for Chiral Separations in CEC
Pavel Řezanka, Klára Navrátilová, Pavel Žvátora, David Sýkora, Vladimír Král
Institute of Chemical Technology Prague, Dept. of Analytical Chemistry, Technická 5, Prague 6, 166 28, Czech Republic
Gold nanoparticles applicable in separation techniques (ref. 1) can be prepared by several means. The most frequent approaches are:
first, citrate reduction of aqueous solution of a gold(III) salt; second, borohydride reduction of aqueous solution of a gold(III) salt; and
third, two phase (water-toluene) reduction using borohydride as reducting agent and tetraoctylammonium bromide as transfer agent of
a gold(III) salt. Each method provides different concentration and size of the generated nanoparticles as well as the different potential
for their subsequent modification (ref. 2). Many separation applications are related to the open tubular capillary electrochromatography
(OTCEC) where the capillary walls have been coated with gold nanoparticles (ref. 2). The immobilization of the gold nanoparticles onto
the inner surface of a fused-silica capillary can be carried out applying either noncovalent (ref. 3) or covalent (ref. 4) approach.
The proper functionality of many biological compounds stems from their chirality. The chirality plays a crucial role in living systems. Thus, enantioselective separations represent a very important branch of the separation science. Surprisingly, to the best of our
knowledge, there are only two papers dealing with the use of nanoparticles (polystyrene) to enhance enantiomeric separation by CE
(ref. 5,6).
In this work gold nanoparticles were prepared by citrate reduction of a gold(III) salt. The resulting nanoparticles were characterized by
absorption spectroscopy and transmission electron spectroscopy. Fused-silica capillaries were pre-derivatized by (3-mercaptopropyl)
trimethoxysilane that allows for a subsequent covalent modification of the capillary walls with the synthesized gold nanoparticles followed by the modification of the immobilized gold nanoparticles with thio derivatives of b-cyclodextrin. The obtained capillaries were
used to separate chiral aromatic compounds (e.g., binaphtol acids and Tröger bases). The effects of the capillary preparation method,
concentration of the immobilized nanoparticles, pH and the type of a running buffer as well as other factors are discussed.
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Poster Abstracts
Financial support from The Ministry of Education, Youth and Sport of the Czech Republic, no. MSMT6046137307, and from The Czech
Science Foundation, no. 203/09/0675, are gratefully acknowledged.
1 P. Řezanka, K. Záruba, V. Král, Chem. Listy, 101 (2007) 881-885
2 C. Nilsson, S. Birnbaum, S. Nilsson, J. Chrom. A, 1168 (2007) 212–224
3 W. Wang, L. Zhao, F. Zhou, J.-J. Zhu, J.-R. Zhang, Talanta, 73 (2007) 534–539
4 L. Yang, E. Guihen, J. D. Glennon, J. Sep. Sci., 28 (2005) 757-766
5 N. Na, Y. P. Hu, J. Ouyang, W. R. G. Baeyens, J.R. Delanghe, T. De Beer, Analytica Chimica Acta, 527 (2004) 139-147.
6 N. Na, Y. P. Hu, J. Ouyang, W. R. G. Baeyens, J.R. Delanghe, Y.E.C. Taes, M. Xie, H. Chen, Y. Yang, Talanta, 69 (2006) 866-872
P149:
Interaction between PEGylated poly(alkyl cyanoacrylate) nanoparticles and amyloid peptide highlighted by Capillary Electrophoresis
Romain R. Verpillot1, Davide D. Brambilla2, Karine K. Andrieux2, Benjamin B. Le Droumaguet 2, Julien J. Nicolas2, Matteo M. Canovi6,
Marco M. Gobbi7, Mario M. Salmona 8, Valérie V. Nicolas9, Patrick P. Couvreur2,1 Myriam M. Taverna
1 Univ Paris-Sud 11, UMR CNRS 8612, LPNSS, Faculté de Pharmacie, 92296 Châtenay-Malabry, France
2 Univ Paris-sud 11, UMR CNRS 8612, Faculté de Pharmacie, 92296 Chatenay-Malabry, France
6 Istituto Ricerche Farmacologiche “Mario Negri” Milano, Italy
9 Univ Paris-Sud 11, IFR 141, microscopy plateform. Faculté de Pharmacie, 92296 Châtenay-Malabry, France
Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive functions and specific pathological changes in the brain, evidenced by the formation of extracellular ß-amyloid (Aß) peptide aggregates (or fibrillar plaques) and
tangles of hyperphosphorylated Tau protein inside neurons. Aß peptide aggregates are widely regarded as the main cause of neuronal
cell degeneration and oligomeric forms are considered as neurotoxic. By adapting a methodology published by Sabella et al. in 2004,
we propose here a capillary electrophoresis (CE) method able to monitor the different states (monomeric and oligomeric species) of an
in vitro aggregation process of the Aß amyloid 1-42 as a function of time. The ability of nanoparticles (NPs) to slow down or disrupt
the aggregation process has been investigated through kinetic studies performed on mixtures containing the Aß 1-42 peptide and
biodegradable NPs generally developed for drug delivery in the field of therapeutic strategies for cancer treatment. The investigated
PEGylated poly[(hexadecyl cyanoacrylate)-co-methoxypoly(ethylene glycol) cyanoacrylate] (PEG-PHDCA) NPs have not only a relatively
high in vivo long circulation in blood but also the ability to overpass the blood brain barrier (Calvo et al. 2001, Brigger et al. 2002).
Assignment of the peaks in the CE profile was achieved by performing identical experiments but on a CE-LIF system and using rhodamine B-tagged nanoparticles (Brambilla et al. 2009) or FITC labeled Aß 1-42 peptide. Surface plasmon resonance was used to check the
affinity between PEG-PHDCA NPs and Aß peptide 1-42 while confocal microscopy clearly confirmed the accumulation/aggregation of the
peptide onto the surface of NPs and the formation of Aß 1-42 coated NPs with various sizes depending on the peptide concentration.
Finally, all these experiments clearly demonstrated the ability of our NPs to link the Aß peptides. This interesting feature could prevent
or slow down the formation of Aß toxic oligomers or aggregates under physiological conditions. This finding opens an avenue to new
therapeutic strategies based on nanotechnologies in the field of Alzheimer‘s disease.
Sabella S, Quaglia M, Lanni C, Racchi M, Govoni S, Caccialanza G, Calligaro A, Bellotti V (2004) Electrophoresis, 25, 3186–3194
Brigger I, Morizet J, Aubert G, Chacun H, Terrier-Lacombe MJ, Couvreur P, and Vassal G (2002) J. Pharmacol. Exp. Ther. 303:928.
Calvo P, Gouritin B, Chacun H, Desmaele D, D'Angelo J, Noel, JP, Georgin D, Fattal E, Andreux JP, and Couvreur P (2001) Pharm. Res. 18:1157.
Brambilla D, Nicolas J, Le Droumaguet B, Andrieux K, Marsaud V, Couraud PO, Couvreur P (2009) submitted.
P150:
Characterization of ultrathin-layer chromatography on thin films nanoengineered with macropore anisotropies
Steven R. Jim*1, Michael T. Taschuk1, Gertrud E. Morlock3, Louis W. Bezuidenhout1, Michael J. Brett5
1 Department of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada
3 Institute of Food Chemistry, University of Hohenheim, Stuttgart, Germany
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Poster Abstracts
5 NRC National Institute for Nanotechnology, Edmonton, Canada
The ability to quickly analyze many sample mixtures in parallel makes ultrathin-layer chromatography (UTLC) appealing in food chemistry, pharmaceutical analysis, environmental testing, and a variety of other rapid screening applications1, 2. UTLC stationary phases are
typically comprised of thinner monolithic silica gels with finer pore sizes than thin-layer chromatography (TLC) or high-performance TLC
media. UTLC media therefore permit faster separations over shorter distances with better limits of detection3. The strong dependence of
separation behaviour on stationary phase microstructure motivates continued UTLC plate design optimization efforts. Recent advances
include electrospun nanofibrous media1 and nanostructured glancing angle deposited macroporous thin films2.
Glancing angle deposition (GLAD) is a single-step vacuum deposition process for fabricating thin films with porous columnar microstructures suitable for sensing, optical, microfluidic, and other applications4. The method precisely controls film porosity and morphology, and can produce structural anisotropies in the plane or in the normal direction of a thin film. GLAD’s ability to engineer structural
anisotropies over large areas distinguishes it from other approaches to fabricating UTLC stationary phases.
Initial anisotropic GLAD UTLC plates possessed 5 µm thick chevron (“zig-zag”) architectures and channel-like features2. Analyte migration along these features was more rapid than across them. Here, we further investigate these anisotropic effects on the original chevron
media and on two new media: isotropic vertical posts and anisotropic blade-like films. GLAD stationary phase performance is quantified.
Early estimates based on Dimethyl Yellow dye separated from a lipophillic dye mixture over migration distances less than 12 mm suggest theoretical plate numbers of 540 ± 50, 500 ± 100, and 170 ± 40 for GLAD media with isotropic vertical post, anisotropic blade-like,
and anisotropic chevron structures, respectively. The limits of detection are approximately 7 ng ± 2 ng for the vertical posts and 12
ng ± 3 ng for the blade-like media. The limits of detection for the chevron plates are similar to those of the blade-like media but precise
measurement is currently limited by poor resolution. Further quantification of nanoengineered GLAD separation media performance
continues and recent experimental results will be presented.
1 J. E. Clark and S. V. Olesik, Anal. Chem. 81 2009
2 L. W. Bezuidenhout and M. J. Brett, J. Chromatogr. A 1183 2008
3 H. E. Hauck et al, J. Planar Chromatogr. - Mod. TLC 14 2001
4 M. M. Hawkeye and M. J. Brett, J. Vac. Sci. Technol. A 25 2007
P151:
Carbon nanotechnology-based gene delivery system for plant cells
Maged Fouad, Noritada Kaji, Yukihiro Okamoto, Manabu Tokeshi, Yoshinobu Baba
Nagoya University
Carbon nanotechnology, including fullerene and nanotube, is one of promising technology for advanced manufacturing and engineering
materials. Although biological materials such as DNA have been reported to assist carbon nanotube dispersion and separation, there
still remains challenging problems in biological applications of the carbon nanotechnology. In this presentation, we will demonstrate
the usefulness of cellulase-immobilized carbon nanotubes (CNT) as a gene delivery tool for intact plant cells.
Various gene transfection techniques such as particle-mediated gene gun and agrobacterium-mediated transfection have been developed. However, there are several drawbacks; low transfection efficiency and severe damage to plant cells especially the methods which
require preparing protoplasts in prior to gene transfection. So mild but high efficiency transfection techniques are anticipated.
In this experiment, cellulase enzymes which could digest cell wall were immobilized on the surface of cup-stacked carbon nanotubes
(CSCNT). The cellulase-immobilized CSCNT could make a nanometer-sized hole and penetrate cell wall. Since DNA molecules were strongly adsorbed onto the CSCNT surface, the DNA molecules were translocated to cytoplasm. Several plant cells including Arabidopsis
thaliana were used for this carbon nanotube-based DNA delivery system. GFP gene-coded plasmid was translocated into nucleus and
GFP expression was confirmed by confocal microscope. Transfection efficiency was not so high (~10%) compared with conventional PEG
or virus-based system but a small quantity of DNA (~ng) was enough to transfection due to strong adsorption of DNA on the surface of
CSCNT. Toxicity assay of CSCNT to plant cells was also performed. But no obvious toxicity was observed in this system.
We have proved the possibility of gene delivery of cellulose-immobilized CSCNT. This system could skip the process of cell wall removal
to prepare protoplasts and no severe damage for further regeneration of cells. Therefore this system could be a promising tool for plant
cells DNA transfection and plant cell biology studies.
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Poster Abstracts
P152:
Capillary electrophoresis detector based on surface-enhanced Raman spectroscopy
Jan Přikryl1, Karel Klepárník2, František Foret2
1 Institute of Analytical Chemistry of the ASCR, v.v.i., Veveri 97, 602 00 Brno, Czech Republic
Masaryk University, Faculty of Science, Department of Chemistry, Kotlarska 2, 611 37 Brno, Czech Republic
2 Institute of Analytical Chemistry of the ASCR, v.v.i., Veveri 97, 602 00 Brno, Czech Republic
Surface-Enhanced Raman Spectroscopy (SERS) is interdisciplinary method covering physics and chemistry; vibrational spectroscopy,
plasmonics and nanotechnologies. This method is based on the possibility to enhance the weak Raman scattering of molecules attached to metal nanomaterials, such as metal nanoparticles, roughened surfaces or metal tips. Under the optimal conditions, both
the excitation and emission radiation are enhanced. The enhancement factor reaches a value up to 1012, and thus SERS becomes a
high sensitive analytical method. The concentration profile of analytes can be registered at vibration bands with specific Raman shifts.
Various techniques of SERS have already proved the potential for qualitative and quantitative analyses.
The laboratory-built Raman detection system consists of a epifluorescence microscope body (JENALUMAR, Carl Zeiss Jena, Germany)
equipped with a 50× (0.95 NA) microscope objective and a He-Ne laser (λ=632.8 nm, 15 mW). The separation capillary is irradiated by
the laser in epifocal arrangement. The Raman scattering collected by microscope objective passes through a dichromatic mirror (λ=650
nm) and a Raman notch filter (λ=632.8 nm). The scattered light is then dispersed by a spectrograph (Shamrock SR-303i, Andor, UK)
equipped with diffraction gratings (300 l/mm, 600 l/mm or 1200 l/mm, blazed for 500nm) and collected by a 16-bit deep cooled backilluminated CCD camera (iDus DU420A BR-DD, Andor, United Kingdom). Silver nanoparticles prepared by Lee-Meisel procedure are added to background electrolyte. Raman-active compounds are separated in silica-fused capillary (ID=50 μm, OD=375 μm) and detected
after a passage of 12 cm long migration path. Application of several types of nanoparticles, their concentrations and an optimization
of aggregating agents will be demonstrated. Limit of detection of some important compounds will be determined.
This work was supported by The Grant agency of Academy of Science of the Czech Republic (KAN400310651 a KJB400310709), Grant
Agency of the Czech Republic (GA203/08/1680, GA301/07/0490), Ministry of Education, Youth and Sports (LC06023, MEB060821) and
institute research plan AV0Z40310501.
P153:
Detection of Cu2+ Ions through Their Suppression of Fluorescence Quenching of Silver/Copper Nanoclusters by Mercaptopropionic
Acid
Yu Ting Su, Guo Yu Lan, Huan Tsung Chang
Department of Chemistry, National Taiwan University
We have developed a simple, homogeneous, and “turn-on” fluorescence assay—comprising 3-mercaptopropionic acid (MPA) with DNAAg/Cu nanoclusters (NCs) in aqueous solution—for the determination of Cu2+ ions. The sensing mechanism is based on the Cu2+
induced suppression of fluorescence quenching of the DNA-Ag/Cu NCs by MPA. The fluorescence quenching is due to the interaction
of MPA with Cu and/or Ag atoms in the DNA-Ag/Cu NCs, which had been supported by the circular dichroism and X-ray photoelectron
spectroscopic data. From Stern-Volmer plots, we found that MPA relative to 2-mercaptoethanol and cysteine had a greater SternVolmer constant, revealing its greater quenching efficiency. Copper ions formed anionic complexes with MPA, which reduced the
interaction of MPA with the DNA-Ag/Cu NCs. The fluorescence of the DNA-Ag/Cu NCs increased upon increasing the concentration
of Cu2+ over the range 5-200 nM. The DNA-Ag/Cu NC probe provided the limit of detection at a signal-to-noise of 3 of 3.5 nM Cu2+,
with high selectivity (by at least 2300-folds over tested ions). The practically of this approach has been validated by the analyses of
Montana soil and water samples (3 replicate measurements), with results of the concentration of Cu2+ ions to be 2.60 (± 0.23) μM and
not detected, respectively.
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Poster Abstracts
P154:
Exploring the Interactions between Gold Nanoparticles and Analytes through Surface-Assisted Laser Desorption/Ionization Mass
Spectrometry
Yang-Wei Lin, Wen-Tsen Chen, Huan-Tsung Chang
National Taiwan University
Surface assisted laser desorprion/ionization mass spectrometry (SALDI-MS) is applied to provide strong evidences for the chemical reactions of functionalized gold nanoparticles (Au NPs) with analytes—Hg2+ ions induced mercaptopropionic acid (MPA)-Au NPs aggregation in
the presence of PDCA (2,6-pyridinedicarboxylic acid) and H2O2 induced fluorescence quenching of 11-mercaptoundecanoic acid (11-MUA)Au NDs. PDCA-Hg2+-MPA coordination is responsible for Au NPs aggregation, while the formation of 11-MUA disulfide compounds that
release to bulk solution is for H2O2 induced fluorescence quenching. In addition to provide the information for the chemical structures, the
SALDI-MS is also selective and sensitive for the detection of Hg2+ ions and H2O2. The LOD for Hg2+ ions and H2O2 through SALDI-MS were
300 nM and 250 mM, respectively. The spot-to-spot variation in the two studies were both less than 18 % (50 sample spots). Our results
reveal that the SALDI-MS has been demonstrated for the study of the analytes induced changes in the surface properties of nanoparticles.
P155:
Fabrication of free-standing metallic microstructures; modifications and applications
Petra Jusková*1, 2, František Foret1
1 Institute of Analytical Chemistry, ASCR, v.v.i., Brno, Czech Republic
2 Masaryk University, Faculty of Science, Department of Chemistry, Kotlarska 2, 611 37 Brno, Czech Republic
Lithographic microfabrication with selective etching and thin film deposition play crucial role in incorporating metal structures into various
kinds of miniaturized devices in electronics. High resolution pattern transfer makes microlithography a promising tool for mass-production
of two dimensional (2D) and three dimensional (3D) free-standing structures with potential bioanalytical use. Depending on the selected
shape and material, particles can serve as signal enhancement components of conventional devices or can be used for biomolecule targeting and delivery.
In our work we have utilized microfabrication methods for preparation of free standing, high aspect ratio metal structures for use as
interaction and sensing elements in microfluidic devices. Optimized fabrication process allows easy and flexible changes in material composition and properties of the structures. Mono and multimetallic particles of various dimensions (from tenths of nanometers to microns)
and shapes were prepared. Combination of these structures with magnetic materials opens up new possibilities for their controllable and
addressable positioning in the microfluidic system and self-assembling into directed topologies. Combination of their unique features with
proper surface modification makes them a suitable tool for variety of clinical applications.
Supported by: KAN400310651, Research Centre LC06035, AV0Z40310501
P156:
Surface modification of lipid bilayers in extended-nano channel for investigation of liquid properties in intercellular spaces
Kazuma Mawatari*1, Hisaki Emon1, Takehiko Tsukahara3, Takehiko Kitamori1
1 The University of Tokyo
3 Tokyo Institute of Technology
Several tens nanometer spaces between cell membranes and synaptic gaps are believed to play very important roles in cell functions
(signaling, tissue formation etc). Some specific solution properties are suggested close to cell membranes such as high viscosity and fast
proton transfer. So it is necessary to make a tool for investigating solution properties between them, but there has been no experimental
tool. In our laboratory, we made the extended-nano space in glass substrates and found many specific liquid properties. For example,
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Poster Abstracts
properties of water such as viscosity, dielectric constant and proton transfer in the extended-nano space are different from those in a bulk
space. It is suggested that there are similar properties between the extended-nano space and intercellular space. Therefore, we inspired
the biomimetic extended-nano space where cell membranes were modified. To realize this, we needed to modify lipid bilayers in the space.
We have succeeded in modifying phospholipid bilayers in a few hundreds nanometer-sized channel and creating “the biomimetic extended-nano space” which mimics an intercellular spaces (from several tens to several hundreds nanometer-spacing). Vesicle-fusion
method (diameter of vesicles: 30nm) were utilized. As phospholipids, we used 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) including dye-conjugated lipids (Texas-Red DHPE) 1 %. Micro- and nano-channels were fabricated on glass substrate (equivalent diameter
R = 390-620 nm). The vesicle solutions were driven by air-pressure. After introducing the vesicle solution and washing the extended-nano
channel by buffer solution, the modification was confirmed by observing the fluorescent intensity, though there observed some difference
of fluorescent intensity.
P157:
High Efficient Separation by Chromatography in Extended-nano Space Using Pressure Driven Flow
Ryo Ishibashi1, Masanori Inaba1, Masaru Kato3, Kazuma Mawatari1, Takehiko Kitamori1
1 Graduate School of Engineering, The University of Tokyo
3 Center of NanoBio Integration, The University of Tokyo
Introduction: Our focus has been centered on the well-defined 101-103 nm-scale (so-called extended-nano space) channels on a quartz
microchip, since it is expected to be an ideal experimental tool for characterizing the mesoscopic property of molecules. We have
demonstrated that the water properties in the extended-nano space show a higher viscosity, lower dielectric constant, and higher
proton mobility than that of bulk water. Chromatography in this extended-nano space has the potential of; separating ultra low volume
of sample, efficient separation by the unnecessity of the packed columns due to the high specific interfacial area, and new mode of
separation due to the specificity of this space. In this study, we developed a chromatography system in extended-nano channels using
pressure driven flow, and evaluated its separation efficiency.
Principle: In the extended-nano space, the effect of electric double layer (1~300 nm) generated by the negative channel surface must
be taken into account, which is usually small enough to ignore in the bulk space. The cations gather to the wall to form the double
layer, while anions are repelled from the wall to the center of the channel. Thus, mobility difference will be generated with the charge
difference, due to the parabolic profile of pressure driven flow, which leads to separation.
Experimental: The crossed-shaped extended-nano channels (910 nm wide, 220 nm deep) were fabricated on a quartz glass plate by
electron beam lithography and plasma etching. Nano-channels were linked to micro-channels (300 mm wide, 3 mm deep). Two pressure
controllers were used for the fluid control, and the pinched injection was performed by controlling the pressure from the four directions
of the nanochannels. Fluorescent images were taken by a fluorescence microscope for detection.
Results and discussion: Mixture of fluorescein and sulforhodamine B (50 mM each) in a 100 mM phosphate buffer (pH = 8) was used
as a sample. Small amount of sample band (550 aL) was successfully injected to the separation channel. By the fluorescence detection
at 1300 mm downstream from the injection point, the two compounds were successfully separated. The maximum theoretical plate
number of fluorescein was 172,000 plates/m, which was two times larger than that of conventional HPLC, even though the specific
interfacial area is smaller in nanochannel. Elimination of eddy diffusion by the absence of packed column should be one contribution
to this fact. Another possibility is the decrease of effective diffusion coefficient in extended-nano space. Interaction of samples to the
wall is enhanced, and the diffusion will be suppressed. Also, viscosity in extended-nano space is increased, which leads to suppression
of diffusion. The high efficient separation by the specific properties in extended-nano space would be a powerful tool for the separation
of ultra small volume of sample, such as in single cell proteomics.
P158:
Conjugation techniques to link biologically important molecules with quantum dots
Marcela Lišková1, Karel Klepárník2, Ivona Svobodová2, Věra Hezinová2, Jan Přikryl2, František Foret2
1 Institute of Analytical Chemistry of the AS CR, v.v.i., Veveri 97, 602 00 Brno, Czech Republic
Brno Masaryk University, Faculty of Science, Centre for Syntheses at Sustainable Conditions and their Management, Kotlarska 2, 611 37 Brno, Czech Republic
2 Institute of Analytical Chemistry of the AS CR, v.v.i., Veveri 97, 602 00 Brno, Czech Republic
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Poster Abstracts
Quantum dots (QDs), semiconductor inorganic nanocrystals, show a great potential as photoluminescent probes at cellular level for in
vivo immunolabeling or cellular tracking. The main advantages of quantum dots, when compared with the conventional organic fluorescent dyes, are practically no photobleaching, wide excitation and narrow emission spectra and size dependent emission maximum
wavelengths. Typical materials for the preparation of quantum dots are elements of II – VI or III – V group. QDs have a big potential in
single-cell analyses with LIF detection.
We have prepared CdTe water soluble quantum dots of size 3.5 nm by the chemical reaction between CdCl2 and NaHTe at the presence
of 3-mercaptopropionic acid. Bioconjugation reactions are used for linking of two or more molecules to form a compound with new
desirable properties. A lot of biologically active molecules with various reactive groups can be conjugated using a variety of different
cross-linkers. The conjugation of QDs with different biomolecules will be demonstrated. The conjugation reaction via zero-length crosslinkers such as 1-ethyl-3-(3-dimethyl-3-aminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) is
convenient through its simplicity, fastness and selectivity. This reaction binds carboxyl group on the QD surface and a primary amino
group in a molecule, which is to be stained. A high reaction yield of the conjugation of QDs with anti-ovalbumin and a fast formation
of immunocomplex with ovalbumin are checked by the capillary zone electrophoresis with LIF detection. This work was supported by The Grant agency of Academy of Science of the Czech Republic
(KAN400310651 a KJB400310709), Grant Agency of the Czech Republic (GA203/08/1680, GA301/07/0490), Ministry of Education,
Youth and Sports (LC06023, MEB060821) and institute research plan AV0Z40310501. Monday, March 22nd, 2010
P159:
Luminescence Quantum Yield of CdTe Quantum Dots
Ivona Svobodova1, Marcela Liskova2, Petr Taborsky2, Karel Kleparnik1, Frantisek Foret1
1 Institute of Analytical Chemistry of the ASCR, v.v.i.
2 Faculty of Science, Masaryk University, Brno
Semiconductor nanocrystals – quantum dots (QDs) exhibit extraordinary optical properties as narrow and symmetrical emission
spectra, wide excitation spectra, good chemical- and photo-stability and fluorescence emission wavelength dependent on particle size.
These properties are prerequisites for applications as fluorescent markers in analytical chemistry, molecular biology and medicine. The
spectroscopic and physicochemical properties of QDs are controlled by materials of their core, particle sizes and size distributions,
coatings and surface chemistry including inorganic passivation layer, silica and organic ligands bound to surface atoms. Water soluble CdTe QDs are synthesized by reaction of NaHTe with CdCl2 in the presence of organic ligands. Different preparation
processes of NaHTe and various pH of reaction mixture are tested to optimize QDs synthesis. The effect of surface coatings formed
by inorganic salts (CdS and ZnS) and organic ligands (3-mercaptopropionic acid, thioglycolic acid, 11-mercaptoundecanoic acid and
cysteamine) is tested.
Quantum yields (QY) is defined as a percentage of the number of emitted per number of absorbed photons. The changes of QY of
different QDs prepared by different synthetic processes with different surface coatings are measured by the secondary method and
compared. A solution of fluorescein in ethanol is used as a standard. It is found out that QY of QDs strongly depends on pH of the
reaction mixture. In the case of QDs with 3-mercaptopropionic acid and cysteamine, the optimum pH of reaction mixture is found to
be 11 and 3, respectively. The coatings of CdTe core by inorganic salts CdS and CdS/ZnS significantly increase QY.
P160:
Electrospun cellulosic meshes for use in thin layer chromatography and blotting
Samuel C. Ligon
Medical University Innsbruck
141
Poster Abstracts
Electrospinning is a versatile technique for producing nano to micro sized fibers from a variety of synthetic and natural polymers.
Processing conditions such as polymer concentration, flow rate, and applied voltage may be adjusted to effect fiber diameter and homogeneity. While the mode of collection may be controlled to provide fiber alignment, deposition on flat plate collectors is preffered for
mechanically flexible and randomly oriented meshes. Although electrospinning was first developed more than a hundred years ago, a
renewed interest in the process has arisen in the last 15 years led chiefly by applications in tissue engineering. Excitement in this area
of research has been fueled by the semblance of the nanoarchitechture of electrospun matrices and natural extracellular matrices in
humans. Thus, elctrospun meshes of natural polymers such as collagen, gelatin, and chitosan may be seeded with cells from suspension
or surgically implanted to support tissue growth. Although not as heavily studied as tissue engineering, electrospun meshes may also
be used in a number of filtration and separation applications relevant to the bioanalyst. In particular, electrospun cellulosic meshes
have higher surface areas and better mechanical properties than traditional cellulosic membranes. Thus electrospun meshes of cellulose acetate, cellulose nitrate, and regenerated cellulose may be used in filtration, thin layer chromatography and Western blotting from
PAGE gels. The meshes may be used directly or derivatized with antigens for immunoassays with sensitivity and transfer efficiencies as
good as or better than commercially available blotting membranes. In addition, electrospun cellulosic membranes may be collected on
glass microscope slides and used as stationary phases in thin layer chromatorgaphy. Separation efficieny is dependent on mesh material, thickness, and fiber diameter, and thus on processing conditions. The influence of polymer concentration, voltage, and mode of
collection has been seen to effect fiber properties of cellulose acetate meshes. Use of these membranes for chromatographic separation
of electrophoresis stains and as blotting papers for improved transfer of proteins from PAGE gels is the subject of current investigations.
P161:
A comparative analysis between native fluorescence CELIF and absorbance CE/UV in pathological antibodies in plasma
Audrey Boutonnet1, Elisabeth Caussé2, Bernard Feurer3, Pierre Naccache3, François Couderc1
1 Laboratoire des IMRCP, Université Paul Sabatier, Université de Toulouse, 31062 Toulouse, France
2 Laboratoire de Biochimie CHU Rangueil, 31052 Toulouse France
3 Picometrics, 18 rue Ste Cécile, 31100 Toulouse
A new optical arrangement for laser induced fluorescence (LIF) detection (Rodat et al Electrophoresis, 2008, 29, 740-6) is able to detect
antibodies at considerably lower concentrations than silver staining in slab gels. As an example, this arrangement increased the sensitivity
antibodies labelled with fluorescein isothiocyanate or TAMRA by a factor of 100.
An alternative to labelling the molecule is to use the intrinsic fluorescence of these antibodies. Since the fluorescence yield of Trp and Tyr
(which are the fluorophores of the proteins) is lower than that of the dye, the sensitivity obtained from native fluorescence is lower than
that of an assay using a dye. Although the sensitivity is lower than with the use of a dye, the major advantage is that the analytical procedure is much shorter and simpler and the sensitivity still is greater than when the absorbance is measured. with a significant increase of
sensitivity compare to the absorbance. We modified the previous detector to be used with a UV pulsed 266nm diode laser and the lowest
detected concentration was identical with silver staining.
An important question is “are fluorescence and UV-absorption giving the same results in term of number of compounds detected?” keeping in mind that fluorescence is more sensitive than UV absorption.
In this poster we will show the comparison of CE/UV-LIF with classical CE/UV absorption and demonstrate that CE/UV-LIF is 50 times more
sensitive than the CE/UV method. In addition, we compared CE/UV-LIF and CE/UV absorption and pathological antibodies from plasmas,
and show that the same compounds are detected. Monday, March 22nd, 2010 P162:
Investigations on the stability of poliovirus in the presence of chemical interfering agents
Iuliana Oita1, Hadewych Halewyck2, Bieke Dejaegher1, Bert Thys2, Bart Rombaut2, Yvan Vander Heyden1
1 Vrije Universiteit Brussel-VUB, CePhaR, Department of Analytical Chemistry and Pharmaceutical Technology
2 Vrije Universiteit Brussel-VUB, CePhaR, Department of Pharmaceutical Biotechnology & Molecular Biology
Nowadays, developing separations to study biological samples, and particularly virus particles, is one of the most emerging interdisciplinary
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Poster Abstracts
challenges. Poliovirus (PV) is among the best studied viruses, but its replication and how the different subviral particles emerge during
replication of the virus is still not completely understood. PV is a member of the Picornaviridae. It is a relative stabile virus. It is stabile
one to three hours in pH 3-5, and the wildtype virus is relatively heat resistant (when stabilized by magnesium cations), and also resistant
to many common detergents and disinfectants. However, PV is readily inactivated by drying, UV, high heat, formaldehyde, pH above 10
and free chlorine.
A simple capillary electrophoresis (CE) separation was initially developed as a tool for PV studies, and a further 16 times signal enhancement
was obtained adjusting the separation conditions. The possible use of CE for separation of subviral particles, building-up (morphogenesis
related) or breaking-down (degradation related) particles is currently under investigation. Since reference materials are out of question for
the moment, and the amount of information concerning the stability of these particles is limited, forced degradation of poliovirus seemed
an option to generate breaking-down particles and to improve the ability of the CE methods to detect these particles.
PV was exposed to extreme pH values, sodium dodecylsulphate (SDS) and diluted formaldehyde but also to magnesium chloride. The
extreme pH values were generated in-line injecting hydrodynamically solutions of 0.1 M hydrochloric acid, 0.05 M acetic acid, 1M sodium
hydroxide, or 1M ammonia. To assess the formaldehyde effect, PV was incubated for 12 days with formaldehyde 1:4000 in water, thus the
same conditions used during inactivated PV vaccine manufacturing. The effects of magnesium chloride and SDS were studied both off-line
and in-line. The in-line experiments were performed injecting both 1% and 5% PV sample plugs.
At extreme pH values, PV was faster and almost completely degraded by 0.1 M hydrochloric acid within the acidic range and by the 1 M
ammonia, within the alkaline range. Compared to the extreme pH, the SDS exposure had much less effect. Magnesium chloride showed a
certain improvement of the PV separation from the neighbouring peak by increasing peak height and resolution. Formaldehyde treatment
had a strong effect on PV; the peak corresponding to PV completely disappeared from the second incubation day. During all experiments,
the reduction in PV peak area was accompanied by the generation of new peaks.
P163:
Monitoring changes in ligand binding properties and aggregation state of proteins using capillary electrophoresis and Taylor dispersion analysis
Jesper Østergaard*, Henrik Jensen
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen
Protein pharmaceuticals constitute a special challenge in drug development and manufacturing. Development of protein pharmaceuticals
requires a detailed knowledge of physical and chemical stability properties since therapeutic effect is closely associated to the conformational structure. In addition, biomolecular processes often involve molecular recognition and ligand binding by proteins. Characterization of
proteins and their interactions is essential to improve our understanding of this interesting class of molecules. In this context, integration
of the biophysical and functional characterization into one method would be advantageous.
The objective of the present study was to develop a capillary based method capable of performing simultaneous detection of protein
aggregation through size measurements (Taylor dispersion analysis) and characterization of protein function in terms of ligand binding.
Bovine serum albumin (BSA) incubated in phosphate buffer at pH 7.4 and 70°C in the presence/absence of the ligands ibuprofen, L-tryptophan and warfarin was selected as a model system allowing the effect of aggregation on BSA ligand binding properties to be studied.
Capillary electrophoresis (CE) and Taylor dispersion analysis (TDA) were conducted on an Agilent 3D CE coupled to an Actipix D100 UV
area imager (detection at 200 nm). Fused silica capillaries of 100 cm in total length (50 µm ID) with 30 and 50 cm to the first and second
detection window, respectively, were applied in all CE and TDA experiments. A 0.067 M sodium phosphate buffer (pH 7.4) was used as
sample buffer as well as back ground electrolyte in the CE and TDA. Average protein size (Rh) was measured by TDA (mobilization pressure
300 mbar) and ligand binding was determined using frontal analysis CE (23 kV) at 25°C. Additional characterization experiments were
conducted using size exclusion chromatography (SEC) and capillary zone electrophoresis.
From TDA experiments (and SEC) it is apparent that BSA aggregation starts immediately upon elevation of the incubation temperature to
70°C. The increase in apparent hydrodynamic radius (Rh 10-15 nm) determined from temporal variances of the BSA peaks upon storage at
70°C for 3 h was irreversible, i.e. incubation at 25°C did not result in a decrease in Rh to 3-4 nm. Frontal analysis CE experiments showed
that BSA aggregation was associated with a loss of the ability to interact with the ligands ibuprofen, L-tryptophan and warfarin.
In conclusion, Taylor dispersion analysis (TDA) is feasible for detection of protein (BSA) aggregation and estimation of the size of aggregates.
Affinity CE on the same samples revealed that BSA function in terms of ligand binding ability deteriorates upon storage at 70°C due to
the aggregation. Capillary based techniques combining measurements of diffusivity (size), ligand binding and electrophoretic mobility may
constitute and attractive approach for characterizing proteins with small amounts of sample.
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P164:
On-line concentration of tobacco-specific N-nitrosamines by cation-selective exhaustive injection-sweeping-micellar electrokinetic
chromatography
Youyou Yang1, Chenchen Li1, Jie Liao3, Huwei Liu1
1 Department of Chemistry, Peking University, Beijing 100871, China
3 Medical Experiment & Analysis Center, General Hospital of Chinese PLA, Beijing 100853, China
In this paper, an MEKC method combined with cation-selective exhaustive injection (CSEI) and sweeping was developed to separate and
concentrate four tobacco-specific N-nitrosamines (TSNAs) including N’-nitrosoanabasine (NAB), and 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL).
Experimental parameters affected separation efficiency and enhancement factors were investigated in detail. Under the optimum MEKC
condition, NAB, NNK, NNAL and iso-NNAL were baseline separated with high efficiency and good peal shape. Furthermore, with the preconcentration by CSEI-sweeping-MEKC, the detection limit (LOD) of four TSNAs was improved by almost four orders of magnitude compared
with that obtained from usual MEKC. The LODs (S/N=3) were ranged from 0.004 to 0.016 μg/mL. In addition, this method had fairly good
repeatability, and the RSDs of retention time and peak area were less than 1% and 5%, respectively. Finally, this method showed promising
capability in the application of detecting and analyzing TSNAs in human urine samples.
P165:
Optimization of the fluorescence labeling of Poliovirus and analysis with laser induced capillary electrophoresis
Hadewych M Halewyck*1, Iulia D Oita2, Bert Thys1, Bieke Dejaegher2, Yvan Vander Heyden2, Bart Rombaut1
1 Department of Pharmaceutical Biotechnology & Molecular Biology, Pharmaceutical Institute, VUB
2 Department of Analytical Chemistry and Pharmaceutical Technology, Pharmaceutical Institute, VUB
One of the main features of samples of viral origin consists in a very low amount of detectable analyte, represented by the virus itself or
the sub-viral particles. For decades, radioactive labeling was one of the most popular approaches to lower the limit of detection for viral
samples. Within a context of increasing awareness for environmental aspects, researchers cannot afford to neglect the switch towards
the use of green and eco-friendly research alternatives. Radioactivity is not only an immediate hazard, but also a threat for the future
giving the waste needing years of storage before it can be thrown away. A nice alternative for the radioactive labeling is the use of fluorescent labels. For the analysis of poliovirus, a separation method using capillary electrophoresis with UV detection has been developed
(ref Iulia). The same method will be used to analyze the poliovirus with laser induced fluorescence capillary electrophoresis. The signal
was increased by adjusting the separation conditions in UV analysis, but the use of the LIF detection can bring a further increase of
the analytical signal and supplementary gains in terms of selectivity. The main disadvantage is the lack of the natural fluorescence of
the poliovirus that has to be overcome by a pre-column derivatization using a fluorescent dye. The aim of this study is to optimize the
labeling of poliovirus with fluoresceine isothiocynate (FITC). The derivatization reaction obeys the rules of a nucleophilic substitution,
but due to the nature of the sample, the reaction parameters need to be adjusted to obtain an efficient labeling in a rational timeframe.
Preliminary experiments showed a need for high molar excess of fluorescent dye to obtain efficient virus labelling. Different dye to virus
ratios were tested to select the most efficient ratio. There are only few alternatives to characterize the labeling efficiency. The most
used one calculates the number of dye molecules attached to one analyte molecule based on the ratio between the absorbances of
conjugated and dye. An indirect method, based on the quantification of the FITC excess using CE was proposed. To validate the method,
albumin labeled by the same protocol used for poliovirus was used. The labeling conditions were adjusted to obtain the best labeling
efficiency within the shortest time. As there are no virus standards available, FITC-labeled albumin was used to set up a calibration line.
From this calibration line, free FITC and, in an indirect way, labeled virus can be quantified.
P166:
A model of in vitro drug metabolism testing by in-capillary enzymatic reaction utilizing capillary electrophoresis
Marta Zeisbergerová*, Aleš Mádr, Zdeněk Glatz
144
Poster Abstracts
Department of Biochemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic
In vitro drug metabolism studies serve as an adequate screening mechanism to reveal potential metabolites of new drug candidates,
elucidate their pathways and make prediction of their in vivo clearance. Further, high throughput screening methods are desired as a
number of potential new drug compounds is large. Recombinant human cytochrome P450 (CYP) enzymes belong to favorable in vitro
test system. The metabolism pathway of dextromethorphan was chosen as a model system of drug transformation under the action of
cytochrome P450 2D6 (CYP 2D6). This enzyme belongs to important isoforms of hepatic CYP enzymes as it is involved in metabolism of
about 25% of all commonly prescribed drugs. Nowadays, most of drug metabolism studies is performed by liquid chromatography-mass
spectrometry methods characterized by off-line setup. However, capillary electrophoretic techniques offer direct automated assays.
Moreover, capillary electrophoresis enables to conduct in-line homogenous enzymatic reactions1. The reaction and the separation can
take place in capillary at the same time or consecutively. The aim of our study was to develop in-capillary CYP 2D6 enzymatic assay.
The optimal conditions assuring the reaction and separation were reached thank to a partial filling method. The successful separation
of standards was reached in tetraborate buffer (80 mM, pH 9.8) with addition of 2-propanol (8 %, v/v) at 37°C. The UV detection of
analytes was performed at 200 nm. Setting of the sequential injection scheme was a key part of method development. The model assay
was focused on characterization of initiation of enzymatic reaction to reach the maximal metabolite production.
The authors thank for financial support Grant Agency of Czech Republic (grant No. P206/10/0057) and Czech Ministry of Education
(research project No. MSM0021622413 and research center LC06023).
1 X. Hai, J. Konečný, M. Zeisbergerová, E. Adams, J. Hoogmartens, A. Van Schepdael, Electrophoresis 29 (2008) 3817
P167:
Capillary Electrophoresis with Indirect Ultraviolet Detection for Pharmaceutical Counterion Analysis
Cynthia Boardman1, Johannes Dewald1, Jean-Bernard Falmagne3, Francois de l‘Escaille3
1 Beckman Coulter, Inc
3 Analis sa/nv
Capillary electrophoresis (CE) with indirect UV detection is a proven technology for the analysis of counterions. CE offers short analysis
and cycle times, broad tolerance for sample matrices, small sample- and buffer-volume requirements, and low waste production. These
traits make it a desirable alternative or complementary analysis method to ion-chromatography. We present here the characterization
of a counterion analysis method based on CE with indirect UV detection. The method characterization includes determination of the
linear range of quantitation and limit of detection for commonly employed organic and inorganic counterions. We additionally demonstrated the ability of the method to accurately analyze samples prepared in several organic solvents commonly used to address drug
solubility.
P168:
Capillary Electrophoresis with LIF Detection for Characterization and Quantitation of Mitochondria Based on the Cardiolipin Content
Wenfeng Zhao1, Yingsing Fung1, Waisum O3
1 Department of Chemistry, the University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
3 Department of Anatomy, the University of Hong Kong, Pokfulam Road, Hong Kong SAR, China
It is important to characterize and quantify mitochondria for cell research because they play a significant role in cellular functions
including electron transport, oxidative phosphorylation and apoptosis1. As mitochondria exist in cells as particles of folding membrane
structure with variable shape and geometry, tedious electron microscopic counting procedures are needed to quantify mitochondria
after elaborate sample pretreatment techniques. As the cardiolipin content of mitochondria is unique and fairly constant (about 20% of
total lipids) amongst a given cell line [2], its feasibility for use as biomarker for quantifying mitochondria is investigated in the present
work. The relationship between cardiolipin content with mitochondria number is established by comparison of results obtained from
145
Poster Abstracts
TEM count and cardiolipin content of specified mitochondria numbers. To reduce interference from other cellular components and
enhance the detection sensitivity for assessing cells with low mitochondria counts, a new CE-LIF procedure is developed based on a
sensitive detection of cardiolipin by the formation of a specific 1:1 complex between cardiolipin and a specific dye, 10-N-nonyl acridine
orange.. The pros and cons of the procedure developed as compared to existing CE-UV method are assessed. The results from the
applicability study of the optimized CE-LIF procedure for determining the mitochondria number based on cardiolipin content for HepG2
cell with and without oxidative damage will be presented and discussed at the meeting.
1 Saraste, M. Science, 1999, 283, 1488-1493
2 Daum, G, Biochimica et Biophysica, 1985, 822, 1-42
P169:
CE/LIF and HPLC/LIF MS of polysaccharides labelled by APTS
Audrey Boutonnet1, Véréna Poinsot1, Jalloul Bouajila1, Pierre Naccache4, François Couderc1
1 Laboratoire des IMRCP, Université Paul Sabatier, Université de Toulouse, 31062 Toulouse, France
4 Picometrics, 18 rue Ste Cécile, 31100 Toulouse
9-Aminopyrene-1,4,6-trisulfonate (APTS) is a well known dye that is used for derivatizing mono or oligo saccharides (Fraysse et al
Electrophoresis. 2003, 24, 3364). The derivatization process for labelling the sugars is very well known and involves the reductive amination of the reductive function of the mono or oligosaccharides. This reaction can be done on very small sample volumes (e.g. 5µL).
In this poster, we discuss the detection of these derivatives with a number of lasers as excitation sources which provide different power.
We will show that the lowest detected (LOD) concentration is highly dependent on the power of the light power. In addition, the LOD
also depends on the stability of the light source.
An attempt to employ µHPLC/LIF/MS to separate sugars labelled with this high fluorescence sensitivity dye and to compare the sensitivity of these two detection means using µHPLC separation is described. This mode of separation is used as LIF detection is at least
100 times as sensitive than MS when using µHPLC separation. While µHPLC may be more familiar than CE for many users, it should be
noted that µHPLC provides lower resolution than CE for APST sugar compounds.
An application of this strategy will be shown using derivatized biological samples, such as plasma and milk. Wednesday, March 24th, 2010 P170:
CE-LIF Protein detection based on RCA
Cai Tie, Xin-Xiang Zhang
The Key Laboratory of Biochemistry of MOE, College of Chemistry, Peking University, Beijing 100871, China
CE-LIF becomes a wide applied high throughput bio-sample analysis technique, since it shows lots of advantages including separation
condition friendly to bio-molecules, high sensitivity, low sample consumption and high separation speed,. We developed a method
combines the Specificity of Aptamer, the signal amplification of the RCA (Rolling Circle Amplification) and the strong separation capability of CE-LIF to detect platelet-derived growth factor (PDGF). Single stand DNA probe contains PDGF Aptamer was applied and formed
circle template with PDGF. After the RCA process, the amplified products was digested into small fragments and analyzed by CE-LIF.
With this method, 40pM PDGF can be detected. The method also shows potential to be a high throughput method by applying several
different probes.
P171:
Characterization of a PEGylated liposomal drug formulation by capillary electrophoresis
Ulrik Franzen1, Charlotte Vermehren2, Jesper Østergaard1
146
Poster Abstracts
1 University of Copenhagen, Faculty of Pharmaceutical Sciences, Department of Pharmaceutics and Analytical Chemistry, Universitetsparken 2, DK-2100 København Ø
2 LiPlasome Pharma A/S, Diplomvej 378, DK-2800 Lyngby
Liposomes consisting of phospholipid bilayers have been studied extensively in the biological and pharmaceutical sciences. Due to their
similarity with biological membranes, they have been used as models for studying solute-membrane interactions. Liposomes have also
found use as drug delivery systems with a few marketed products.
In this work, the potential of using capillary electrophoresis (CE) for characterizing a novel liposomal drug formulation, of the anti-cancer
agent oxaliplatin, was examined.
The liposomal drug formulation were composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioctadecanoyl-sn-glycero3-phospho-(1’-rac-glycerol) (DSPG) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] DSPEPEG 2000 in the ratios (DSPC/DSPG/DSPE-PEG2000 70/25/5 mol%) in a solution consisting of 10 % (w/v) sucrose and 1 mM calcium
gluconate.
The characterization of the drug formulation by CE comprised of interaction studies between three model drug compounds (oxaliplatin,
paracetamol and propranolol) and the empty drug formulation. The interaction studies were performed by capillary electrophoresis
frontal analysis (CE-FA) and electrokinetic chromatography (EKC). Furthermore, estimation of the zeta potential of the liposomal drug
formulation, calculated from electrophoretic mobilities, was performed.
By using the CE-FA methodology it was possible to detect interactions between one of the model drug compounds, propranolol, and
the empty liposomal formulation, whereas this methodology were not able to reveal any interactions between the two other model
compounds and the empty formulation. Initial EKC experiments showed that interactions between propranolol and the empty liposomal
formulation were also detectable.
It is envisioned that CE could be a helpful tool in the characterization of nanoparticulate drug delivery systems (including liposomes) by
requiring small sample volumes, being fast and easily automated.
P172:
Contactless Conductivity Detection for Capillary Electrophoresis and Microchip Electrophoresis
Marko Stojkoviæ, Worapan Pormsila, Benjamin Bomastyk, Thanh Duc Mai, Thitirat Mantim, Peter C. Hauser
eDAQ Pty Ltd., 6 Doig Ave, Denistone East, NSW 2112, AUSTRALIA
Contactless conductivity detection can be considered as an electro-analytical technique, which has ability to detect any charged analyte
irrespective of whether it contains an electro-active group or not. The only requirement is that the migrating analyte zones possess a
conductivity that is different from that of the background electrolyte. Many inorganic and organic ions, as well as biological species
have been analyzed using CE with C4D. The method might be of benefit to the areas of genomic researches and investigations, genetic
analysis, solving forensic problems, medical and pharmaceutical researches and handling of diseases.
P173:
Electromigration techniques as a useful tool for the identification of similar strains of microorganisms.
Marie Horká1, Jaroslav Horký2, Anna Kubesová1, Karel Mazanec1, Hana Matoušková2, Karel Šlais1
1 UIACH, Brno, Czech Republic
2 SRS, Olomouc, Czech Republic
The detection and identification of pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic procedures to modern molecular-based methods. The wide range of methods employed reflects the great diversity
of microorganisms and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop
ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to
complement traditional techniques. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal
health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for pathogen identification,
particularly of unknown samples, has been less rapid. In this study similar strains of different Pseudomonas syringae pathovars were
identified by the gas chromatography of fatty acid methyl esters used in the routine laboratory and compared with results from the
147
Poster Abstracts
developed electromigration and mass spectrometry techniques. Acording to our experiments appropriate combination of electromigration techniques appear to be useful for fast detection the correct results rapidly and economicly.
This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic No. IAAX00310701 and by the Institutional research plan AVO Z40310501.
P174:
Liquid-phase microextraction and capillary electrophoresis of zwitterionic cetirizine in biological samples, as a main metabolite of
hydroxyzine
Ali Reza Fakhari Zavareh, Saeed Nojavan, Arezoo Moharami
Cetirizine hydrochloride, an antihistamine, is a major metabolite of hydroxyzine, and a racemic selective H1 receptor inverse agonist
used in the treatment of allergies, hay fever, angioedema, and urticaria. In this work porous hollow fiber liquid-phase microextraction
(LPME) was evaluated for the capillary electrophoresis (CE) of the cetirizine as a main metabolite of hydroxyzine in human plasma.
Cetirizine was extracted from 2 ml plasma sample through n-octanol immobilized in the pores of a porous polypropylene hollow fiber
and into 20 μl of 10 mM hydrochloric acid (pH 2.0) or 10 mM sodium hydroxide (pH 12.0) present inside the hollow fiber (acceptor
phase). Prior to extraction, the samples were made neutral (pH 7) in order to promote LPME of the zwitterions compounds. Owing to
the high ratio between the volumes of sample and acceptor phase, and owing to high partition coefficients, cetirizine was enriched by
a factor of 25 to 30. In addition, sample clean-up occurred during LPME since salts, proteins and the majority of endogenic substances
were unable to penetrate the octanol layer. Since the extracts were aqueous, they were injected directly into the CE instrument. Limit
of quantification for cetirizine in plasma was 5 ng/ml, while the limit of detection was 0.5 ng/ml. This enabled cetirizine to be analyzed
within the therapeutic range by LPME–CE and detection limit was comparable with previously reported methods1,2.
1 P. Kowalski, A. Plenis, Biomed. Chromatogr. 21 (2007) 903
2 S. Azhagvuel, R. Sekar, J. Pharm. Biomed. Anal. 43 (2007) 873
P175:
Preliminary Evaluation of Carbohydrate Determination with Direct UV Detection
François de l‘Escaille, Fabrice Benoit, Jean-Bernard Falmagne
Analis R&D Diag
Carbohydrates are important elements in living organism as energetic reservoir, as part of structural component and they play an
important role in several biological processes. Furthermore they are an important constituent in several food products.
There are different methods to analyze carbohydrates such as GC (Gas Chromatography) and HPLC (High Performance Liquid Chromatography). Capillary Electrophoresis has demonstrated to be also an interesting analytical technique due to their high selectivity and
low sample consumption. However carbohydrates do not absorb in the UV and are ionized only at high pH. For this reason carbohydrates need a derivatization process for detection with an UV or LIF detector. A good example is Beckman Coulter method using APTS
derivatization.
Stella Rovio1,2 and coworkers have developed a simple method for determination of monosaccharide by capillary electrophoresis using
a high pH buffer and direct UV detection.
We will present an adaptation of this method by introducing dynamic double coating3 of the capillary wall and perform a preliminary
evaluation of this technique.
1 Stella Rovio, Jari Yli-Kauhaluoma, and Heli Sirén (2007). Determination of neutral carbohydrates by CZE with direct UV detection. Electrophoresis 28, 3129 -3135.
2 Stella Rovio, Helena Simolin, Krista Koljonen, and Heli Sirén (2008). Determination of monosaccharide composition in plant fiber materials by capillary zone electrophoresis. J. Chromatogr. A 1185, 139 – 144
3 US Patent 5,611,903
148
Poster Abstracts
P176:
Selective preconcentration of amino acids and peptides using single drop microextraction
Yang-Ki Park1, Kihwan Choi1, Jihye Kim1, Ahmed Y. Badjah-Hadj-Ahmed4, Zeid A. Al-Othman4, Doo Soo Chung1
1 Department of Chemistry, Seoul National University, Korea
4 Department of Chemistry, King Saud University, Saudi Arabia
Single drop microextraction (SDME) using a hanging drop at the capillary tip was developed recently. Analytes in the aqueous donor
phase were enriched to the drop of an aqueous acceptor phase through a thin organic layer. However, preconcentration of amino acid
and peptide is difficult since their zwitterionic property prevents them from being partitioned to the organic phase. When amino acids
are derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadialzole (NBD-F), amino acids without a charged side chain were converted to simple
carboxylic acids. In the pH 1.0 donor phase, NBD-amino acid complexes are predominantly neutral, and they were successfully concentrated to a basic acceptor phase. On the other hand, amino acids with a charged side chain were not concentrated through SDME.
Therefore, selective SDME was possible, and an enrichment factor of about 550-fold was obtained within 10 min at 25°C. Furthermore,
this method was applied to the selective preconcentration of peptides.
P177:
Separation of biologically active pyridines using capillary- and chip-based electrophoresis with various detection modes
Nantana Nuchtavorn1, Leena Suntornsuk1, Marketa Ryvolova3, Mirek Macka3
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Rd., Rajathevee, Bangkok 10400, Thailand
3 National Centre for Sensor Research, Irish Separation Science Cluster and School of Chemical Sciences, Dublin City University, Dublin 9, Ireland
The detection of biologically active pyridines (i.e. nicotine, cotinine, nicotinic acid and nicotinamide) plays an important role in diagnostics of smoking related diseases. Capillary electrophoresis (CE) with UV detection has been previously shown to be a useful separation
technique and the separation selectivity has been studied in various electrolytes1. Chip-based CE offers even lower sample consumption
and shorter analysis time and therefore could serve as an efficient high throughput routine screening method. In this work, the separation of pyridine based compounds has been studied by both CE and chip-based CE using a variety of detection techniques including
absorbance (UV), capacitively coupled contactless conductivity (C4D) and fluorescence detection (FD). Separations by CE have been
investigated in acidic background electrolytes including phosphate buffer pH 2.1 and acetic acid pH 3.1 using CE-UV and C4D. In order
to achieve LODs required for biological samples, a substantial increase in the detection sensitivity is needed, which can be achieved by
CE coupled to FD. However, due to the lack of natural fluorescence of the target analytes, suitable pre-column derivatization methods
had to be investigated. Studies of the derivatisation reactions through separations of the reaction products were carried out using
both capillary- and chip-based CE. The results were first compared to the validated CE-UV method for the determination of nicotine,
nicotinamide and nicotinic acid in pharmaceutical formulations.
1 Nuchtavorn N, Suntornsuk L, Analysis of selected biologically active pyridines by capillary zone electrophoresis, Proc. 21st International Ion Chromatography Symposium (IICS 2009), Malahide, Co. Dublin, Ireland, 21-24 September 2009, poster presentation P504-T, p.142
P178:
CZE determination of activities of some enzymes involved in cytokinin biosynthesis and degradation
Tibor Beres*1, Petr Tarkowski2, Vitezslav Maier3, Marketa Gemrotova1, Marta Kowalska2, Karel Dolezal1, Miroslav Strnad1
1 Laboratory of Growth Regulators, Palacky University & Institute of Experimental Botany AS CR, Slechtitelu 11, 78371 Olomouc, Czech Republic
2 Department of Biochemistry, Faculty of Science, Palacky University, Slechtitelu 11, 78371 Olomouc, Czech Republic
3 Department of Analytical Chemistry, Faculty of Science, Palacky University, Tr. 17. listopadu 12, 77146 Olomouc, Czech Republic
Cytokinins (CKs) are plant hormones involved in various aspects of plant growth and development. Prenylation of the exocyclic amino
149
Poster Abstracts
group of ATP/ADP by adenosine phosphate isopentenyltransferase (IPT) is considered to be the first, rate-limiting step of their biosynthesis.
Nine putative genes (AtIPT1-AtIPT9) were identified in Arabidopsis thaliana genome. However, the products of IPT action, intact di- or
triphosphorylated CKs have not been identified in plant material, yet. On the other hand cytokinin oxidase/dehydrogenase (CKX), is an
enzyme catalyzing the cleavage of the isoprenoid side chain of N6-(2-isopentenyl)adenine (iP) and zeatin (Z), converting them to adenine
and corresponding unsaturated aldehydes. In Arabidopsis thaliana, seven CKX isoenzymes are present (AtCKX1-AtCKX7). Their substrate
specificity towards various CK phosphates has never been determined.
In this work an off-line CE assay method was developed to study some in vitro reactions of adenosine and prenylated adenosine (CK) nucleotides using recombinant enzymes involved in CK biosynthesis (AtIPT1) and degradation (AtCKX1, 3, 7). The main studies were focused
on the substrate specificity determination and reaction kinetics monitoring. The reaction conditions were taken from previously used
assays or slightly modified to fulfil the demands needed for efficient CE separation. The measurement itself was performed by means of a
homogenous enzyme assay. The reaction was initiated by adding an enzyme solution to the reaction mixture containing the substrates and
injected to the capillary immediately. All the substrates and reaction products of off-line enzymatic reactions were separated by CE with UV
detection as anions in alkaline pH buffer using strong electroosmotic flow at positive separation voltage. Separation optimization included
appropriate buffer (co-ion as well as counter-ion) selection. The optimal buffer was chosen with respect to the best resolution, highest peak
efficiency and the lowest current generation. The quantification was done using UMP as an internal standard. The kinetics was determined
by repeated injection without stopping the enzymatic reaction. Currently, the products of the CK biosynthesis and degradation are assayed
immunochemically (ELISA) or by LC/MS. Both methods demand an enzymatic dephosphorylation step prior to detection. CZE overcomes
this problem and is capable of intact nucleotide separation. Despite the fact of higher detection limits, the need for minimal sample treatment together with small material consumption made this method a cheaper and much more rapid alternative to currently used methods. Wednesday, March 24th, 2010
P179:
Possibilities of pITP-FT-SERS approach in the study of buserelin concentration levels in human urine
Václav Ranc1, Andrea Staňová2, Vítězslav Maier1, Joanna Znaleziona1, Radim Knob1, Juraj Ševčík1, Dušan Kaniansky2, Jozef Marák2
1 Department of Analytical Chemistry, Faculty of Natural Sciences, Palacký University, Třída Svobody 8, CZ-77146 Olomouc, Czech Republic
2 Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University,Mlynská Dolina CH2, SK-842 15 Bratislava, Slovak Republic
FT-SERS represents one of the potential identification method for an analysis of species at low concentration levels. The main limitation
of only few using of this instrumental method in analytical practice lies in its complicated ability to identify compounds present in complex mixtures. This is a place where preparative isotachophoresis (pITP) can help. Preparative isotachophoresis can be used as sample
pretreatment technique for complex samples, often of biological origin, and provides the possibility to isolate ionogenic compounds
into the several discrete fractions. Fractions can be further analyzed by an orthogonal separation technique (CE, HPLC, GC) or via
coupling the separation technique with a spectral technique providing essential structure information, e.g. CE-MS, HPLC-MS, GC-MS, GCAED. When utilizing techniques which are not fully compatible, off-line coupling of such systems presents a simple and straightforward
solution. The abilities of proposed analytical system were tested on the analysis of buserelin in the diluted human urine. Buserelin is one
of the potential drugs for a treatment of prostate and breast cancer. This nona-peptide (5-oxoPro–His–Trp–Ser–Tyr–D–Ser(t-Bu)–Leu–
Arg–Pro–NHC2H5; Mr = 1238.66 g.mol-1, pI = 10.5) represents a synthetic analogue of natural gonadotropin-releasing hormone agonist
(GnRH agonist). The study was performed using the set of 9 model samples containing 3 concentration levels of analyte (no buserelin,
8 × 10-8M, 8 × 10 -7M) and 3 levels of human urine (no urine, 100× diluted, and 10× diluted). Samples were separated using pITP analyzer
and 6 fractions were collected in one run. Each fraction was lyophilized, diluted by 100 uL of deionized water and measured using
FT-SERS. Results for each fraction were compared with the spectrum of buserelin standard. Using this way of evaluation, buserelin
was successfully detected in the 4th fraction of each model samples containing buserelin. The next steps taken on this project will be
focused on a careful study of influence of experimental parameters on identification and quantification approach based on the FT-SERS.
This work was supported by a grant from the Slovak Research and Development Agency (No. VVCE-0070-07), by the Research Project
of the Ministry of Education of the Czech Republic No. MSM6198959216 and the grant of Slovak Grant Agency, No. 1/0882/09. Monday, March 22nd, 2010 P180:
Preliminary results of 15-F2-Isoprostane Analysis using CE-MS
Koen Raedschelders, Yu Hui, Xufei Zhong, David M. Ansley, David D.Y. Chen
150
Poster Abstracts
The University of British Columbia
Oxidative stress incurred during aortocoronary bypass grafting with cardiopulmonary bypass is a significant factor in intraoperative ischemia reperfusion injury. Oxidative stress is a pathophysiological state where reactive oxidative species (ROS) overwhelm endogenous
antioxidant defense. 15-F2-Isoprostane (15-F2-IsoP) is a sensitive and specific marker of in vivo phospholipid oxidation and oxidative
stress. Quantitative analysis of 15-F2-IsoP can be complicated by the existence of regioisomers, stereoisomers and similar prostaglandinlike structures in plasma. While GC-MS and LC-MS have shown good specificity and sensitivity for the analysis of 15-F2-IsoP, CE-MS may
provide additional benefits in terms of separation efficiency and simplicity of sample preparation and instrumentation.
We are currently developing a CE-MS based method for the quantitative analysis of 15-F2-IsoP. Our ultimate goal is the routine incorporation of a CE-MS based 15-F2-IsoP analysis into a parallel clinical study of patients undergoing aortocoronary bypass grafting. The first
step in this technique involves immunoaffinity column purification of 15-F2-IsoP. The sample is subsequently dried and resuspended in
50% methanol. The sample is injected into the capillary for 30 seconds at 1psi. The analyte is preconcentrated online using dynamic pH
junction velocity-difference induced focusing with a backround electrolyte consisting of 1% ammonium hydroxide in 50% methanol. The
ion source is an ESI interface developed by our lab with a low flow modifier of 1% ammonium hydroxide in methanol.
Our preliminary data suggest that 1) a relatively large volume can be injected in conjunction with pH-junction focusing; 2) the limit
of detection for this technique so far is below 10ng/mL; 3) along with the pre-concentration effect of the immunoaffinity column, we
predict the detection limit of 15-F2-IsoP in plasma to be below 100pg/mL, which would be comparable to LC-MS based detection limits.
Preliminary results from head-to-head experiments comparing CE-MS and LC-MS show an advantage in sample consumption and analysis time using CE-MS.
P181:
Study on the glucose oxidase enzymatic hydrolysis in capillary enzyme microreactor with capillary zone electrophoresis
Siming Wang, Ping Su, Hongjun E, Yi Yang
College of Science,Beijing University of Chemical Technology
As a kind of biomacromolecule with catalytic function, enzymes have been widely used in the fields of food and medicine industry, and
biochemical analysis, etc. Comparing with free enzymes, the immobilized enzymes have better stability, high enzymatic efficiency and
can be reused and separated easily. The capillary enzyme microreactor is a new developed immobilized enzymes microreactors which
have attracted widespread attention in recent years. Due to less sample consumption, easy separation and automation, and suitable
for multiple detectors, the capillary enzyme microreactor has a potential application in high-throughput screening enzyme reaction and
enzyme inhibitors. In this study, a capillary enzyme microreactor was prepared by immobilizing glucose oxidase (GOD) onto inner wall
of fused-silica capillary with glutaraldehyde as a cross-linking agent. The enzymatic hydrolysis of ß-D-Glucose in the prepared microreactor was evaluated by capillary zone electrophoresis. Based on fused silica capillary modified with 3-aminopropytriethoxysilane, a
kind of bonding immobilized GOD capillary microreactor was prepared by the glutaraldehyde approach. According to the peak area of
the enzymatic product of gluconic acid, the enzymolysis conditions that include substrate concentration, temperature, pH environment
and flow velocity were investigated, respectively. The results showed that high efficiency of the enzymatic hydrolysis could be obtained
at pH 8.0 and 40 % for 1 mg/ml ß-D-Glucose with flow rate of 0.83 μL min-1. The enzyme micro-reactor was stable while its activity was
decreased less than 10 % within a week. The Experimental data provided some evidence for the development and application of online
immobilized capillary enzyme microreactor.
P182:
Transferrin immunoextraction for determination of CDT in human serum by CZE
Jitka Caslavska*, Wolfgang Thormann
Department of Clinical Pharmacology and Visceral Research, University of Bern, Bern, Switzerland
The high-resolution capillary zone electrophoresis (CZE) assay for carbohydrate-deficient transferrin (CDT) in which serum is mixed with
an iron-containing solution prior to analysis, was found to be an effective approach for the determination of CDT in patient sera1. Sera of
patients with progressed liver cirrhosis, however, can have a low transferrin level and/or are prone to interferences in the b-region which
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Poster Abstracts
prevent the proper determination of CDT by CZE. Previous work revealed that immunopurification of transferrin is the best approach to
pretreat such samples2. The need of a simple and economic approach to immunoextract transferrin from human serum prompted us to
investigate the use of a laboratory-made anti-Tf centrifugal column with an immunoaffinity medium composed of polyclonal anti human
transferrin antibodies linked to Sepharose 4 Fast Flow beads. A Pierce Centrifuge column # 89896 (5 ml total volume) with a screw cap
on top and a press-on bottom cap served as container to hold 1 ml immunoaffinity medium and the applied sample. Lipemic sera are
delipidated using a mixture of di-isopropyl ether and butanol (60:40)3. Five-fold diluted untreated or delipidated serum (400 mL diluted
to 2000 mL with pH 7.4 Tris/HCl buffer containing 150 mM NaCl ) is applied to the column and incubated for 1 h at room temperature
while gently full end-over-end mixing on a rotator with a fixed speed of 20 rpm. After immunocapture, the column is rinsed with the
same buffer used for dilution to remove non-specifically bound proteins. Transferrin is stripped from the beads into a neutralizing Tris
buffer containing BSA using an acidic glycine buffer. Prior to analysis by CZE, the extract is concentrated and the buffer is replaced
with 50 mM pH 8.4 Tris/HCl buffer containing 150 mM NaCl using Vivaspin 2, an ultrafiltration spin column with a 30000 Da cutoff.
The developed procedure was applied to a number of relevant patient samples and could thereby be shown to represent an effective
method for transferrin extraction and concentration. CDT could unambiguously be determined in all pretreated samples.
This work was supported by the Liver Foundation (Bern, Switzerland) and by the Swiss National Science Foundation.
1 J. Joneli, C. Lanz, W. Thormann, J. Chromatogr. A 1130, 272-280 (2006).
2 C. Lanz, J.-B. Falmagne, F. de l’Escaille, U. Marti, W.Thormann, J. Chromatogr. A 1206, 33-40 (2008).
3 B.E. Cham, B.R. Knowles, J. Lipid Research 17, 176-180 (1976).
P183:
High-quality Screening of Pharmaceutical Chaperones by Capillary Electrophoresis
Meera Shanmuganathan, Philip Britz-McKibbin
The proposed goal of this research project is to develop a simple, sensitive yet selective inhibition assay based on capillary electrophoresis (CE) for high-quality screening of pharmaceutical chaperones (PCs) that target ß-glucocerebrosidase (GCase). To date, several
types of in-vitro GCase enzyme assays have been reported for drug screening using fluorescence methods, which are constrained by
interferences caused by substrate autofluorescence and/or fluorescence quenching. In this work, the rate of GCase-catalyzed hydrolysis
of 4-methylumbelliferyl-ß-D-glucopyranoside (MUG) will be quantified directly by CE with UV detection using several different classes
of orthosteric and/or allosteric inhibitors as a model system. The pH and surfactant concentration dependence of inhibitor binding on
GCase activity will also be examined by CE. PCs that can stabilize the conformational stability of mutant GCases upon binding under
neutral pH conditions of the endoplastic reticulum (ER) offer a promising therapeutic strategy for treatment of lysosomal storage
disorders, such as Gaucher’s disease.
P184:
Capillary electrophoresis can assess the effect of a bis-tacrine derivative on abeta-protein oligomerization
Raffaella Colombo1, Stefania Butini2, Egeria Guarino2, Samuele Maramai2, Sandra Gemma2, Ved Chauhan6, Giuseppe Campiani2,
Ersilia De Lorenzi1
1 Department of Pharmaceutical Chemistry, University of Pavia, Pavia, Italy
2 European Research Centre for Drug Discovery and Development (NatSynDrugs), and Department of Pharmaceutical and Applied Chemistry, University of Siena, Siena,
Italy
6 Institute for Basic Research in Developmental Disabilities, Forest Hill Road, Staten Island, NY, USA
Alzheimer’s disease is a form of amyloidosis, a disorder where the conformational changes of a protein ultimately lead to the deposition of insoluble fibrils. The formation of amyloid fibrils is a dynamic and complex process, as it implies the formation of different
intermediate species at equilibrium, including non-covalently associated soluble oligomers, protofilaments and protofibrils, according
to a nucleation-elongation mechanism. The characterisation of such a process can be partially elucidated by a variety of spectroscopic
techniques that nevertheless yield population-average data. In this respect capillary electrophoresis is an excellent complementary tool
that enables the separation and the detection of these species during their formation and just before fibril deposition. We exploited
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Poster Abstracts
this potential of CE with Abeta42 peptide, which is found in the brain of Alzheimer’s disease patients. The availability of a CE method
capable of monitoring these processes (1) is extremely valuable, as it can be used as a starting point for co-incubation studies with small
molecules, to investigate the action exerted on the equilibrium of these intermediate species, and to verify, by electron microscopy,
whether there is a delay or an inhibition of fibril formation. Our previous results have already demonstrated the applicability of this CE
method, by assessing the effect of two antitumorals on a toxic oligomeric intermediate of the fibrillogenesis of Abeta42 (2, 3).
In an effort to develop potential disease-modifying anti-Alzheimer’s tools and on the basis of our previous experience in the field (4), we
disclosed a bis-tacrine-elongated compound (NF1056) as a potent reversible inhibitor of human acetylcholinesterase (hAChE) catalytic
and non catalytic function, endowed with antiamyloidogenic properties. The effect exerted on neurotoxic forming oligomers, as found
by using CE, provides further clues on the mechanism of action of NF1056 and could make this compound a promising candidate to
develop a new class of Multi-Target Directed Ligands with a peptidomimetic structure. Wednesday, March 24th, 2010
P185:
Analysis of calix[4]arenes by nonaqueous capillary electrophoresis
Yi-Ping Lee, Hsiu-Li Su, Huan-Yi Hung, You-Zung Hsieh
National Chiao Tung University, Hsinchu, Taiwan
In this study, we developed a simple and efficient nonaqueous capillary electrophoresis (NACE) method to analyze calix[4]arenas. Calixarenes are macrocyclic products of phenol-formaldehyde condensations which inner cavity can accept various molecules. The phenolic
hydroxyl groups of calixarenes influence the inner cavity of calixarenes. The less phenolic hydroxyl groups are, the smaller radius of cavity is. For instance, calixarenes with four phenolic units are called calix[4]arenes, which have better selectivity for small metal ions than
others do. The effects of buffer composition, nonaqueous solvent composition and proportion, and concentration of nonaqueous buffer
on the electrophoretic mobility of calix[4]arenes were examined. These analytes were successfully separated by using an acetic acid/
ammonium acetate buffer in formamide/methanol nonaqueous solvent. The changes on analytes’ mobilities are due to the different
degree of deprotonation on phenolic hydroxyl groups of calix[4]arenes. The most important factor on analytes’ mobilities is nonaqueous
solvent composition. The highest separation efficiency was achieved in an acetate buffer solution with formamide/methanol (50:50, vol).
The relative standard deviations of the analytes’ migration times were less than 1.0% under the optimized separation condition. The
limits of detection of the analytes also were satisfactory. Experimental results reveal that nonaqueous capillary electrophoresis exhibits
adequate separation efficiency and satisfactory quantitative data for analysis of calix[4]arenes.
P186:
Detection of trace number of risk pathogens in real samples using electromigration separation techniques and MALDI
Marie Horká1, Anna Kubesová*1, Jiří Šalplachta1, Dana Moravcová1, Eva Zapletalová5, Jaroslav Horký5, Karel Šlais1
1 Institute of Analytical Chemistry of the ASCR, v. v. i., Veveří 97, 602 00 Brno, Czech Republic
5 State Phytosanitary Administration, Division of Diagnostics, Šlechtitelů 23, 77900 Olomouc, Czech Republic
Abstract: Plant, animal and human diseases initiated by the particles of biological origin have had major economic and social impacts
during history. Thus the prediction of the unexpected outbreaks of diseases by sensitive detection of pathogens is necessary. Rapid
genotyping identification techniques have precedence over slow traditional phenotyping techniques for the development of strategies
to minimize the risk of pandemics. Preconcentration techniques play here important role. Modern molecular diagnostics have made it
possible to rapidly detect, identify and quantify traces of pathogen now. However, great potential have electromigration techniques,
which can integrate preconcentration, separation, detection and make possible subsequent cell cultivation. Combination of these
techniques with MALDI mass spectrometry fingerprint of the whole cells can predict the risk pathogens. For our experiments conidia
from fungi were selected as a model bioparticles. The brown rot or mummified fruit produced by Monilinia species, causes major losses
in agrosystems. The basic unknown electromigration properties of the conidia from Monilinia laxa, Monilinia fructigena and Monilinia
fructicola were determined. These knowledges have been used at their preconcentration by the free-flow isoelectric focusing techniques
from the real sample extracts. The efficiency of the preconcentration and a preliminary identification were verified by the electromigration techniques and MALDI and by the phenotyping technique after several week cultivation of the focused sample of conidia.
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Poster Abstracts
This work was supported by the Grant Agency of the Academy of Sciences of the Czech Republic No. IAAX00310701 and by the Institutional research plan AVO Z40310501.
P187:
Determination of drug stability against human liver microsomes by utilization of short-end injection capillary electrophoresis mode
Roman Řemínek1, Jochen Pauwels2, Jos Hoogmartens2, Ann Van Schepdael2, Zdeněk Glatz1
1 Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
2 Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Catholic University of Leuven, O&N II Herestraat 49,3000 Leuven, Belgium
During a new drug development, the pharmaceutical companies endeavour to predict action of enzymes on the given new drug candidate
within human body. This knowledge enables to omit the substances with the inappropriate qualities and thus simplify implementation of
the following tests. The current methods of drug stability assessment based on incubations with the whole hepatocytes or even liver slices
are laborious and time-consuming nevertheless. In this regard, the human liver microsomes (HLM) provide possibility of unfavourable state
circumvent, because they offer advantage of fast analysis combined with the preservation of realistic simulation of conditions inside the
liver.
The goal of this study was to introduce a new method based on the capillary electrophoresis (CE) allowing assessment of new drug candidate stability against HLM. Since, HLM contain high concentrations of membrane-bond proteins, the short-end injection CE mode was used
to reduce the time spend by sample inside the capillary. A replaceable polymeric gel as a background electrolyte was used either to avoid
the adsorption of sample components onto inner capillary wall in order to ensure the high reproducibility of the method.
Considering the large variety of possible new drug substances, the method based on NADP+ production monitoring was hired rather than
usually used measurement of substrate consumption or product creation respectively. In this manner, the method allows analysis of every
compound which does not co-migrate with NADP+.
As result the new generic method enabling fast determination of new drug candidate stability was established. The method’s validation
showed an RSD of 1.77 % for migration time (n = 6), an RSD of 2.04 % for relative peak areas (n = 6), an LOD of 6.5 μmol∙l-1 (S/N = 3), an LOQ
of 20 μmol∙l-1 (S/N = 10) and a recovery of 100.63-103.91 % (n = 6) for NADP+. Finally, method’s potentiality was proved by carrying out of
fast screening of 12 chosen probe drugs.
This work was supported by research scholarship from Flemish Ministry of Education, by research project No. MSM0021622413 and research centre No. LC06023 both from Czech Ministry of Education and by grant No. P206/10/0057 from The Czech Science Foundation. Wednesday, March 24th, 2010
P188:
Focusing and mobilization of bacteria by capillary isotachophoresis
Farid Oukacine1, Laurent Garrelly2, Bernard Romestand3, David M. Goodall4
1 Institut des Biomolécules Max Mousseron, Université de Montpellier 2, place Eugène Bataillon, 34095 MONTPELLIER, France
2 COLCOM, Cap Alpha Avenue de l’Europe, Clapiers, 34940 Montpellier, France
3 Laboratoire Ecosystèmes Lagunaires, place Eugène Bataillon, 34095 MONTPELLIER, France
4 Paraytec Ltd, 1a St Georges Place, YORK YO24 1GN, United Kingdom
For more than twenty years, the analysis of bacteria by capillary electrophoresis (CE) has been studied with great interest. The goal was
generally to develop a fast and straigthforward method for the separation and quantification of microbial food contamination or of microorganisms in biological samples. This approach based on separation techniques does not require the use of time-consuming microbial
cultures. However, CE analysis of bacteria is complicated by the heterogeneity in composition of the bacterial suspensions, by the formation
of bacterial aggregates of different sizes, and by the size and shape dependence of the orientations of the microbes under the electric field.
These issues that are characteristic to the CE analysis of microbes lead to poor repeatability with multiple small peaks corresponding to different bacterial aggregates.Three main CE methods were reported in the literature in order to limit the emergence of multiple peaks: (i) the
use of a neutral polymer (polyethylene oxide, PEO) in a TRIS / boric acid/ EDTA buffer at low ionic strength1, 2, (ii) the Large Volume Sample
Stacking with polarity switching (LVSS) in a TRIS / boric acid/ EDTA buffer in presence of PEO, and (iii) capillary isoelectric focusing (CIEF)1.
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Poster Abstracts
In this work, we propose the use of isotachophoresis mode (ITP) for the focusing and mobilization of bacteria. This ITP mode allows the
detection and quantification of Micrococcus lysodeikticus (also called Micrococcus luteus, studied as a model of Gram+ bacteria) in only
one single peak of high efficiency without spikes that could come from the formation of irregular clusters and aggregates of bacterial cells.
This method has been successfully applied on both fused silica and coated (hydroxypropylcellulose and polyethylene glycol) capillaries. It
leads to very short migration times while maintaining very low electrical currents in order to minimize the overheating of the capillary and
the lysis of bacteria.
The use of a UV detector with multiple detection points and UV imaging (ActiPix D100, Paraytec) allowed us to visualize in real-time the
moving of the focused bacterial zone along a 9 mm long window without sacrificing spatial resolution of the detector (70 µm). Furthermore,
the implementation of four successive UV detection points located at 15 cm one each other permitted to check if the steady state of the
ITP process was reached according to the experimental conditions.
1 Armstrong, D.W.; Schulte, G.; Shneiderheinze, J.M.; Westenberg, D.S.; Anal. Chem. 1999, 71, 5465-5469
2 Armstrong, D.W.; Girod, M.; He, L.; Rodriguez, M.A.; Wei, W.; Zheng, J.; Yeung, E.S.; Anal. Chem. 2002, 74, 5525-5530
3 Yu, L.; Li, S.F.Y.; J..Chromatogr. A 2007 ,1161, 308-313 Monday, March 22nd, 2010
P189:
Determination of pharmaceutical residues in river water and wastewater by solid phase extraction and high performance liquid
chromatography with mass spectrometric detection
Lucie Vydrová*, Milada Vávrová, Hana Vítečková, Josef Čáslavský, Ludmila Mravcová
Brno University of Technology, Faculty of Chemistry, Brno, Czech Republic
Pharmaceutically active compounds are complex molecules with different functionalities, physicochemical and biological properties.
The reason of their development and high consumption is more or less specific biological activity of these compounds. They have often
basic or acidic functionalities. Pharmaceuticals are continually escaping into the environment as a result of their industrial production
and domestic use. Since pharmaceuticals are designed with the intention of performing defined biological effect, this concept may be
very important for these specific substances in terms of fate and effects toward non target organisms in the environment, even at low
concentrations.
In our study, following pharmaceuticals were chosen: Diclofenac, Ketoprofen, Ibuprofen, Naproxen, Salicylic acid, Mefenamic acid and
Paracetamol. All of them belong to a group of analgesics which is one of the most used groups of pharmaceuticals. Drugs were analyzed
in surface water from relatively clean region (Křetínka River situated in Bohemian-Moravian Highlands) and in wastewater taken in the
large scale waste water treatment plant situated in the Brno – Modřice.
Selected non-steroidal anti-inflammatory drugs were extracted from waters by solid phase extraction (SPE). High performance liquid
chromatography with mass spectrometric detection was the method of choice for the analysis of isolated compounds. This combination
of instruments together with preparation procedure based on SPE gives us a highly sensitive analytical method with the possibility
of determination at the levels of ng/L. Analytes were separated using reversed phase system, electrospray was used as the ionization
method, single and tandem mass spectrometry on spherical ion trap was employed for their detection and identification.
This study was conducted within the activities related to the project COST Action 636, No. OC-183 and project FRVS No. 2604/2009. Tuesday, March 23rd, 2010
P190:
An Alternative Approach for Characterization of Impurities and Site-specific Modifications in Protein Drugs
Martin Gilar, Hongwei Xie, John C. Gebler
Waters Corp.
Sequence variants and posttranslational modifications (PTMs) such as glycosylation, deamidation, and oxidation are common in recombinant protein pharmaceuticals. They potentially affect the safety, activity and stability of protein drugs. Effective monitoring of these
variants and PTMs requires sensitive and reproducible methods. Traditionally, LC-MS and multiple tandem mass spectrometry (either
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Poster Abstracts
DDA-MS/MS or targeted MS/MS) measurements are required for elucidation of such impurities and covalent modifications. In this study,
we have evaluated an alternative approach for mapping protein digests, employing LC coupled with Data Independent Acquisition (DIA)
Mass Spectrometry with alternated high-energy and low energy scanning (LC-MSE). The peptide precursor and fragmentation information acquired in MSE mode was utilized for effective identification of peptide sequences, and site-specific modifications within a single
LC run. The stoichiometry of PTM modifications was inferred from the relative ratio of the MS signal intensities between modified and
unmodified peptides. Nearly complete sequence coverage was obtained for yeast enolase (96 %), ADH (98 %) and IgG ( > 97 % for both
heavy and light chain) proteins. Minor protein contaminants were found to be detected in enolase at levels between 1.4 % and 13.4 %
(normalized to enolase 1). M-oxidation and N-deamination sites were characterized and quantified. Synthetic peptide standards were
utilized to confirm the identified deamidations in “PENNY” motif peptide of IgG heavy chain, and to distinguish aspartic and isoaspartic
isoforms of N-deamidations.
P191:
Capturing drugs by lipid dispersions
Jana Lokajova1, Jaana Laine2, Jevgeni Parshintsev2, Juha M. Holopainen4, Susanne K. Wiedmer2
1 Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Albertov 2030, 128 43 Prague 2, Czech Republic
2 Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland
4 Helsinki Eye Lab, Department of Ophthalmology, Haartmaninkatu 4 C, University of Helsinki, 00290 Helsinki, Finland
The number of drug overdoses and other kinds of accidental poisoning leading to severe intoxication or even death is still today unfortunately growing rapidly. Our long-term aim is to develop a sensitive, efficient, and non-harmful lipid-based formulation to specifically
trap harmful substances in vivo. We have investigated the use of synthetic lipid mixtures for this purpose and compared the efficiency
of these for capturing drugs with a commercial lipid emulsion (Intralipid). Intravenous infusion of Intralipid dispersion has lately attained increasingly attention because of its promising use for resuscitation of patients suffering from severe, systemic drug toxicity. The
methods we used for in vitro interactions between drugs and lipid dispersions included EKC, CZE, direct infusion ESI-MS, immobilized
Intralipid chromatography-UV, immobilized Intralipid chromatography-APCI-MS(IT), and fluorescence polarization measurements.
The drug retention factors and distribution constants were calculated from the EKC data and the results demonstrate that there is
higher interaction between the drugs and the negatively charged liposome dispersion than with the commercial Intralipid dispersion.
The electrophoretic mobilities of liposomes were calculated from data obtained previously by LEKC-LIF runs using a fluorescent lipid
marker. Since labeled Intralipid was not accessible, the electrophoretic mobility of the Intralipid dispersed phase was calculated using
the iterative procedure and a homologous series of alkyl phenyl benzoates (C1-C6), and the retention factors for the analytes were
determined.
Our results demonstrate that the studied techniques are promising tools for in vitro study on interactions between lipid dispersions
and low-molar mass drugs.
Interactions between local anesthetics and lipid dispersions studied with liposome electrokinetic capillary chromatography, J. Muhonen, J.M. Holopainen, S.K. Wiedmer, J. Chromatogr. A, 1216 (2009) 3392-3397;
Liposomes for entrapping local anesthetics. A liposome electrokinetic chromatographic study, J. Lokajová, J. Muhonen, E. Puukilainen, M. Ritala, J.M. Holopainen,
S.K. Wiedmer, Electrophoresis, in press;
Interaction of commercial lipid dispersion and local anesthetics in human plasma. Implications to drug trapping by ‘lipid-sinks’, J. Laine, J. Lokajová, J. Parshintsev,
J.M. Holopainen, S.K. Wiedmer, Anal. Bioanal. Chem., in press Monday, March 22nd, 2010
P192:
Innovation of special study programs aimed at analysis of biological materials at University of Pardubice
Lucie Korecká*1, Karel Ventura2, Zuzana Bílková1
1 University of Pardubice, Department of Biological and Biochemical Sciences, Studentska 573, 530 03 Pardubice, Czech Republic
2 University of Pardubice, Department of Analytical Chemistry, Studentska 573, 530 03 Pardubice, Czech Republic
Operational program for education and competitiveness funded by European Union sponsors also the project Innovation of study pro156
Poster Abstracts
grams „Special Chemical and Biological Fields of Study“ at University of Pardubice. The main resolver is the Department of Biological
and Biochemical sciences and duration time of the project is 3 years.
The Department of Biological and Biochemical Sciences at the Faculty of chemical technology, was established in 1996 to precise
the education and practical preparation of students for analysis of biological materials. The department has committed to provide
the specialists in diagnostic assays in medicine – clinical biochemistry, immunochemistry, microbiology, molecular genetics etc. The
department is in a constant need for qualified personnel who shall oversee and assist in the construction and developing of diagnostic
methods and instruments.
The subject of the project is to renovate existing two bachelor (Clinical biology and chemistry, Medical laboratory technician) and one
master level (Analysis of biological materials) in frame of study programmes Special chemical-biology branches. Innovation will be held
according new trends in clinical laboratory diagnostics and analysis of biological materials. The main aim is to provide interdisciplinary
education with possible occupational chance of graduates not only in Czech Republic but even in other countries of European Union.
Graduates acquire theoretical and practical knowledge in clinical laboratory and diagnostic branches and know how to operate with
the relevant analytical devices controlled by computing technology and use modern laboratory instrumentation, following up the actual
trends as miniaturization and automatization. Project team forms our academics, specialists from clinical laboratories, external lecturers, partners from Hospital of Pardubice and laboratory company SEDIUM.
Authors would like to acknowledge to the ESF project “INCHEBIO” CZ.1.07/2.2.00/07.0139.
P193:
Increasing activity of bacterial biocatalyst with toxic anthropogenic substrate using methods of protein engineering
Pavel Dvořák*, Martina Pavlová, Martin Klvaňa, Jan Brezovský, Radka Chaloupková, Zbyněk Prokop, Jiří Damborský
Loschmidt Laboratories, Institute of Experimental Biology and National Centre for Biomolecular Research
Faculty of Science, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic
Haloalkane dehalogenases (HLDs; EC 3.8.1.5.) are microbial enzymes that catalyze hydrolytic conversion of haloalkanes to the corresponding alcohols and hydrogen halides1. Some synthetic halogenated alkanes, like 1,2-dichloroethane (DCE), are produced worldwide
and are classified as persistent environmental pollutants. DCE is widely used as solvent and intermediate in chemical industry. Its toxic
and carcinogenic effects are well recognized. HLDs have been investigated for possible applications in detoxification of halogenated
pollutants including DCE2,3. It remains a challenge to provide HLDs that could degrade these substrates efficiently. Here we report
construction of a mutant dehalogenase with improved catalytic activity towards DCE. Dehalogenase DhaA from Rhodococcus erythropolis Y24 has only minimal activity with DCE. Large cavity of DhaA active site and wide mouth of the main tunnel cannot effectively
accommodate small DCE molecule and results in non-productive binding. We hypothesized that reduction of size of DhaA active site and
tunnels together with elimination of non-productive binding could lead to the increased activity with DCE. Initially, computer modeling,
site-directed and saturation mutagenesis were applied to target the passage of ligands through the access tunnels5. Up to seven bulky
substitutions were introduced into the walls of DhaA tunnels and in vicinity of the active site. Closing up the tunnels influenced the
substrate specificity of DhaA and improved the enzyme’s activity towards DCE 8-times. This DhaA variant was used as a template for
the second round of in vitro evolution. Other four residues were randomized with the aim to reduce the size of DhaA active site and
to influence the non-productive binding of DCE above the catalytic histidine. Screening of four thousand clones from resulting mutant
libraries revealed one positive DhaA variant which showed 12-times higher specific activity with DCE compared to the wild-type DhaA.
The substitutions showing the most significant impact on DhaA activity with DCE will be inserted to another HLD to validate their effect.
Understanding the structure-function relationships in constructed mutants is important to design next generation of biocatalysts for
degradation of anthropogenic substrate.
1 Janssen, D.B. (2004) Curr. Opin. Chem. Biol. 8: 150-159
2 Stucki, G., Thuer, M. (1995) Environ. Sci. Technol. 29: 2339-2345
3 Pavlova, M., Klvana, M., Prokop, Z., Chaloupkova, R., Banas, P., Otyepka, M., Wade, R., Tsuda, M., Nagata, Y., Damborsky, J. (2009) Nat. Chem. Biol. 5: 727-733
4 Kulakova, A.N., Larkin, M.J., Kulakov, L.A. (1997) Microbiology 143: 109-115
5 Klvana, M., Pavlova, M., Koudelakova, T., Chaloupkova, R., Dvorak, P., Stsiapanava, A., Kuty, M., Kuta-Smatanova, I., Dohnalek, J., Kulhanek, P., Wade, R.C., Damborsky,
J. (2009) J. Mol. Biol. 392: 1339-1356
157
Poster Abstracts
P194:
New sample preparation technique for HPLC analysis of alpha-tocopherol in erythrocyte membrane
Jiří Plíšek1, Hana Vlčková2, Lenka Krčmová1, Markéta Kašparová1, Dagmar Solichová5, Petr Solich2, Luboš Sobotka5
1 Univerzita Karlova v Praze, Farmaceutická fakulta v Hradci Králové, Katedra analytické chemie, Heyrovského 1203, 500 05 Hradec Králové
Fakultní nemocnice, Klinika gerontologická a metabolická, Sokolská 581, 500 05 Hradec Králové
2 Univerzita Karlova v Praze, Farmaceutická fakulta v Hradci Králové, Katedra analytické chemie, Heyrovského 1203, 500 05 Hradec Králové
5 Fakultní nemocnice, Klinika gerontologická a metabolická, Sokolská 581, 500 05 Hradec Králové
Free oxygen radicals influence aging process, tissue damages and cancerogenesis. Alpha-tocopherol (vitamin E) is antioxidant which is
stored in cell membranes in all body tissues and contributes to the general protection of the organism. Imbalance between production
and elimination of free radicals rises as result of alpha-tocopherol deficit. Consequently oxidation stress, which affects integrity of
membranes and endangers a lot of physiologic processes dependent on membrane integrity, is formed. Monitoring of alpha-tocopherol
levels in erythrocyte membranes contributes to clear up mechanisms of action and antioxidant balance changes for example in LDL
aphaeresis treatment and in anticancer therapy.
In this study the new and modern method using combination of gradient ultracentrifugation and solid-phase extraction for sample
preparation before HPLC analysis of alpha-tocopherol in erythrocyte membrane is presented. Firstly, erythrocyte ghosts were prepared
in ultracentrifuge TL-100 with rotor TLA-100.2 (Beckman Coulter, Palo Alto, USA) in gradient of 50 mM D - mannitol and 5 mM HEPES. Second step was solid–phase extraction of alpha-tocopherol using Spe-ed columns C18/18%, 500 mg/3 ml/ 40 μm /60 Å (Applied
Separations, Allentown, USA). Then after elution of alpha-tocopherol by n-hexane, the extract was analyzed by HPLC method using
monolithic stationary phase Chromolith™ Performance RP-18e, 100 × 4.6 mm (Merck, Darmstadt, Germany).
Combination of ultracentrifugation and solid-phase extraction allows effective, easy and fast manipulation with large sequences of the
blood samples in shorter time which are the main aspects in routine clinical analysis.
Supported by: GAUK124809/2010, MSM0021620822, MZO00179906, MSM0021620820
P195:
Analysis of minute volumes of tick saliva samples using MALDI TOF/TOF mass spectrometry
Jan Sterba1, Marie Vancova2, William R. Alley, Jr.3, Benjamin F. Mann3, Lenka Bucinska3, Jarmila Dupejova1, Libor Grubhoffer2,
Milos V. Novotny3
1 Faculty of Science, University of South Bohemia, Ceske Budejovice, Czech Republic
2 Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, v.v.i., Ceske Budejovice & Faculty of Science,
University of South Bohemia, Ceske Budejovice, Czech Republic
3 National Center for Glycomics and Glycoproteomics, Department of Chemistry, Indiana University, Bloomington, IN, USA
A number of emerging infectious diseases is transmitted by blood-feeding organisms. Blod-feeding ticks from the genus Ixodes are the
main vector of tick-borne bacteria, viruses, and protozoa. The parasites influence the host defence systems and processes through
pharmacologically-active molecules in the tick saliva that are released into the wound in small volumes.
The carbohydrate/glycan-protein interactions play a pivotal role in the parasite-pathogen , pathogen-host, and parasite-host interactions. To date, the glycome of tick (or any other blood-feeding arthropod) saliva has not been studied. Any experimental study is limited
by the microliter amount of material available – tick saliva.
Data on protein glycosylation in ticks are known only for one protein, the lectin Dorin M from the tick Ornithodoros moubata. Its glycosylation sites are modified by high-mannose type truncated glycans with up to nine mannoses and a core-bound fucose. Generally
core-fucosylated, truncated high-mannose structures lacking terminal sialic acids were identified in closely related organisms from the
subphylum Chelicerata (scorpions, spiders etc.). Similar glycans have been found in other insects as well.
In the current work, we have studied the saliva glycome of the tick Ixodes ricinus using a multimethodological approach. Enzymatically
released glycans from 2–4 microliters of tick saliva were purified, solid-phase permethylated, and analysed by MALDI TOF/TOF. We have
analysed samples from saliva of unfed ticks as well as blood-fed ticks.
We found that tick saliva glycans are predominantly modified by core-bound fucose. Core-fucosylated tick glycans are released only
by combination PNGase A/PNGase F enzymes, but not by PNGase F alone, suggesting α1,3 bonding of L-fucose. Similar results were
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obtained for glycans isolated from both saliva and salivary gland extracts and also from tick hemolymph.
The most abundant N-glycans are the high-mannose type with up to nine mannose residues, similar to glycan structures found to date
in insects and other arthropods.
The most surprising finding was the presence of core-fucosylated hybrid glycans, which have not been reported in similar organisms.
These structures contain fucosylated cores with attached hexoses and N-acetylhexosamines (HexNAc6Hex3dHex, HexNAc5Hex6dHex).
Fucosylated hybrid glycans have been found in salivary glands of unfed ticks as well as in blood-fed ticks. The presence of core-bound
fucose suggests their tick origin.
P196:
Novell method of protein separation – native red electrophoresis
Tomáš Dráb, Jana Kračmerová, Ivana Tichá, Eva Hanzlíková, Marie Tichá, Jiří Liberda
Department of Biochemistry, Charles University, Prague, Czech Republic
Blue-native polyacrylamide gel electrophoresis (BN-PAGE), which uses negatively charged protein-bound Coomassie Blue dye to impose
a charge shift on the proteins, is a method for the separation of intact protein complexes. This separation method is characterized by
a high resolution and can be used to analyze abundant, stable protein complexes from 10 000 to 10 MD. It allows the determination of
the size, the relative abundance, and the subunit composition of a protein complex.
In the present communication we have investigated, an application of two anionic reversible protein stains - Ponceau S and Ponceau
2R – in the native electrophoretic separation of proteins and protein complexes, analogously as Coomassie Blue-dye. Contrary to the
previously used dye, the reversible dyes (Ponceau S and Ponceau 2R) adsorbed to proteins can be easily removed by washing with
buffer and thus the enzyme activity of separated proteins or protein complexes can be detected or they can be analyzed without further
restrictions.
Native polyacrylamide electrophoresis in the presence of two reversible protein anionic stains (Ponceau S and Ponceau 2R) was used
to the study oligomeric states of soluble proteins. A gentle binding of the used protein stains to non-dissociated protein oligomers
imposed a charge shift on the proteins leading to separation of protein species essentially according to their size under physiological
conditions. Adsorbed stains could be easily removed after electrophoresis by washing polyacrylamide gels with buffer and protein complexes could be visualized either by the detection of their enzyme activity or with a non-specific protein stain. The specific detection
of enzyme activity was shown on examples of glycosidases , lactate dehydrogenase or phosphatases.
This work was gently supported by GACR, grant No. 42-440080.
P197:
Data Quality in Gel Electrophoresis – a Necessity for Quality Control and Proteomics
Xi Deng1, Simone Schröder1, Sabine Redweik1, Asita Brandmüller1, Aftab Ahmed5, Hermann Wätzig1
1 Institute of Pharmaceutical Chemistry, University of Technology Braunschweig, Braunschweig, Germany
5 RI-INBRE Research Core Facility, College of Pharmacy, University of Rhode Island, Kingston, USA
Gel electrophoresis (GE) is, due to its unrivalled selectivity, one of the major techniques in proteome research. However, it is still known
for its often unsatisfactory precision. Percent relative standard deviations (RSD%) up to 60 % have been reported. An improvement of
precision and sensitivity is absolutely essential here, for proteome research and particularly for the quality control of biopharmaceuticals. Our work reflects the remarkable and completely irregular changes of the background signal from gel to gel. This irregularity was
identified as one of the governing error sources. Fortunately this is strongly reduced by using signal detection in the near-infrared (NIR)
range. In order to further investigate variance components in GE, an experimental Plackett-Burman screening design was performed.
Out of seven investigated parameters, just four showed a significant effect on some proteins, namely destaining time, staining temperature, changes of detergent additives (SDS and LDS) in the sample buffer, and the age of the gels. In later experiments, the effects of
the temperature and the shaking speed during the staining and destaining procedure were investigated in particular. Employing precise
control of these parameters, we have now achieved RSD%s of approximately 3 % for spot areas for one-dimensional separations in repetitive experiments under favourable circumstances. For our analysis two different NIR detectors were used.
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P198:
Extending The Dynamic Range Of Protein Quantification And Identification With A Novel Two-Dimensional LC/MS Approach
Mark A. McDowall
Waters Corporation
Purpose
We propose a novel 2D LC/MS strategy to increase coverage in quantitative protein profiling. Exact mass LC/MS of complex protein
digests can detect>150,000 unique ions. Deconvolution of such data reveal >20,000 non-redundant precursor ions for MS/MS interrogation. It is advantageous to captureinformation from as many tryptic peptides (precursors) as possible to maximize sequence coverage
enabling the quantitative mapping of modifications andisoforms. LC/MS/MS requires multiple injections to additively compile a quantitative inventory of peptides when analyzing complex digests. In additionconventional 2D chromatographic strategies (SCX+ RP) cause a
significant number of peptides to be diluted across multiple fractions.Methods and MaterialsWe have developed a data independent LC/
MS methodology using a mass spectrometer optimized for resolution (≤50,000 FWHM), mass accuracy (1ppm)and spectral acquisition
rate (20 spectra/sec) to enable multiple precursor ions to be interrogated in parallel. This approach has been extended to cover aneven
wider quantitative range of protein abundance by implementing a novel 2D (RP+RP) separation strategy, employing differential pI.ResultsWe have generate precise relative quantification values for proteins contained in biological systems and have constructed protein
abundance curves fortissues, cell lysates and bio-fluids. These data have been shown to be transferrable between laboratories and
instrument independent. This quantitativeapproach allows on-column sample loading to be determined and optimized, to ensure that
ideal LC and MS performance is obtained. This results in themaximum number of peptide and proteins being quantitatively determined.
Results obtained from such studies will be compared to theoretical models of thegiven proteome; considering complexity, dynamic
range and the inherent physiochemical properties of tryptic peptides in solution and the gas phase.ConclusionData independent 2D
(RP × RP) LC/MS provides enhanced quantitative coverage in bottom-up protein profiling studies.
P199:
Field-Enhanced Sample Injection for High Sensitive Analysis of Proteins by Capillary Electrophoresis-Matrix Assisted Laser Desorption/Ionization Mass Spectrometry
Mohammad Reza Pourhaghighi, Jean-Marc Busnel, Hubert H. Girault
Ecole Polytechnique Federale de Lausanne (EPFL)
Capillary electrophoresis (CE) is a simple and fast separation technique combining very high separation efficiency with low sample requirement and high sensitivity. In combination with Mass Spectrometry (MS), sensitive detection and characterization of peptides and proteins
is also possible. As compared to chromatography based techniques, CE, due to its miniaturized format, generally suffers from a low loading
capacity, which induces rather low concentration sensitivity. To circumvent this drawback, simple on-line preconcentration techniques can
be applied. If used properly, stacking techniques indeed enable a significant improvement of CE sensitivity. As a result, the development
of preconcentration methodologies and the assessment of their applicability have generated a considerable interest over the last years.
These approaches are moreover rather straightforward to integrate in analytical workflows as only simple manipulation of the background
electrolytes (BGEs) and sample matrices are required.
Herein, field enhanced sample injection (FESI) was evaluated to improve the sensitivity of protein analysis by CE. After having optimized
the stacking conditions, a test protein mixture (cytochrome C, lysozyme, bradykinin, ribonuclease A, ribonuclease S, ß-lactalbumin, ß-lactoglobulin, lactoferrine, bovine serum albumine, aldolase and myoglobin) was analyzed to demonstrate the achievement of sensitivity
enhancement factors higher than 3000. In spite of the use of a UV detector combined with 50 mm internal diameter capillaries, analysis
in the very low nanomolar range was permitted as a result of the significant sample depletion occurring during the extensive electrokinetic
injection process.
While rather conductive BGE solutions were used to permit the integration of FESI, only volatile BGE constituents have been chosen. Therefore, to widen the applicability of the developed methodology, the system was further evaluated to know to which extent it was compatible
with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). In this context, a recently developed iontophoretic fraction collection interface has been used1. With the use of optimized preconcentration, separation and spotting conditions, we demonstrate,
as in the case of UV detection, the possible achievement of limit of detection in the low nanomolar range by CE-MALDI-MS.
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Poster Abstracts
1 Busnel, J.-M., Josserand, J., Lion, N., Girault, H.H. Analytical Chemistry 2009, 81 , 3867-3872
P200:
Development and Validation of a UPLC Hydrogen/Deuterium Exchange Mass Spectrometry System
Michael Eggertson, Keith Fadgen, Martha Stapels, Joomi Ahn
Waters Corporation
Hydrogen/deuterium exchange mass spectrometry (HXMS) has proven to be a useful analytical method for the study of protein dynamics
and changes to protein conformation. In order to maximize deuterium signal recovery during LC/MS analysis, rapid chromatographic
separations at 0°C must be utilized. Recently, an ultraperformance liquid chromatograpy (UPLC) system has been developed capable of
high resolution separations at 0°CZ In this study, we describe a comprehensive HXMS workflow which is capable of chromatographic
separations ranging from 300 µm to 1.0 mm i.d. columns. Online pepsin digestion was utilized for rapid, reproducible protein digestion.
Peptides were fragmented by a simple scheme of alternating scans with low and high collision energy (MSE). To validate the system hardware performance, a standard protocol has been developed to evaluate all aspects of the workflow. MSE analyses show high confidence
peptide identification, up to 100 % linear sequence coverage, and reproducible peptic peptide peak area (less than 10 % RSD) in large scale
replicate analyses of Phosphorylase B. Results using an automated robotics platform utilized for sample preparation will also be discussed. Monday, March 22nd, 2010
P201:
HPLC-MS and CE-MS analysis of non-enzymatically modified bovine serum albumin
Zdenka Zmatlikova*1, Ivan Miksik2
1 Institute of Physiology, Academy of Sciences of the Czech Republic, (v.v.i.), Vídeňská 1083, 142 20 Prague 4, Czech Republic
Department of Analytic Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice,
2 Institute of Physiology, Academy of Sciences of the Czech Republic, (v.v.i.), Vídeňská 1083, 142 20 Prague 4, Czech Republic
Posttranslational modifications of proteins are important reactions, which significantly affect the function of proteins in the body. In principle,
they can be divided into enzymatic and non-enzymatic modifications. Non-enzymatic reactions include glycation, which plays important role
in the chronic complications of diabetes mellitus, uremia, in the process of aging and degeneration of the brain.
This work deals with the study of non-enzymatic posttranslational modifications of bovine serum albumin (BSA). Four various oxo-compounds
(glucose, ribose, glyoxal and glutardialdehyde) were used for modification. Modified BSA was digested by three enzymes (trypsin, pepsin,
proteinase K) with different cleavage specificity. This approach enabled to achieve a high coverage of protein and detected location of posttranslational modifications.
The main method for analyzing of arising modifications is ion-trap mass spectrometry (MS) coupled to high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). An appreciable problem in CE-MS is the use of sheath liquid to the coupling, because this
liquid induces the dilution of the samples and the subsequent reduction in sensitivity. The suitable solution can be the sample preconcentration approach. We used dynamic pH junction technique. This method is based on the pH change between backgroung electrolyte and sample.
HPLC-MS was the suitable analytical method for peptide mapping used. Carboxymethylation of lysine (CML) was the main type of detected
modification. Extent of modification and coverage of protein depended on the selected oxo-compound. Reactivity increased in order from
glucose to glutardialdehyde (glucose < ribose < glyoxal < glutardialdehyde). Glucose, being a mild modifier, offers a rich peptide profile, while
ribose treatment led to a moderately modified protein more resistant to enzymatic cleavage than native BSA. Reactions with dioxo-compounds
(glyoxal, glutardialdehyde) provide modified proteins highly resistant to enzymatic cleavage. This resistance is caused by cross-linking reactions
of the dioxo-compounds with free amino-groups of two proteins.
Very reactive position of BSA is peptide sequence 548-557 (KQTELVELLK). Lysine 548 is prone to carboxymethylation. Other reactive sites
are lysines in position 88, 248, 256, 401, 412, 455, 495 and 597. CML were not found in samples modified by glutardialdehyde, but oxidized
methionines occurred.
CE-MS technique was used for analysis of BSA glycation after trypsin digestion. The coverage of protein sequence was lower and subsequent
less CML were detected by this method (compared with HPLC-MS). Dilution of the analyte by sheath liquid caused a significant reduction in
sensitivity. Preconcentration technique (dynamic pH junction) insufficiently increased protein sequence coverage.
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Poster Abstracts
P202:
Possibilities of protein additives identification in historical mortars by peptide mass mapping method
Michaela Crhova, Stepanka Kuckova, Radovan Hynek, Milan Kodicek
Institute of chemical technology Prague
Since the ancient times a natural organic materials have been added into mortars to improve their mechanical properties. For example the
addition of eggs, curd, blood even beans could slow down or conversely accelerate their setting and consequently the dry mortars could
possess higher hardness or porosity. Natural materials have been used as admixtures to mortars until nineteenth century, when industrial
production of cement was introduced and the natural additives receded into the background. The recipes for preparation of such mortars
have always been handed down only verbally and therefore they were lost in this period. Recovery of these recipes is very beneficial, not only
from the historical aspect, but it could help with restorations of historical buildings too.
Identification of protein additives in mortars is very difficult, because of their very low concentrations. Most of the analytical methods used
exhibit too high detection limits. Only the mass spectrometry techniques - MALDI-TOF and nanoLC/nanoESI/Q-q-TOF, and several immunochemical methods can be practically used.
In this work a method called peptide mass mapping utilizing MALDI-TOF mass spectrometry is presented. This method was originally
worked out for identification of protein components contained in color layers of art works. Using this method an enzymatic solution is
added to the solid powdery sample without any other preliminary treatment. Resulting peptides are isolated and concentrated using
reversed-phase chromatography. Purified peptides are then analyzed by mass spectrometry. Obtained spectrum is compared with those of reference materials (eggs, curd, etc.) and thus the unknown protein additive can be identified. The detection limit of this method,
in the application on historical mortars, is about hundred times lower than in the case of other analytical methods.
Some examples of real samples analysis is presented.
Financial support from the Czech Ministry of Education (grant number 6046137305) and from the Grant Agency of Czech Republic
(grant number 230/07/P360) is kindly acknowledged.
P203:
Method Development for Improved Chromatographic Resolution and Throughput for Proteomics
Martha Stapels, Keith Fadgen, James Langridge
Waters
A typical goal of many bottom-up proteomics experiments is to characterize as many proteins as possible over a wide dynamic range.
Latest trends in proteomics point to the need to analyze multiple biological and technical replicates in order to confidently identify
proteins or measure statistically significant changes in quantitative experiments. One of the bottlenecks in running nanoflow LC/MS of
proteomics samples is the traditional long gradient length (two hours or more). Additionally, sample loading time and gradient delay in
which no peptides elute from the column at low flow rates also limit sample throughput. In this study, LC conditions will be optimized
to maximize chromatographic resolution and sample throughput to identify and quantify as many proteins as possible per day. Key to
this work is a new column chemistry (1.8 μm HSS T3), which is more retentive than the packing material used in the trapping column
(5 μm Symmetry C18) and even more retentive than the analytical column material currently used (1.7 μm bridged ethyl hybrid (BEH)).
This new chemistry allows for better re-focusing of the peptides on the analytical column after trapping, yielding narrower peak widths
and better peak capacity. With the T3 column, higher column temperatures can be used without sacrificing peptide focusing, so higher
flow rates can be used to increase throughput and resolution without increasing pressure on the system. The reduction in gradient
delay due to higher flow rate, along with optimized loading, trapping, and re-equilibration conditions, means that peptides elute over
the majority of the chromatogram. Data from multiple sample types, including different rat organs, will be shown. Two-dimensional
chromatography using high-low pH RP/RP will also be evaluated using the optimized method for the second dimension separation.
162
Poster Abstracts
P204:
Intact protein analysis by CE-ESI-TOF/MS, from method optimization to quantitation
Aline Staub, Julie Schappler, Serge Rudaz, Jean-Luc Veuthey
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Bd d’Yvoy 20, 1211 Geneva 4, Switzerland and Swiss
The on-line combination of capillary electrophoresis (CE) with mass spectrometry (MS) is an attractive option for intact protein analysis
(i.e., no digestion, no derivatization). On the one hand, CE presents features such as high speed, great efficiency, and low solvent and
sample consumptions. On the other hand, MS provides selectivity and ability to identification. Time-of-flight (TOF) analyzer is particularly well suited to protein analysis, due to its high mass range and mass accuracy. This lecture presents a CE-ESI-TOF/MS development
for intact protein analysis and quantitation, with human insulin as model protein.
A CE-ESI-TOF/MS method was developed and optimized taking into account major issues such as adsorption onto the capillary wall and
ionization in the electrospray source. The method was fully validated according to ICH guidelines. The choice of the internal standard
was not trivial and a new approach was tested using an ECHO peak technique. The latter is based on successive injections of a reference
standard and the sample in one single run. The complete methodology was applied to pharmaceutical formulations with success. This
analytical strategy may be useful in the context of biopharmaceuticals quality control, as well as counterfeits, often illegally available.
P205:
An Automated Online Two-Dimensional Reverse-Phase Capillary Liquid Chromatographic System Utilizing Online Sampling Fractionation and Gradient Mixing Strategies for Shotgun Proteomics
Ivan K Chu, Maggie P. Y. Lam, Shiu On Siu, Edward Lau
Department of Chemistry, The University of Hong Kong
n this study we describe the first online coupling of high-pH reverse-phase(RP) and conventional low-pH RP in a two-dimensional capillary liquid chromatography (2DLC) separation system for shotgun proteomics analysis. The system features a peak-parking sampler for
online sample fractionation and a downstream partial-loop injector for solvent mixing and reconditioning of the mobile phase between
the high pH RP separation in a high-organic eluate and the low-pH RP separation in a high aqueous eluate. The elution and injection
methods were examined initially using well-characterized tryptic-digested protein standards; the orthogonality of separation under
high- and low-pH RP conditions led to effective increases in peak capacity, with remarkably few peptide overlaps across the fractions.
We demonstrated the applicability of this online RP-RP 2DLC system through analyses of complex samples, namely mouse embryonic
fibroblast cell and zebrafish embryo lysates, resulting in the identification of 1955 and 1752 proteins, respectively, from low-microgram
amounts of samples. Because this effective system is fully automatable and requires minimal sample handling, it provides a robust
methodology for other possible online 2DLC combinations in MS-based proteomics applications.
P206:
Multi-Dimensional pI-based Separations Allow Increased Peptide and Protein Coverage for Large-Scale Proteomic Studies
Stephanie M. Cologna*, William K. Russell, Gyula Vigh, David H. Russell
A major challenge for mass spectrometry –based proteomics for studies of large proteomes, i.e., fungi, bacteria, mammalian, etc., is
related to the dynamic range of protein expression, ranging from a few copies per cell to 106 copies per cell1. Sample prefractionation,
where proteins are sorted on the basis of interactions with substrates (as in metal affinity chromatography, hydrophilic and hydrophobic interactions and charge based interactions) or electrophoretic properties, followed by liquid chromatography (LC)-MS or LC and
tandem mass spectrometry (MS-MS) has proven to be effective at mitigating the problem. Possibly, one of the most generally applicable
prefractionation methods is based on the pI values of proteins. A small-scale isoelectric trapping (IET) device termed ‘membrane separated-wells for isoelectric focusing and trapping’ (MSWIFT) was developed and used for the isolation of small molecules2. Recently
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Poster Abstracts
we demonstrated the utility of MSWIFT for the separation/isolation of specific peptides from protein digests, followed by the analysis
of the isolated peptides by MALDI-MS, LC-MS and LC-MS-MS. Yeast tryptic peptides were separated into six fractions and analyzed by
LC-MS, resulting in the assignment of 800 proteins at the 99.99 % confidence level3.
Currently, we are implementing a multi-dimensional fractionation platform using MSWIFT prior to LC-MS analysis for large-scale proteomic studies. To test the approach, a tryptic digest of yeast proteins was first separated into six fractions in the 2.9 < pH <11 range,
followed by the re-separation of the contents of the 4.0 < pH < 5.4 well into three additional fractions. From LC-MS/MS analysis and
database searching, 1257 distinct peptides were identified in the 4.0 < pH < 5.4 fraction, leading to the identification of 112 protein at
the 99 % confidence level. The second isoelectric trapping separation that gave three sub-fractions increased the number of distinct
peptides to 2448 and the number of identified proteins to 177. Protein concentration varied widely, e.g., the protein PRTB_YEAST is
present at 1600 molecules/cell compared to KPYK1_YEAST which is known to be expressed at 291,000 molecules/cell in log phase SD
medium4. Several unique peptides were observed only after the second isoelectric trapping separation: the 4.0 < pH < 4.3 well yielded
216 unique peptides while the 4.3 < pH < 5.0 and pH 5.0-5.4 fractions resulted in 145 and 61 peptides respectively, at the 99 % confidence level. These peptides were not identified after the first fractionation. We are currently utilizing this multi-stage IET separation
platform for the proteomic analysis of various organisms.
1 S. Ghaemmaghami, W. Huh, K. Bower, R.W. Howson, A. Belle, N. Dephoure, E.K. O'Shea, J.S. Weissman, Nature 425 (2003) 737
2 P.J. Lim, R.Y. North, G. Vigh, Electrophoresis 28 (2007) 1851
3 S.M. Cologna, P.J. Lim, W.K. Russell, G. Vigh, D.H. Russell, in preparation
4 www.uniprot.org
P207:
Potential of CE-MS for proteomics
Věra Hezinová*1, Carolina Simó2, Alejandro Cifuentes2, Karel Klepárník4, František Foret4
1 Brno University of Technology, Faculty of Chemistry, Brno, Czech Republic; Institute of Analytical Chemistry of the ASCR v.v.i., Brno, Czech Republic
2 Institute of Industrial Fermentation, Madrid, Spain
4 Institute of Analytical Chemistry of the ASCR v.v.i., Brno, Czech Republic
Capillary electrophoresis coupled with mass spectrometry become to be an important technique in proteomic analysis because of its
good separation efficiency, low running cost or small consumption of samples. The main problem when using capillary electrophoresis
- mass spectrometry in proteomics is the lack of sensitivity. This drawback could be improved using various concentration techniques.
Several methods of on‑line as well as off-line concentration techniques are usually used for the improvement of sensitivity in peptide
analysis. One of the off‑line concentration methods is solid phase microextraction, where peptides or proteins are bound on the appropriate stationary phase in a tip and eluted afterwards with fewer amounts of solution containing organic solvent. Using this approach
we have been able to decrease the limit of detection of selected peptide 20 times.
Another possibility to increase sensitivity of this method is the selection of interface. Mostly, the sheath-flow interface is used when
coupling capillary electrophoresis with mass spectrometry. This type of interface, that is the only one commercially available, has one
important drawback. The sheath liquid flow dilutes the analytes, and thus decreases the sensitivity. Another choice is the liquid junction
interface with nano-electrospray, where separation and spray capillaries are placed opposite of each other in the liquid reservoir. This
interface, because of its lower flow of spray liquid, provides lower limits of detection. One the other hand, its disadvantage could be a
poorer stability of spray.
In this work, the possibility of using capillary electrophoresis combined with mass spectrometry for detection of the proteins from twodimensional gel electrophoresis was studied. Generally, the protein spot was cut off the gel, digested by trypsin and originated peptides
are concentrated by solid phase microextraction and separated by capillary electrophoresis. Although the CE-MS sensitivity was still
not sufficient to detect all the peptides, tandem mass spectrometry of collision induced fragmentation enabled the identification of
some of the separated peptides.
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Poster Abstracts
P208:
Proteomic analysis of human dentin
Adam Eckhardt, Statis Pataridis, Kateřina Lacinová, Pavla Sedláková, Zdeňka Zmatlíková, Michal Jágr, Ivan Mikšík
Institute of Physiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4, Czech Republic
The tooth is the hardest part of the human body with specific construction and constitution. It consists of enamel, dental pulp, and
dentin. Dentin is the main part of the tooth lining the inner parts of the roots and crown. It is constituted by anorganic material (about
70 %), organic material (about 20 %), and water.
The aim of this study was to identify dentin proteom. Six healthy permanent human molars from six adults were cut, pulverized, denaturated (by guanidine) and demineralized (by EDTA). Denaturation and demineralization steps were repeated twice. Extracted proteins
were cleaved (by trypsin), separated, and detected using liquid chromatography-tandem mass spectroscopy (LC-MS/MS). For protein
separation was used SDS-PAGE, and the ZipTip pipette tips containing reverse phase media too.
We identified 58 proteins with at least two identified peptides, and another 10 proteins on the basis of only one characteristic peptide.
The main part of the dentin proteins represents collagens (type I, III, IV, V, VII, X, XI, and XXVII), albumin, biglycan, vimentin, coagulation
factors, alpha-2-HS-glycoproteins, and dentin sialophosphoproteins. We found several proteins that have never been detected before in
human dentin (titin, collagen type X, and collagen type XXVII).
This study is one of the first featuring the list of proteins detected in human dentin.
P209:
Determination and quantification of proteins originating from activated T-cell derived membrane vesicles by mass spectrometry
Lilla Turiák1, Oliver Ozohanics1, Petra Misják3, Edit Buzás3, Károly Vékey1
1 Hungarian Academy of Sciences, Chemical Research Center H-1025, Pusztaszeri 59-67., Budapest, Hungary
3 Semmelweis University, Department of Genetics, Cell and Immunobiology H-1089. Nagyvárad tér 4., Budapest, Hungary
Membrane vesicles (e.g. exosomes) are particles deriving from several cell types during apoptosis or under physiological conditions.
Recently their functions in cell-to-cell communication through membrane cell-surface elements have been reported. Also, membrane
vesicles are thought to play an important role in the regulation of immune response. The features of these vesicles could be described
more precisely if their composition were known.
In the literature a number of proteins originating from different types of microvesicles (e.g. B-cell, platelet) have been identified by mass
spectrometry. Our goal is to reveal the proteome of T-cell derived membrane vesicles. We have investigated the protein profile of resting
and activated Jurkat T-cell derived membrane vesicles by nano liquid chromatography coupled to mass spectrometry and developed
a method for the extraction of proteins from the vesicles. The peptide composition of the in-solution and the in-gel digested samples
were also compared to determine which method is more effective.
Jurkat T-cells were activated by different concentrations of Concanavalin A. The membrane vesicles were separated by ultracentrifugation and several washing steps from other cell particles. During the sample preparation two methods were compared. The membrane
vesicle fraction was exposed to 7 freeze-thaw cycles then the samples were digested in-solution by trypsin. Another method was direct
SDS-PAGE electrophoresis of the samples, followed by in-gel digestion. Following the digestion of the proteins the peptide mixture was
separated by nano HPLC.
The peptide composition of the resting and activated Jurkat membrane vesicle samples was then compared and differences in their
protein expression could be detected. The content of some proteins in the samples was also determined by label fee quantification
which showed good correlation with the amount of activation.
P210:
In-gel detection of phosphoproteins by selective staining: possibilities and comparison
Barbora Jankovicova*1, Tomas Kroupa1, Petra Spidlova3, Lucie Korecka1, Lenka Hernychova3, Zuzana Bilkova1
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Poster Abstracts
1 Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, Czech Republic
3 Institute of Molecular Pathology, University of Defence, Hradec Kralove, Czech Republic
Protein phosphorylation represents one of the most studied post-translational modifications (PTM), which in particular plays a significant role in a wide range of cellular processes, in both prokaryotic and eukaryotic organisms. The identification of phosphoproteins and
the characterization of their phosphorylation sites are necessary for understanding of their regulatory roles. Phosphoproteins analysis
is not straight-forward, various analytical methods for their detection and characterization have been developed which are more or
less powerful and have also some limitations1. In this work we focused on selective detection of phosphoproteins directly in 1-D or 2-D
electrophoretic gels using different staining techniques. In-gel detected phosphoproteins can be consequently analyzed in detail by
mass spectrometry emerged as a reliable and sensitive method for the characterization of protein phosphorylation sites2. We compared
commercially available fluorescent detection by Pro-Q Diamond (Invitrogen) and Phos-tag Phosphoprotein Gel Stain (PerkinElmer) and
non-fluorescent GelCode Gel Stain (Pierce). Mentioned dyes were utilized for selective detection of phosphoproteins in model protein
mixtures, cell lysates of Francisella tularensis or in dephosporylation studies. Parameters such selectivity, sensitivity, required instrumentation, time-consumption or compatibility with MALDI-TOF-MS were evaluated.
This work was supported by the Ministry of Education of Czech Republic (ME08105 and MSMT 0021627502) and by Czech Science
Foundation (GA 203/09/0857 and GA203/08/1536).
1 F. Delom and E. Chevet, Proteome Sci. 4 (2006) 15
2 A. Sickmann and H.E. Meyer, Proteomics 1 (2001) 200
P211:
Novel functional brush coatings: applications to microfluidic devices
Gabriele Di Carlo, Marina Cretich, Laura Sola, Francesco Damin, Marcella Chiari
Istituto di Chimica del Riconoscimento Molecolare, C.N.R Milano, Italy
Thin polymeric coatings applied to the surface of different materials can dramatically improve the properties of lab-on-chip devices as they
allow to control the interaction of the surface with its environment.
Examples of interfacial phenomena that can be controlled by a polymeric coating include the electroosmotic flow and the adsorption of
macromolecules in microchannels. Polymeric coatings can be obtained by “grafting-onto” or by a “grafting from” approach. The former technique that involves reacting an appropriate functional group of the polymer with the surface results in low graft densities as the steric hindrance
imposed by the chains, already grafted to the surface, obstacles the diffusion of the incoming polymer chains. In contrast, the grafting from
method leads to higher graft densities as the existing grafted polymers does not hinder the diffusion of the small-sized monomers on the
surface. The work presented here was aimed at developing high density, brush coatings. The goal has been achieved by combining surfaceinitiated and a controlled polymerization technique, the so called Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization. A
major advantage of the RAFT polymerization process over other processes for living/controlled free-radical polymerization is its compatibility
with a wide range of monomers including functional monomers. In this work we report on two applications of brush coatings obtained by
RAFT polymerization: suppression of electroosomtic flow in capillary electrophoresis and covalent attachment of biological molecules to the
active area of sensing devices. Examples will be shown in both fields of application.
P212:
Electric field gradient focusing in microchannels with ebbedded bipolar electrode
Dzmitry Hlushkou1, Robbyn K. Perdue2, Derek R. Laws2, Richard M. Crooks2, Ulrich Tallarek1
1 Philipps-Universität Marburg, Germany
2 The University of Texas at Austin
Multifunctional microchip devices for chemical separation and analysis provide significant advantages over conventional systems through
faster analysis times and low sample and reagent consumption. However, the use of smaller geometries reduces the number of analyte
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molecules such that their detection becomes a challenging task. Analyte preconcentration before (off-line) or after (on-line) sample injection
is an alternative to the use of highly sensitive detectors. Recently, we reported a novel electric field gradient focusing technique for use in a
straight microchannel containing an embedded floating gold electrode (i.e., without galvanic connection to any part of the system). 1-3 When
a sufficiently high driving voltage is applied across a buffer-filled microchannel, a bipolar electrode (BPE) situated at the center of the channel
provides a secondary current path, i.e., in the vicinity of the BPE, current can be carried by electrons (through the electrode) as well as ions
(through the solution above the BPE). This results in complex ion transport due to the combined actions of electromigration of buffer ions and
electroosmotic flow (EOF), which in turn leads to an electric field gradient in the solution. If charged analytes are introduced to the system,
their migration velocity depends on their respective electrophoretic mobilities. The EOF direction in the studied system is toward the cathode,
so that negatively charged (anionic) analytes which are transported by the cathodic EOF are met with an opposing force from their anodic
migration. At the location in the channel where the two opposing forces are equal in magnitude analytes achieve a stationary position. Analytes
accumulate at different locations in the channel accoding to their respective electrophoretic mobilities, resulting in several analyte-enriched
zones along the channel. Utilizing an array of microband floating electrodes, analytes may be passed from one stationary position to another by
relocating the BPE through external connection of different microbands. Our work presents experimental results and interpretive simulations
that provide new fundamental and applied insights into bipolar electrode focusing.
P213:
Determination of imipramine and trimipramine by capillary electrophoresis with electrochemiluminescene detection
Caixia Yu1, Baiqing Yuan1, Tianyan You3, Yingsing Fung4
1 State Key Laboratory of Electroanalytical Chemistry/Graduate School of the Chinese Academy of Sciences,
Changchun Institute of Applied Chemistry, Chinese Academy of Science
3 State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Science, , Changchun , China
4 Department of Chemistry, Hong Kong University, Pokfulam Road, Hong Kong SAR , China
Imipramine and trimipramine are tricyclic antidepressant (TCA) drugs, the most effective drugs presently available for the treatment of depression and other psychiatric disorders by blocking the reuptake of norepinephrine at nerve terminals1. To enable their clinical applications to achieve optimum therapeutic effects and minimize side effects, analytical method with high sensitivity and free from interference is needed. Many
methods have been developed for detection of imipramine and trimipramine such as HPLC2, GC3, capillary electrophoresis (CE)4 and NACE5. As
both imipramine and trimipramine contain a tertiary amines group, electrochemiluminescene (ECL) can provide a sensitive detection method
and thus the coupling of CE with ECL detection is investigated in the present work. The addition of ß-CD to the running buffer was found to
enable base-line separation of the two analytes and the addition of acetonitrile (ACN) as an organic additive to improve the repeatability and
sensitivity of the CE method. Under the optimized conditions with buffer in the ECL detection cell containing 2 mM Ru (bpy)32+ and 50 mM
PBS (pH = 7.0) and CE running buffer composed of 20 mM Tris (pH = 2.0), 0.2 mM ß-CD and 20% (v/v) ACN, wide linear ranges of 0.1 - 5 μM
and 0.1 - 5 μM, with a low detection limit of 5 nM and 1 nM (S/N = 3) respectively for imipramine and trimipramine are shown to be achieved.
1 R.A. de Toledo, M.C. Santos, K.M. Honorio, A.B.F. da Silva, E.T.G. Cavalheiro, L.H. Mazo, Anal. Lett. 39 (2006) 507-520
2 M. Woźniakiewicza, R. Wietecha-Posłusznya, A. Garbacika, P. Kościelniak. J. Chromatogr. A 1190 (2008) 52-56
3 K.Y. Tserng, R.J. McPeak, I. Dejak, K. Tserng, Ther. Drug Monit. 20 (1998) 646-651
4 C.S. Liu, X.F. Li, D. Pinto, E.B. Hansen, C.E. Cerniglia, N.J. Dovichi, Electrophoresis 19(1998) 3183-3189
5 X. L. Xu, G. L. Zhou, H. X. Li, Q. Liu, S. Zhang,, J. L. Kong, Talanta 78 (2009) 26-32
P214:
Coupling continuous micro-evaporator to capillary electrophoresis for determination of organophosphorus pesticides in vegetables
Qidan Chen, Josephine L. L. Mak, Yingsing Fung
Department of Chemistry, The University of Hong Kong
The increasing use of organophosphorus pesticides (OPs) to replace environmental persistent organochlorine pesticides leads to health concern for residual OPs which exhibit high acute toxicities with poisoning cases reported in developing countries1. This leads to demand for methods with high sensitivity to detect OPs in food products. As the whole vegetable is required to be assessed for residual OPs as stipulated by
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Poster Abstracts
the Codex Alimentarius Commission, a large volume of organic solvent is often used for pesticide extraction, and detection of sub-ppm levels
of OPs in a complex sample matrix is needed2. To improve evaporation efficiency and reduce analyte loss on evaporator surface, a continuous
micro-evaporator (CME) is developed in the present work as an essential step in a carefully designed route for full solvent evaporation within a
reasonable time of 30 minutes by CME with minimal solvent contacting surface area to produce a 10µl sample in buffer/methanol containing
OPs to be determined by CE separation. A total enrichment factor of about 2000 times has been achieved in a 4-step clean-up/enrichment
procedure using solvent extraction, solid phase extraction, rotary evaporation and CME with the major contribution of 100 times enrichment
from CME. In addition to greatly enhance the enrichment factor, the use of CME has been shown to improve repeatability and recovery of OPs.
Using commonly occurred OPs such as mevinphos, phosalone, methidathion and diazinon, their separation in a fused silica column of 40/65
cm´50 mm are optimized as follow: BGE, 20 mM borate, 50 mM SDS, pH 9.6, 3 % methanol; applied voltage, 25 kV; detection wavelength,
202 nm. The separation is completed in less than 12 min with baseline resolution. Details on the design of CME and its coupling with the CE
procedure will be presented and discussed at the meeting.
1 Mayank Bhanti, Ajay Taneja, Chemosphere, 69 2007, 69, 63-68.
2 FAO/WHO Food Standards Programme, Codex Alimentarius Commission, MRLs for spices adopted by 28th Session of the Commission (Rome 4-9 July 2005),
http://www.codexalimentarius.net/
P215:
Separation selectivity enhancement through complex formation in capillary zone electrophoresis
Jitka Široká*, Klára Petrů, Jan Honegr, Miroslav Polášek, Marie Pospíšilova
Charles University, Faculty of Pharmacy, Department of Analytical Chemistry, Heyrovského 1203, CZ-50005 Hradec Králové, Czech Republic
Separation selectivity enhancement has fundamental importance in the field of separation techniques. There exist several approaches
(mostly based on selective interactions between the separated compounds and components of background electrolyte, BGE) how to
attain the improvement of separation selectivity in capillary zone electrophoresis (CZE). One of them exploits the capacity of charged
or even electro-neutral analytes to form anionic or cationic complexes varying in stability and electrophoretic mobility depending on
pH and on the concentration of appropriate complex forming agents in the BGE.
Analytically important complexes are formed due to weak charge-transfer interactions between inorganic or organic ligands as donors
of electron pairs and metal (“central”) ions which can accept the electrons. If the complexes of separated compounds are formed rapidly, vary in stability and carry different charge, separation selectivity improvement can be expected.
The process of complex formation can take place prior to the CZE separation as a step of sample pre-treatment or just during the
separation process inside the capillary if the complex forming agent is present in the BGE.
In last decade a number of papers dealing with the use of complex formation for the CZE analysis of metal ions as well as for separations
of organic ligands were published. The communication presented brings a critical survey of the most important applications of complex
formation for the separation of biologically active organic ligands by the addition of metal ions into the BGE.
Financial support by the Czech Ministry of Education (grant MSM 0021620822) is gratefully acknowledged.
P216:
Glycomic profiling of sera from tularemia infected patients
Lenka Hernychova*1, Lucie Balonova1, Zuzana Bilkova3, Milos V. Novotny4, Jiri Stulik1
1 Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic
3 Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
4 National Center for Glycomics and Glycoproteomics, Indiana University, Bloomington, IN
Introduction: Protein glycosylation plays a key role in protein stability, folding, and cell-to-cell interaction. It has been shown that
aberrant, additional, or missing glycosylation is correlated with cancer diseases. While the presence of bacterial glycoproteins was
discovered, little is known about the variation of glycans in immune sera. Therefore, the profiles of N-glycans released from blood sera
of tularemia infected patients were the aim of this work.
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Poster Abstracts
Methods: The glycomic profiles were generated for the human blood sera obtained from (1) healthy individuals, and (2) patients infected
by intracellular bacterium Francisella tularensis. The immune sera had a positive agglutination titer. The N-glycans derived from glycoproteins of human blood sera were enzymatically released using Asn-GlnNAc-specific PNGase F. The released N-glycans were purified
from deglycosylated/nonglycosylated proteins by solid-phase extraction procedure, in which released glycans were separated from the
protein pool and also desalted using activated graphite. The conversion of native oligosaccharides to their methylated counterparts was
achieved through spin-column permethylation. The permethylated glycans were subjected to the MALDI-TOF MS analysis in ion-positive
reflectron mode.
Results: The acquired MS spectra were processed using DataExplorer 4.0 (Applied Biosystems) to generate ASCII files listing m/z values
and their corresponding intensities. The peak intensities of N-glycans were then extracted using the software tool PeakCalc NET.2.0
(in-house software of Indiana University). Data were statistically analyzed using a principal component analysis (PCA) and a single factor
Analysis of variance (ANOVA). A difference between the groups (1) and (2) was considered as statistically significant when p-values were
less than 0.05.
Conclusion: The PCA showed a distinct clustering with a significant difference between the healthy and immune sera. In addition,
significant differences were observed for two core-fucosylated glycan structures, for which p-values were less than 0.05. This study
shown interesting preliminary observations and further validation of this approach will necessitate a large number of sera from patients
suffering from different infections.
This work was financially supported by the Czech grants: MSMT0021627502, ME08105, FVZ0000604, and GA203/09/0857. The glycan
profiling methodologies have been developed at the National Center for Glycomics and Glycoproteomics at Indiana University under the
center grant P41 RR01894 from the National Center for Research Resources, U.S. Department of Health and Human Services.
P217:
HPLC column-switching determination of antiflogistics in biological materials
Ivana Brabcová, Dalibor Šatínský, Petr Solich
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
Column-switching techniques for on-line preparation and simultaneously analysis of biological samples are modern trends of analytical
process implementation to one step . Biological samples contain the large number of substances that are incompatible with chromatographic system. Therefore sample clean-up before the analysis is a crucial step of whole analysis. To widely used extraction techniques
belong: solid-phase extraction (SPE) and liquid-liquid extraction (LLE). These methods work in the off-line mode and they are slow and
laborious. In addition, there is a greater risk of loss of analyte.
On the other side, column-switching techniques use restricted-access materials (RAM) which enable direct and repetitive injection
complex matrix into HPLC system.
Automated column-switching HPLC method was developed for specific and sensitive determination of non-steroidal anti-inflammatory
drugs (ketoprofen, ibuprofen, sodium diclofenac and flurbiprofen) in human serum using high-performance liquid chromatography
with UV detection. Determination of pharmaceuticals was carried out on column Chromolith Performance RP 18e 100 × 4.6 mm with
precolumn Chromolith Guard Cartridge RP-18e 10 × 4.6 mm. The mobile phase used was 0.085 % phosphoric acid and acetonitrile in
gradient mode. Flow rate of the mobile phase was 1 ml min-1 and temperature 45°C, sample volume injection was 200 μl. UV detection
was at 210 nm.
Composition of the various flushing eluents with a low ratio of organic solvent on RAM column was optimized.
Low content of organic solvent prevents to denaturation of proteins and ballast components from human serum are simultaneously
washed out to waste. The time of valve switching was optimized. There was observed influence of flow rate on the washing process.
The valve was switched to the analytical column and to the detector after purification and preconcentration of the sample drugs. Valve
switching time was set at 7 minutes.
The sample was pretreated on RAM column Merck LiChrospher ® RP-18 ADS. Optimal compositions of the flushing eluent were 10 %
acetonitrile with 0.085 % phosphoric acid at a flow rate 2.5 ml min-1. Developed method was validated. Parameters of chromatographic
system suitability test (SST) (repeatability, resolution, peak asymmetry and efficiency of chromatographic columns) were determinated.
Validation parameters of the method (accuracy, precision, linearity in the tested concentration range, selectivity and limit of detection)
were carried out.
The authors gratefully acknowledge the financial support of the Grant Agency of the Ministry of Education the Czech Republic MSM
0021620822 and Grant Agency of Charles University Project No. 34609/2009.
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Poster Abstracts
P218:
Single DNA molecule detection by on-bead rolling circle amplification
Kae Sato1, Yuri Kitamura1, Kiichi Sato3, Kazuma Mawatari3, Mats Nilsson5, Takehiko Kitamori3
1 Japan Women's University
3 The university of Tokyo
5 Uppsala University
Introduction: Ultra sensitive DNA detection from complex matrixes is of great importance for a variety of diagnostics. One powerful
analytical reagent is a padlock probe, which utilizes rolling circle amplification (RCA) for generation of 1-µm-sized fluorescent dots
from single DNA templates in a solution1. However, counting the fluorescent dots in bulk scale is difficult. We have implemented single
molecule detection by on-bead RCA.
Methods: Polystyrene or agarose beads were used for on-beads RCA. 5'-amino-modified primer was immobilized on NHS-activated
Sepharose beads (34 µm). 5'-biotin-modified primer was immobilized on streptavidin modified beads (19 µm). Sample DNA solution
was added to the beads, followed by ligase to create circular padlock DNA, and DNA polymerase with an excess of dNTPs to extend the
DNA. Fluorophore-labeled antisense DNA was hybridized to the extended DNA to facilitate detection.
Results and Discussion: The enzymatic reactions were realized on the both beads successfully. Individual RCA products on the beads
were visible with a fluorescence microscope as a bright object. Using 2.5 f mol DNA with 10,000 agarose beads in a PCR tube, 1400
countable RCA products per one bead could be detected, corresponding to a detection rate of ~1%. This is far superior to the previously
reported value of 0.02% (7 × 108 circles, approximately 105 counted objects)1.
1 Jarvius, J., et al. (2006). Digital quantification using amplified single-molecule detection. Nature Methods 3, 725-727
P219:
Searching for Francisella tularensis FTT1103 substrates by combination of immunoprecipitation and LC-MS/MS
Petra Spidlova*1, Iva Senitkova2, Marek Link1, Jiri Stulik1
1 Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic
2 Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic
Conserved hypothetical lipoprotein FTT1103, essential virulence factor of Francisella tularensis, shares homology with proteins of disulfide
oxidoreductase DsbA family. These proteins catalyze formation of disulfide bonds which are necessary for folding, activity and stability of many
proteins exported from the cytoplasm. Secreted and surface proteins, virulence factors of pathogenic bacteria, often contain disulfide bonds
and thus the inactivation of DsbA proteins leads to the pathogen attenuation. The aim of this study was to identify proteins which could
interact with FTT1103 and thus play role in F. tularensis pathogenesis.
In our study we used F. tularensis FSC200/ΔFTT1103 deletion mutant strain complemented in trans by introduction of replicative plasmid
carrying ftt1103 gene fused with FLAG-tag. In order to purify protein FTT1103 and its substrates immunoprecipitation using Anti-FLAG M2
Affinity Gel (Sigma) was performed. Three different types of elution were tested and the most effective one, elution using 3× FLAG peptide,
was applied. After methanol/chloroform precipitation to remove excess of 3× FLAG peptide and subsequent in solution digestion with trypsin, samples were analyzed by 1D RP LC-MS/MS. Strain F. tularensis FSC200/ΔFTT1103 with inserted empty replicative plasmid was used as
negative control.
Data we have obtained showed several proteins unique for the eluate from F. tularensis FSC200/ΔFTT1103 strain complemented in trans and
therefore missing in negative control. One of them D-alanyl-D-alanine carboxypeptidase family protein (gi|89144226) was previously found to
be putative substrate of FTT1103 by comparative proteomic analysis. Another interesting protein, we have identified, was HlyD family secretion
protein (gi|89143316). Predicted localization in periplasm, presence of four cysteine residues and the role in secretion support our hypothesis,
that HlyD family secretion protein might be another substrate of FTT1103.
This work was financially supported by Ministry of Defence of Czech Republic (FVZ0000604, FVZ0000501 and FVZ200808).
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Poster Abstracts
P220:
Interactions of selected cations with dodecylsulphate micelles in electrophoretic systems
Ludmila Müllerová, Jana Lokajová, Martina Riesová, Bohuslav Gaš
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Praha 2, Czech Republic
As interactions in micellar electrophoretic systems are not fully understood yet predictions and simulations of such systems are rather
complicated. The simulation program PeakMaster1 can predict correctly behavior and properties of the electrophoretic systems where
no aggregation occurs. If background electrolyte contains a surfactant in concentration above its critical micelle concentration (CMC)
predictions of the PeakMaster fail and the simulations are not in agreement with experiments. We suppose that this disagreement is
caused by ion pairing of cations present in the system with the micelles. This interaction leads to decreasing of effective mobilities of
these cations. In case of alkaline cations the interaction should differ according to the atomic number and diameter of the cation. The
smaller the cation, the bigger solvation shell. As a consequence, smaller cations exhibit weaker interactions with the micelle and thus
their effective mobility should be less influenced by the presence of micelles.
We measured dependencies of effective mobilities of selected cations (Na+, K+, Cs+, Tris+) on concentration of dodecylsulphate in background electrolyte above its CMC. CMC was determined using the method proposed by Lokajová et al.2.
The effective mobilities of these cations were decreasing with increasing concentrations of the surfactant above its CMC. These dependencies seemed to be linear with different slopes for different cations. To compare interactions of these cations with the micelle
we plotted relative effective mobilities against the surfactant concentration. Steeper decrease of the relative effective mobility reflects
stronger interaction. In addition to the alkaline cations, organic cation Tris+ was also studied and its interaction with the micelle appeared to be slightly stronger than the interaction of Cs+.
The order of the absolute value of the slope of the dependencies of the alkaline cations can be related to their interaction force with
micelles. It was found that the higher absolute value of the slope increase with increasing value on atomic number and decreasing hydrodynamic radius. This was in agreement with our presumptions as well as with the works of other authors3, 4 ,5, in which the authors
used other methods to investigate such interactions.
This work was financially supported by the Grant Agency of the Charles University grant number 51009, the Grant Agency of the Czech
Republic grant number 203/08/1428 and the Ministry of Education, Youth and Sports, Long Term Research Plan MSM0021620857.
1 Gaš B., Jaroš M., Hruška V., Zusková I., Štědrý M.; LC GC Europe 2005, 18, 282-288
2 Lokajová J., Hruška V., Tesařová E., Gaš B.; Electrophoresis 2008, 29, 1189-1195
3 Ahuja E. S., Foley J. P; Anal. Chem. 1995, 67, 2315-2324
4 McLaren D. G., Boulat O., Chen D. D. Y.; Electrophoresis 2002, 23, 1912-1920
5 Joshi J. V., Aswal V. K., Bahadur P., Goyal P. S.; Curr. Sci. 2002, 83, 47-49
P221:
Electromigration oscillations occuring in ternary electrolyte systems
Martina Riesová*, Vlastimil Hruška, Bohuslav Gaš
Charles University in Prague, Faculty of Science, Department of Physical and Macromolecular Chemistry, Albertov 2030, 128 43 Prague 2, Czech Republic
Recently, a new class of electrophoretic systems exhibiting oscillations in concentration profiles of all ions has been discovered in our
lab. These systems had been predicted from a linear model of electromigration. The oscillations are a direct consequence of complex
system eigenmobilities. Binary systems with oscillating properties had been already found and experimentally verified1.
As the next step we searched for ternary oscillating systems, acidic and alkaline, composed of two acids and one base and two bases
and one acid, respectively. We used program PeakMaster2, 3, which was modified to enable easier and faster finding of systems with
complex eigenmobilities. The adapted PeakMaster version also allowed optimization of composition of these systems. We found several
ternary systems with complex eigenmobilities and inspected them experimentally4. Five acidic systems and one alkaline system proved
oscillating behavior. In the experiments we recognize system zones like small gaps in the pattern of oscillation. To compare the theory
and experiment we calculated a theoretical position of the system zones with complex eigenmobilities by the PeakMaster program.
The position of the gap observed in electropherograms was in a very good agreement with its theoretical prediction obtained by the
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Poster Abstracts
PeakMaster. We confirmed that the complete oscillating pattern travels in the capillary with velocity determined by the real part of the
complex eigenmobility.
In addition we investigated behavior of an analyte in the oscillating background electrolytes. The signal of the analyte can be easily
immerged in the signal of the oscillating pattern and then undetectable at the wavelength at which the background electrolyte absorbs.
However, in case we chose wavelenght characteristic for the analyte we obtained well-shaped peak, no unusual peak broadening was
observed.
All these experiments were also simulated by program Simul3.
This work was financially supported by the Grant Agency of the Charles University grant number 51009, the Ministry of Education,
Youth and Sports, Long Term Research Plan MSM0021620857 and the grant of Agilent Technologies Foundation, No. 09EU-648
V. Hruska, M. Jaros, B. Gas, B, Electrophoresis 27 (2006) 513
M. Jaros, V. Hruska, M. Stedry, I. Zuskova, B. Gas, Electrophoresis 25 (2004) 3080
http://www.natur.cuni.cz/gas
M. Riesová, V. Hruška, E. Kenndler, B. Gaš; J. Phys. Chem. B 113 (2009) 12439
P222:
Electrophoretic determination of calystegines in potato
Michaela Jirušková, František Kvasnička
ICT Prague
Calystegines are water-soluble nortropane alkaloids and they are active as glycosidase inhibitors. The most abundant calystegines in
potatoes are calystegine B2 and A3. The distribution of calystegines in potatoes was followed in different varieties (yellow, red and
violet flesh) and parts of potatoes (whole potato, flesh, peel, sprouts). The highest concentrations of calystegines were found in potato
sprouts. Violet variety of potato contains higher levels of calystegines than the yellow and red potatoes. Capillary zone electrophoresis is suitable for fast and sensitive determination of calystegines. The analysis time was approximately 5 minutes. The background
electrolyte for capillary zone electrophoretic analysis was the mixture of 20 mM histidine, 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid and 20% (v/v) methanol in demineralized water. Calystegines were detected by the indirect UV detection at 210 nm.
wWednesday, March 24th, 2010
P223:
A noval microfluidic chip-based LC/MS workflow for N-glycan analysis for biologics
Tom A. van de Goor, Maggie A. Bynum, Hongfeng Yin, Katherine Felts, Yvonne M. Lee
Agilent Technologies
Glycosylation is of great importance for new biologics. Changes in the glycosylation profile can lead to dramatic differences
in glycoprotein pharmaceutical efficacy, immunogenicity and stability. In this presentation, we will describe in detail a new
microfluidic chip designed for N-glycan analysis cleaved off monoclonal antibodies (MAbs). The aim for this novel fully-integrated microfluidic LC/MS chip is to improve the efficiency of enzyme cleavage of glycans off glycoproteins and subsequent analysis of N-glycans. Preliminary data will show the on-chip workflow that involves enzymatic deglycosylation with immobilized
PNGase F enzyme, glycan purification, separation, identification and quantitation of N-linked glycans from MAbs. With this
chip, the experimental time from antibody injection to LC/MS result is reduced to 10 minutes while current routine analysis
via other methods demands at least 2-3 days. We will also show that the GlycoPRO-Chip workflow matches well with current
analysis methods, such as CE-LIF or MALDI MS.
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Poster Abstracts
P224:
Comparison of protein and DNA migrations during capillary electrophoresis in polymer solutions: Effect of analyte chain length
and matrix mesh size
Koichi Mayumi*1, Keiko Sumitomo2, Hiroyuki Minamikawa3, Mitsutoshi Masuda3, Yasuhiro Sakai1, Hideaki Yokoyama1,
Toshimi Shimizu3, Yoshinori Yamaguchi8, Kohzo Ito1
1 Department of Advanced Material Science, Graduate School of Frontier Sciences, The University of Tokyo, Japan
2 SORST, Japan Science and Technology Agency(JST)
3 Nanotube Research Center(NTRC), National Institute of Advanced Industrial Science and Technology(AIST)
8 PARC, Department of Applied Physics, Graduate School of Engineering, Osaka University, Japan
Migration of biological macromolecules such as DNA and sodium dodecyl sulfate (SDS)-bound proteins in capillary electrophoresis is dominated by their molecular size and the mesh size of a sieving polymer network. DNA migration in capillary electrophoresis is classified in three regimes that depend on the ratio of the DNA size R and the mesh size a of the sieving polymer network:
Ogston sieving (R<a), reptation (R>a), and reptation with orientation (R>>a). On the other hand, the migration mechanism of
SDS-bound proteins has not fully been understood.
We have studied the optimization of the protein separation in SDS capillary electrophoresis and reported that the ideal mesh size
for the separation of proteins with molecular weights from 14 300 to 97 200 Da was less than 10 nm1. In the present work, we
investigated the migration mechanism of SDS-bound proteins from a polymer physics point of view. We compared the mobility
of SDS-bound proteins and double-stranded DNA (dsDNA) in hydroxyethyl cellulose (HEC) solutions, focusing on the two physical
parameters, the matrix mesh size a and the chain length L of proteins or dsDNA. The chain length of the proteins (14 300 to 97
200 Da) was from 52 to 350 nm, whereas dsDNA (20 to 15 000 bp) had a wider range of chain length from 6.8 to 5 100 nm.
The mesh sizes of HEC solutions were determined experimentally by dynamic light scattering measurements. We found that the
mobility µ of SDS-bond proteins divided by mesh size a is a function of the ratio of chain length L and mesh size a: µa=f(L/a).
The relationship among µ, a, and L for SDS-bond proteins is similar to that for dsDNA from Ogston regime to reptation regime.
This result was the direct experimental evidence that the migration mechanism of SDS-bound proteins and dsDNA is basically the
same and that the migration of the proteins from 14 300 to 97 200 Da was described by an intermediate mechanism between
the Ogston and reptation models.
1 Sumitomo, K.; Mayumi, K.; Yokoyama, H.; Sakai, Y.; Minamikawa, H.; Masuda, M.; Shimizu, T.; Ito, K.; Yamaguchi, Y. Electrophoresis 2009, 30, 3607.
P225:
Separation buffer composition for the protein separation by capillary sieving electrophoresis with sodium dodecyl sulfate and
poly(ethylene oxide)
Keiko Sumitomo1, Koichi Mayumi2, Hiroyuki Minamikawa3, Mitsutoshi Masuda3, Toru Asahi5, Toshimi Shimizu3, Kohzo Ito2,
Yoshinori Yamaguchi8
1 SORST, Japan Science and Technology Agency
2 Department of Advanced Material Science, Graduate School of Frontier Sciences, The University of Tokyo
3 NTRC-AIST
5 ASMeW, Waseda University,
8 PARC, Department of Applied Physics, Graduate School of Engineering, Osaka University
Poly(ethylene oxide) (PEO) offers several advantages as a sieving polymer for SDS-capillary electrophoresis. The PEO suppresses EOF in
fused silica capillary without chemical synthesis and prevents the physical adsorption of proteins on the capillary wall. Thus the PEO
polymer solution avoids possible contamination by only replacing the polymer solution and it practically eases to precede the consecutive and automatic experiment. In protein separation with capillary polymer electrophoresis, the PEO, however, characterizes the unique
properties that cause the deterioration of separation in capillary polymer electrophoresis. The PEO aggregates itself and forms a cluster
or a polymer domain through inter- and intra- molecular hydrogen bonds intermediated with water molecules. The long chain PEO is
chemically and mechanically cleaved into smaller pieces because of oxidation, photolysis, strong stirreing and other factors. And the
PEO forms polymer-micelle complexes with SDS in aqueous solution, although the driving forces that induce the binding between PEO
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and SDS are still unclear. In this paper, we diagnosed the composition of the PEO running buffer to ensure the separation of proteins in
capillary polymer electrophoresis by measuring the polymer properties with dynamic light scattering (DLS).
The FITC labeled proteins were separated with four kinds of 2% PEO (Mw 300,000) polymer solutions dissolved in different buffer
solutions; 100 mM Tris-CHES, 0.1% SDS buffer pH8.6, 75 mM Tris-Gly, 0.1% SDS buffer pH 8.3, 100 mM Tis-Tricin 0.1% SDS buffer pH
8.3, and 100 mM Tris-HCl 0.1% SDS buffer pH 8.6. The mesh size of polymer solutions, which dominated the mobility of protein and
the separation performance including the number of plates and the resolution, was determined by DLS for direct measurement. SDS
adsorption against PEO was measured by surface tension.
The six proteins with the molecular mass ranged from 14,300 to 97,200 were achieved the baseline separation of 6 proteins in the 100
mM Tris-CHES, 0.1% SDS buffer, while no peak of proteins was recognized in the other buffer solutions. The average mesh size of PEO
polymer network was 7 nm in Tris-Gly, 0.1% SDS buffer, and 5 nm in the other buffer. The deference of the average mesh size in those
buffer solutions was only about 2 nm, thus, the physical sieving process for protein migration was identical in those sieving solutions.
We also measured the surface tension of SDS solutions to quantify the interaction between SDS and PEO in those buffers. From the
surface tension study, we observed no SDS-PEO complexation in 100 mM Tris-CHES up to 60mM SDS.
We will discuss the details of the analytical method and improvement of the resolution of protein separation with PEO polymer as a
sieving matrix.
P226:
Comparison of chromatographic C18 columns packed with 2.6 µm Core-Shell and porous sub-2 micron particles for high-throughput analysis of tetracycline and macrolide antibiotics
Tereza Tylová *1, Zdeněk Kameník1, Miroslav Flieger3, Jana Olšovská3
1 Institute of Microbiology of the ASCR, v.v.i.; Charles University, Faculty of Science
3 Institute of Microbiology of the ASCR, v.v.i.
Chromatographic columns packed with Core-Shell 2.6 µm particles (Kinetex C18, 2.1 × 50 mm) and porous 1.7 µm particles (Acquity
BEH C18, 2.1 × 50 mm) for gradient separation of tetracyclines (oxytetracycline, tetracycline, chlortetracycline, doxycycline) and macrolides (tylosin, clarithromycin, roxithromycin, rapamycin) were studied.
The tested columns were found to be comparable based on evaluation of separation and validation parameters achieved on UPLC system. The time needed for separation of analytes in UPLC mode did not exceed 6.0 minutes with 1.0 min equilibration step.
Further, the compatibility of Kinetex C18 column with HPLC system was investigated. Low pressure gradient chamber and large system
volume of HPLC instrumentation caused the need of the chromatographic conditions adjustment, so that similar separation parameters
in both UPLC and HPLC modes were achieved. Analysis time on HPLC system was 9.5 minutes, but longer equilibration time of 10.0
min was necessary.
P227:
UHPLC-MS/MS method for the determination of atorvastatin in biological samples using microextraction by packed sorbent as
sample preparation technique
Hana Vlčková1, Lucie Nováková1, Milan Bláha3, Petr Solich1
1 Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
3 2nd Department of Internal Medicine, Hematology, Charles University and Teaching Hospital, Sokolská 408, 500 05 Hradec Králové, Czech Republic
Atorvastatin belongs to the group of hypolipidemic drugs named statins. They are inhibitors of microsomal 3-hydroxy-3methylglutarylcoenzym A (HMG-CoA) reductase and reduce the levels of total cholesterol, low-density lipoprotein cholesterol and plasma triglycerides.
UHPLC-MS/MS method for the determination of atorvastatin and its two metabolites in serum was previously developed and validated.
The aim of this work was to develop microextraction by packed sorbent (MEPS) sample preparation for the determination of atorvastatin in biological fluids. MEPS method together UHPLC-MS/MS method will be used for the monitoring of atorvastatin concentrations
in biological samples before and after the extracorporeal elimination (EE).
MEPS is a new miniaturized solid phase extraction and it is implemented by needle and syringe. Approximately 1-2mg sorbents is inserted into the barrel of syringe. Commercially available sorbents are silica based C2, C8, C18 and M1 (mixed of C8 and SCX).The sample
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Poster Abstracts
can be draw through the syringe manually or by an autosampler. MEPS can be on-line connected with LC. This form of solid phase
microextraction makes possible to use lower volumes of sample and solvents. It is fast, simple method and its use is advantageous for
preparation of biological samples.
The optimal conditions of MEPS technique were found. C18 sorbent was used. 50µl of sample was aspirated through the needle. Analytes was eluted with 100µl of acetonitrile: 0.1mM ammonium acetate buffer pH 4.0 95: 5, v:v). Mixture of 0.01mM ammonium acetate:
acetonitrile (85: 15, v: v) was used as the washed solution.
Originally, time-consuming, multi-step and solvent demanding solid phase extraction (SPE) approach was used. On the other hand,
MEPS demonstrated good linearity (r2> 0.9990, recovery (87- 118 %), precision (RSD= 1.43‑ 9.78 %) within much shorter period of time,
using simple procedure and much lower solvent and sample volumes. Therefore formerly used SPE was replaced by MEPS as sample
preparation method for determination of atorvastatin and its two metabolites in serum.
Two microscale methods were optimalized. The new sample preparation approach employing MEPS was developed. Together with
UHPLC-MS/MS method it was used for determination of atorvastatin and its two metabolites in serum obtained from patients with
Familiar hypercholesterolemia before and after EE procedure.
The author gratefully acknowledge IGA NR/9103-4 and MSM 0021620822.
P228:
The Use of capillary electrophoresis for monitoring of glycerol in adipose tissues
Klára Málková, Petr Tůma, Eva Samcová
1 Institute of Biochemistry, Cell and Molecular Biology; Third Faculty of Medicine, Charles University, Ruská 87, 100 00 Prague 10, Czech Republic
Studies on mobilization of adipose supplies and on the possibilities of influencing them are of primary importance for obesity treatment1. The dynamics of degradation of adipose supplies during a sporting action can be monitored by means of microdialysis2. During
lipolysis, the reserve triacylglycerides contained in the adipose tissue are degraded to fatty acids and glycerol. It is possible to continuously sample glycerol, which is readily soluble in water, by a microdialysis probe inserted into the adipose tissue and to determine its
level in the microdialyzate.
A capillary electrophoretic procedure employing contactless conductivity detection has been developed for direct determination of the
glycerol in microdialyzate of adipose tissue. Glycerol is fully separated from the sample matrix within very short time of 3.0, when using
the optimized background electrolyte, 60 mM H3BO3 + 30 mM LiOH (pH 9.1). The limits of detection amount to 0.5 µM is satisfactory
for direct determination of glycerol without sample pretreatment in small volume of microdialyzate about 2.5 µL. The main advantage
of this method is its capability of monitoring rapid changes in the concentrations of glycerol during physiological studies carried out
in vivo.
During the physiological study, the test person was first at rest and then rode an exercise bicycle. The glycerol content is maintained at
a constant basal level of 170 µM during the rest period. At the start of physical effort, the glycerol level begins to gradually increase, up
to ca. 340 µM after one hour of riding the exercise bicycle, i.e., to the double of the basal level. Lipolysis can efficiently be accelerated
by local administration of adrenaline. The combination of adrenaline administration and the exercises bicycle riding leads to an increase
in the glycerol level up to 800 µM, which corresponds to ca. 5times of the basal level.
1 Arner, P., Int. J. Obesity 1995, 19, 435-442.
2 Arner, P., Bolinder, J., Eliasson, A., Lundin, A., Ungerstend, U., Am. J. Physiol. Endocrinol. Metab. 1988, 255, E737-E742.
P229:
Ultra-performance liquid chromatography fingerprinting method for chemical screening of spent fermentation broths
Zdenek Kamenik*1, Dana Ulanova2, Marketa Mareckova3, Jan Kopecky3, Kamila Plhackova2, Jana Olsovska
1 Institute of Microbiology of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
Charles University, Faculty of Science, Albertov 8, 128 40 Prague 2, Czech Republic
2 Institute of Microbiology of the ASCR, v.v.i., Videnska 1083, 142 20 Prague 4, Czech Republic
3 Crop Research Institute, Drnovska 507, 16106 Prague 6, Czech Republic
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Poster Abstracts
Fingerprinting method for chemical screening of secondary metabolites, potential antibiotics, in spent bacteria fermentation broths
is described. The method is based on high-throughput ultra-performance liquid chromatography (UPLC) separation with UV detection
(photodiode array detector). Thirteen various antibiotic standards and four spent actinomycete fermentation broths were used for the
method development. The comparison of several liquid-liquid and solid phase extraction protocols for sample clean-up and pre-concentration revealed that Oasis HLB C18 sorbent gives the best recoveries. The Acquity BEH C18 chromatographic column was chosen for
the samples separation with respect to its universality, selectivity, efficiency and robustness. Further, the evaluation of mobile phases,
gradient elution programs, and column temperatures was performed in order to find the most convenient UPLC conditions. The method
provides two 3D fingerprints obtained under different UPLC conditions (acidic or alkaline aqueous part of the mobile phase) for every
sample. Each pair of 3D fingerprints contains the following physical-chemical information concerning the particular substances: (1)
polarity expressed by retention times; (2) presence of chromophores expressed by UV spectra; (3) compounds concentration expressed
by detector response and (4) acid-basic properties expressed as the influence of different pH of the aqueous parts of mobile phases on
retention times and UV spectra. The method reproducibility including sample extraction was assessed with relative standard deviations
(RSD) under 0.2, 5.0 and 20% for retention times, major peak areas and minor peak areas, respectively.
P230:
Mass spectrometric analysis of ceramides in gastric cancer cells
Yong-Moon Lee, Hun-Young So, Yong-Hyeon Yim, You-Xun Jin, Nam-Young Park, Jun-Young Kwak, Hyun-Seok Lee
Chungbuk National University, 48 San Kaesin-Dong, Hungduk-Ku 361-763 Chongju, Korea
Ceramides and dihydroceramides are pro-apoptotic signaling lipid mediators which are composed of a sphingoid base backbone and
a fatty acid chain. Cellular ceramides are synthesized via de novo sphingolipid biosynthetic pathway which start from the coupling of
palmityl-CoA with L-serine while the apoptotic signal activates sphingomyelinase (SMase) which digests membrane sphingomyelin into
ceramides. in drug-resistant cancer cells, the activation of SMase activity and/or de novo synthesis trigger apoptosis by increasing ceramides. Our final aim is to analyze the diverse ceramides which newly synthesized either SMase activation or de novo synthesis. Here, we
report an analytical methodology by using LC-MS/MS and LC-FTICR-MS. LC conditions of reverse phase Luna C18 column (2.0 x 150mm
ID: 5um) and mobile phase gradient elution of A (1 mM ammonium formate in MeOH (0.2 % formic acid) and B (2 mM ammonium formate in H2O (0.2% formic acid) were successfully analyzed diverse ceramides and dihydroceramides. Ceramides and dihydroceramides with
different fatty acid chains were analyzed with the aid of precise mass measurement by LC-FTICR-MS. Using LC-MS/MS with selective
MRM transitions, diverse ceramides and dihydroceramides in SNU-1 cancer cells were determined. However, LC-MS/MS chromatogram
for real samples frequently showed multiple peaks for a single MRM transition, which necessitates more robust identification of each
ceramide speices. Comparison of LC retention time and retention time relationships of ceramide species provided evidences on the
identity of each ceramide species. Accurate mass measurements from LC-FTICR MS confirmed the elemental composition of each ceramide species identified within ~ 1 ppm mass measurement accuracy. This work is supported by Chungbuk BIT Consortium (CBITRC)
P231:
Creatinine quantification in peritoneal dialysis fluid by capillary electrophoresis and UV detection
Elizabeth Caussé1, Marie Beatrice Nogier2, David Ribes2, Guillaume Echene1, Lionel Rostaing2, Robert Salvayre1, François \Couderc7
1 Biochemistry, Rangueil University Hospital
2 Nephrology, Rangueil Univerty Hospital
7 IMRCP, UMR5623/Picometrics, Toulouse
Measurement of creatinine in peritoneal dialysate is used in the peritoneal equilibration test to assess the performance of ambulatory
peritoneal dialysis. Peritoneal dialysate (PD) solutions often contain glucose, which interferes with the Jaffé creatinine measurement.
Glucose slowly reduces the alkaline picric acid to picramate. Also, enzymatic creatinine assays are routinely used in clinical laboratories
to provide more accurate estimated glomerular filtration rates.
The aim of the study was to evaluate a new method to directly measure creatinine by capillary electrophoresis and possible interference
with glucose. Three methods of creatinine determination in peritoneal dialysate, Jaffé kinetic assay, enzymatic method and capillary
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Poster Abstracts
electrophoresis (CE) were compared.
Methods: 12 PD patients (6 M, 6 F, 66,6 years) from Rangueil hospital dialysis centre were enrolled with 81 fluid collections. Blood serum
creatinine level was measured by standardized Jaffe assay. Creatinine, urea and glucose levels were determined during the Peritoneal
Equilibration Test (PET), the Accelerated Peritoneal Examination test (APEX) or peritoneal clearance for the evaluation of the peritoneal
permeability. Creatinine levels, involved in the above clinical tests, were measured in all fluid samples as follows : using a Jaffé kinetic
(alkaline picrate) method on Olympus AU2700, using an DiaSys Poles enzymatic method adapted on Olympus AU2700 or using a new
direct method by capillary electrophoresis/UV (Picometrics).
Results/Discussion: The CE method had a lower limit of quantification of 3 µmol/L and the intra- and inter-assay imprecision was < 3
% and < 5 %,respectively.
The Jaffé method correlated well with the enzymatic (r2 = 0991) or CE method (r2 = 0983) method. But we confirm an overestimation of
creatinine for PD solutions. For dialysate glucose > 50 mmol/L, the Jaffé biais was 55 µmol/L of creatinine and for glucose < 50 mmol/L
the biais was 30 µmol/L. In all glucose solutions, no creatinine was detected using the CE method. On the other hand, we found 24
µmol/L of creatinine in Nutrineal dialysate by enzymatic method.
Conclusion: This study confirms the overestimation of creatinine measurement using the Jaffé kinetic method in PD solutions, due to
glucose interference. This interference in dialysate is reduced with enzymatic or CE methods. For the first time, the CE method is used
for creatinine determination in PD. This method is simple, rapid, cheap, and could be used for routine determination.
P232:
Separation and characterization of aggregated species of amyloid-beta peptides
Henning Wiberg1, Patrik Ek1, Frida Ekholm Pettersson3, Lars Lannfelt3, Åsa Emmer1, Johan Roeraade1
1 School of Chemical Science and Engineering, Division of Analytical Chemistry, Royal Institute of Technology, SE-10044 Stockholm, Sweden
3 Department of Public Health and Caring Sciences/Molecular Geriatrics, Uppsala University, Rudbeck Laboratory, SE-751 85 Uppsala, Sweden
The onset of Alzheimer’s disease (AD) involves aggregation of amyloid-beta peptides, via soluble intermediate aggregates (protofibrils),
into a non-soluble fibrillar plaque, which is deposited in brain tissue. The analysis and characterization of protofibrils in body fluids such
as CSF is therefore of great importance to obtain a better understanding of the mechanisms in AD, but this is a highly challenging task,
since the concentration of protofibrils in body fluids is anticipated to be extremely low. In the present study, we have separated different
aggregated Aß peptides by means of Isoelectric focusing, followed by immunodetection, using Western blot (IEF-WB). Preparations from
Aß1-40 as well as Aß1-42 peptides yielded a low molecular species (LMW) band at a pI of approximately 5, and a protofibril/oligomer band
at a pI of approximately 6-6.5. The IEF-WB technique was also utilized to characterize protofibrils, prepared from a mixture of monomeric Aß1-40 and Aß1-42 peptides. The results of this study showed that the aggregated species contained both monomers. Protofibril
formation was further confirmed by using a sandwich ELISA with the protofibril conformation-selective antibody mAb158. To characterize the assembly of Aß peptides in the protofibrils, an immunoprecipitation method, followed by nanoelectrospray mass spectrometry
(IP-nESI-MS) was developed. The immunoprecipitation was performed with the protofibril conformation-selective antibody mAb158. The
method also included a step, where the precipitated protofibrils were treated with formic acid, to convert them into their monomers.
The method was used to assess the Aß1-40:Aß1-42 ratio in protofibrils from preparations with various initial monomeric Aß1-40:Aß1-42 ratios.
The effect of different incubation times was also investigated. The results revealed that Aß1-42 in the protofibrils was always present at
an excess level, compared to the level in the initial monomeric Aß1-40:Aß1-42 ratios used. This indicates the importance of Aß1-42 for the
aggregation behaviour of preparations of Aß. Experiments with longer incubation times revealed an increasing incorporation of Aß1-40
in the protofibrils, underlining the importance of dynamics in protofibril assembly. We also used the IP-nESI-MS method to compare
the selectivity of the protofibril conformation-selective antibody mAb158 with the selectivity of the Aß-selective antibody mAb1C3. For
this purpose, the hydrophilic amyloid-beta peptide, Aß1-16, (which is presumed not to co-aggregate), was used as selectivity marker. The
results clearly confirmed the differing selectivities of the antibodies.
P233:
Analysis of bacterial phosphoproteome of Francisella tularensis using different enrichment techniques with subsequent mass
spectrometry analysis
Pavel Rehulka*1, Petra Spidlova1, Lenka Hernychova1, Benjamin F. Mann4, Ales Tichy1, Helena Rehulkova1, Jiri Stulik1, Milos V. Novotny4
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Poster Abstracts
1 Faculty of Military Health Sciences, University of Defence, Trebesska 1575, CZ-50001 Hradec Kralove, Czech Republic
4 Department of Chemistry, National Center for Glycomics and Glycoproteomics, Indiana University, 47405 Bloomington, IN, USA
Phosphorylation of proteins represents a regulatory device, which plays a key role in the control of cellular activities in many organisms
and it is considered a fundamental mechanism for protein function. This post-translational modification has been found to alter also the
function of prokaryotic proteins and could play a pivotal role in bacterial virulence in this way. The innovative proteomics technologies,
combining separation methodology with mass spectrometry analysis, provide capabilities for detection, identification, and characterization
of phosphorylated proteins in bacterial lysates. This work focuses on the phosphoproteome analysis of bacterium Francisella tularensis.
Bacteria were cultivated in the chemically defined Chamberlain medium until they reached OD600 0.8 under the standard conditions. After
harvesting, the whole cell lysates were prepared. Samples containing 270 μg of protein were subjected to kinase reaction with radioactive
γ32P ATP in the presence of Mg2+ and Mn2+ ions and separated on 2D electrophoresis in the pH range 4–7. Gels were exposed to BioMax
MS films for 13 days and then silver stained. Spots corresponding to phosphoproteins were excised from Coomassie blue-stained nonradioactive 2D gels and digested by trypsin. The phosphopeptides were enriched using TiO2 and immobilized metal affinity chromatography
(IMAC) particles according to optimized protocols. Mass spectra were recorded on a 4800 MALDI-TOF/TOF mass spectrometer (Applied
Biosystems).
Autoradiography detection showed the presence of a number of phosphorylated protein spots in the separated whole cell lysate of
Francisella tularensis. Matching gel spots from Coomassie stained 2D gels were selected and some phosphopeptides were identified in
2,3-bisphosphoglycerate independent phosphoglycerate mutase (FTT1329; gi|81597255) using MALDI-TOF/TOF mass spectrometry. Moreover, a similar phosphopeptide in this protein (gi|1790041) was previously identified in Escherichia coli with phosphorylation at the same
serine residue. Other possible candidate phosphoproteins were selected from previously published prokaryotic phosphoproteome projects,
and corresponding protein spots were selected from 2D gels prepared from Francisella tularensis whole cell lysates. These spots were
processed by the optimized methods for TiO2 and IMAC phosphopeptide enrichment and a comparison of both methods was performed.
This work was financially supported by Ministry of Education, Youth and Sports of Czech Republic, grant ME08105 and Ministry of Defence,
Czech Republic, grants FVZ0000604, OVUOFVZ200808, and grant P41 RR018942 from the National Center for Research Resources, U.S.
Department of Health and Human Services.
P234:
Miniaturized analysis of single wood cells performed with preconcentration and CE
Johan Jacksén*, Juliane Beyer, Bernhard Eickenberg, Åsa Emmer
Royal Institute of Technology, KTH, Analytical Chemistry, Stockholm, Sweden
It is important to have the prospect to screen the carbohydrate composition during pulp and paper processes due to the fact that carbohydrate constitution of the fibers settles the chemical and physical properties of the products. A simple and fast analysis method for
studying single wood fibres for bioanalytical purposes is also highly desirable. Preferably chemical pre-modification of the sample such
as labeling should be avoided as that may lead to reduced selectivity and is time consuming. Wood fibres are composed out of hemicellulose, cellulose and lignin. Degradation of cellulose and hemicellulose through acid- or enzymatic hydrolysis results in monosaccharides;
ß-D-glucopyranose (Glu) forms the cellulose, and primarily α-L-arabinofuranose (Ara), ß-D-galactopyranose (Gal), ß-D-mannopyranose (Man)
and ß-D-xylopyranose (Xyl) form the hemicelluloses. The degradation is usually performed in bulk volumes generating a relative average
composition of different carbohydrates. By analysing a single wood cell it is possible to study the absolute distribution between the different monosaccharides for particular cells. In the present work, the monosaccharides were analysed with capillary electrophoresis since
this is a highly efficient, fast method, providing different selectivity options and of low sample consumption compared to other separation
methods. Due to the lack of chromofores on the monosaccharides indirect detection was used. The low monosaccharide amounts derived
from a single fibre and sensitivity disadvantages of indirect detection led to limited detection sensitivity. Therefore, a preconcentration step
was necessary to increase the detectability. The hydrolysis degradation were performed either in conventional bulk scale before analysis or
in microscale with one single wood fibre in an open microchannel with the dimensions 50µm*50µm (length 1 or 3 cm). The channel was
manufactured on a silicon microchip by standard micromachining fabrication methods including photolithography and deep reactive ion
etching (DRIE). For degradation of cellulose and hemicellulose into monosaccharides trifluoroacetic acid (TFA) or an enzymatic cocktail containing cellulase, ß-glucosidase and xylanase were used. The electrical preconcentration of the monosaccharides in the microchannel were
performed in a two electrode setup, and resulted in a significantly enhanced signal intensity of the five separated monosaccharide peaks.
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Poster Abstracts
P235:
Microchip electrophoresis for fingerprinting endotoxin chemotypes from whole-cell lysates
Lilla Makszin, Anikó Kilár, Béla Kocsis, Ferenc Kilár
University of Pécs, Szigeti út 12., 7624 Pécs, Hungary
Endotoxins (lipopolysaccharides, LPSs) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both,
advantageous and harmful biological activity of these microorganisms, are highly immunogenic and directly involved in numerous bacterial
diseases in humans such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The amphiphilic LPS compounds consist of a hydrophobic lipid region (named
Lipid A) covalently linked to the hydrophilic core oligosaccharide with or without the O-polysaccharide region. The differences among the
endotoxins from different bacterial serotypes and their mutants include variations mainly within the composition and length of the O-polysaccharide chains.
The proper assignation of the S or R chemotype of endotoxins is possible by their electrophoretic profiles. The recent microchip electrophoretic methods provide fast characterizations and differentiations of endotoxins directly from whole-cell lysates. The LPS components
are visualized either by the interaction with dodecyl sulphate and a fluorescent dye or by a covalently bound fluorescent dye. The labeled
endotoxin complexes are analyzed in the Agilent 2100 bioanalyzer microchip electrophoresis system applying the Protein 80 LabChip kit or
the High Sensitivity Protein 250 LabChip kit with minor modifications. These chip electrophoretic methods are able to replace the conventional SDS-PAGE with silver staining detection, with the advantage of better sensitivity, high speed and quantification.
The work was supported by the grants GVOP-3.2.1-0168, RET 008/2005 and OTKA-NKTH-NI-68863.
1 Kilár, A., Farkas, V., Kovács, K., Kocsis, B., Kilár, F. Electrophoresis 29 (2008) 1713–1722
2 Kilár, A., Péterfi, Z., Csorba, E., Kilár, F., Kocsis, B. J. Chromatogr. A 1206 (2008) 21-25
3 Makszin, L., Kilár, A., Kocsis, B., Kilár, F. Electrophoresis (2009) – submitted for publication
P236:
Determination of phosphorus content in DNA using capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry
Shin-ichiro Fujii, Kazumi Inagaki, Koichi Chiba, Akiko Takatsu
National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (AIST)
A demand of the quantification of nucleic acid such as DNA and RNA has been increased in many areas. However, the accurate quantification of nucleic acids has not been established yet. Especially, an immediate quantification of the intact large molecule such as
DNA sequencing fragment is necessary. On the other hand, capillary electrophoresis (CE) has advantage in good separation and small
consumption of samples. And inductively coupled plasma mass spectrometry (ICPMS) represents highly sensitive technique for the
determination of element. Therefore, we have developed an interface device for CE hyphenated with ICPMS1. Although the capillary
gel electrophoresis (CGE) is used for the analysis of the DNA sequencing fragment generally, the analysis case of CGE hyphenated with
ICPMS has not been reported. In this study, we have applied the CE-ICPMS with the interface device to the quantification of phosphorus
content in the separated DNA fragment using gel mediated capillary electrophoresis. Then the concentration of DNA was obtained from
the measured value of phosphorus.
This interface device (AIF-01, S.T. Japan Inc.) for CE-ICPMS consists of a three layered nebulizer, a low-volume vaporizing chamber, and an
electrode connector with a sheath solution. The P/ACE MDQ CE system (Beckman-Coulter) with fused silica capillary (100 mm i.d./375
mm o.d.) and Agilent 7500a ICPMS were used. In this study, the 100 bp DNA ladder samples containing 100 to 2000 bp DNA molecules
were analyzed using CGE-ICPMS, and calibrated by the phosphorus standard solution.
The interface realized a few mL/min nebulizing without choking the solution included gel buffer and 0.1 M nitric acid as a sheath solution. And the sample was introduced from CGE to ICP without drain. In the CGE-ICPMS measurement, DNA ladder sample was injected
into the ICPMS, and the element mass of phosphorus content in DNA was detected.
1 S. Fujii, K. Inagaki, A. Takatsu, T. Yarita and K. Chiba, J. Chrom. A, 1216 (2009) 7488–7492
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Poster Abstracts
P237:
Mass spectrometric analyses of bacterial lipopolysaccharides
Ágnes Dörnyei1, Anikó Kilár2, Annamária Bui1, Zoltán Szabó4, Béla Kocsis5, Ferenc Kilár1
1 Institute of Bioanalysis, Faculty of Medicine, University of Pécs, Szigeti út 12., H-7624 Pécs, Hungary
2 Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Ifjúság útja 6., H-7624 Pécs, Hungary
4 Department of Biochemistry and Medical Chemistry, Faculty of Medicine, University of Pécs, Szigeti út 12., H-7624 Pécs, Hungary
5 Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Pécs, Szigeti út 12., H-7624 Pécs, Hungary
Microchip electrophoretic methods provide fast characterizations and differentiations of bacterial endotoxins or lipopolysaccharides
(LPSs). For the mass assignation a mixture of LPSs could be used as mass standards (instead of proteins), if their structures are wellcharacterized. The structural study of intact LPSs by mass spectrometry also contributes to the understanding of processes related to
their physiological effects and bacterial pathogenesis.
The endotoxic LPSs are constituents of the Gram-negative smooth (S) or rough (R) bacterial cell wall and consist of two or three main
parts: (i) a glycolipid region, called lipid A, (ii) an oligosaccharide region, called core, to which (iii) a polysaccharide region, called O-chain,
is attached in the S-type endotoxins. Spontaneous or induced mutations may cause loss of the O-chains resulting in R-type LPSs.
In the poster, the structural variations in the R-type endotoxins of Shigella sonnei mutant strains are discussed. MS and MS/MS investigations were performed. Spontaneous mutation of S. sonnei phase I strain (a pathogenic member of the family Enterobacteriaceae)
leads to the non-pathogenic R-type, S. sonnei phase II (4303), which lacks the O-antigen. From this strain, a series of R-type mutants
were obtained by ethyl-methyl-sulfonate induced mutagenesis. S. sonnei R41, 562H and 4350 strains were isolated and their LPS content, as well as the LPS of 4303 “maternal” strain, were analyzed. A lipid A moiety was obtained by mild acid hydrolysis from LPS of S.
sonnei R41.
The detailed evaluation of the mass spectra indicates heterogeneity in the fatty acid composition in the lipid A part and variability in
phosphorylation stage. The full oligosaccharide cores of the rough mutants contain two 3-deoxy-D-manno-2-octulosonic acid (Kdo)
units. The LPSs of the isogenic rough mutants S. sonnei 4350, 562H, R41 and 4303 are formed in a step-like manner containing 0, 1, 2
and 3 heptoses, respectively, in the inner core region. The outer core region of the LPS from the 4303 mutant contains also 5 hexoses.
P238:
Determination of polyphenolic compounds by LC-MS in the thymus species
Silvia Jakabová1, Borbála Boros1, Ágnes Dörnyei3, Györgyi Horváth4, Attila Felinger1, Ferenc Kilár3
1 Department of Analytical and Environmental Chemistry, Faculty of Science, University of Pécs, Ifjúság útja 6, H-7624 Pécs, Hungary
3 Institute of Bioanalysis, Medical School, University of Pécs, Szigeti út 12, H-7624 Pécs, Hungary
4 Institute of Pharmacognosy, Medical School, University of Pécs, Rókus utca 2, H-7624 Pécs, Hungary
Polyphenolic compounds present the wide group of chemicals, including well known subgroups like phenolic acids, flavonoids, natural
dyes, lignans etc. that are produced in plants. These naturally bioactive compounds possess variety of beneficial properties including
antioxidant, anti-carcinogenic activities, efficiency against coronary diseases and antimicrobial properties.
Thymus species have been reported as an interesting for their content of flavonoids; moreover, composition and content of flavonoids
in thymus species is an important taxonomic marker for the distinction of species.
The objective of this work was separation and quantification of selected polyphenols (gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, dihydroquercetin, ferulic acid, phloridzin, rosmaric acid, apigenin, eriodictyol, naringenin, phloretin, isoliquiritigenin, quercetin,
hesperetin, rutin, hesperidin, catechin and epicatechin) in thymus herbal extracts.
High-performance liquid chromatography (HPLC) coupled with dioda array detector (DAD) and on-line mass spectrometry (ESI – MS)
method was used for analysis.
The most abundant phenolic acids were caffeic acid and its derivative chlorogenic acid. Apigenin, apigenin-7-glucoside, eriodictyol,
rosmarinic acid, naringenin and dihydroquercetin were the most abundant flavonoids. Amounts of eriodictyol, chlorogenic and caffeic
acids were in correlation with thymus species.
180
Poster Abstracts
P239:
Characterization of flavonol glycosides in Allium ursinum by HPLC-MS
Ágnes Dörnyei1, Borbála Boros2, Kinga Rudolf 3, Tamás Morschhauser3, László Márk5, Attila Felinger2, Ferenc Kilár1
1 Institute of Bioanalysis, Faculty of Medicine, University of Pécs, Szigeti út 12., H-7624 Pécs, Hungary
2 Department of Analytical and Environmental Chemistry, Faculty of Sciences, University of Pécs, Ifjúság útja 6., H-7624, Pécs, Hungary
3 Department of Taxonomy and Geobotany, Faculty of Sciences, University of Pécs, Ifjúság útja 6., H-7624, Pécs, Hungary
5 Department of Biochemistry and Medical Chemistry, Faculty of Medicine, University of Pécs, Szigeti út 12., H-7624 Pécs, Hungary
Flavonoids are widespread plant secondary metabolites. They have a wide range of biological and physiological activities and serve
as chemotaxonomical markers. Flavonoid glycosides are predominant forms of naturally occurring flavonoids in plants. The effect of
glycosylation is to render the flavonoid less reactive and more water soluble. Acetylated glycoside in which one or more of the sugar
hydroxyls are esterified with an acid, also occur.
Allium species are amongst the richest sources of dietary flavonoids. The Allium ursinum L. commonly known as wild garlic or ramsons
is widely used as a spice as well as a traditional medicine.
In the poster, the flavonoid composition of Allium ursinum L. studied by LC/UV-DAD/ESI-MSn is discussed. The samples of Allium ursinum ssp. ucrainicum were collected, and 1:1 methanol/water extracts of leaf and crop samples were analyzed. A mass spectrometric
method based on the combined use of electrospray ionization, collision-induced dissociation and tandem mass spectrometry has been
applied to the structural characterization of the flavonoids present in the samples. A large number of kaempferol glycoside and acylated
glycosides has been identified.
P240:
Preparation and investigation of bioactive transferrin-iron complexes formed with different synergistic anions
Judit Gálicza*1, Andrea Vargová2, Viktor Sándor2, Csilla Páger4, Rita Fodor4, Ildikó Miklóssy6, Beáta Ábrahám6, Szabolcs Lányi6,
Ferenc Kilár4
1 POLITEHNICA University of Bucharest, Applied Chemistry and Material Science, Bucharest, 010737, Romania
2 University of Pécs,Department of Analitycal and Environmental Chemistry, Pécs, 7624, Hungary
4 University of Pécs, Medical School, Institute of Bioanalysis, Pécs, 7624, Hungary
6 SAPIENTIA University, Faculty of Technologiacl and Social Siences, Technical Science Department,Miercurea Ciuc, 530104, Romania
Human serum transferrin, a glycoprotein, has two lobes, each having a binding site for iron(III)1. The membrane transferrin receptor-mediated endocytosis of the complex of human serum transferrin bound iron and the transferrin receptor is the major route of cellular iron
uptake. This efficient cellular uptake pathway has been exploited for the site-specific delivery of anticancer drugs2. High performance
capillary electrophoresis and isoelectric focusing (IEF) experiments were performed to determine the differences between human apotransferrin, holo-transferrin, and to control transferrin-iron-anion complex formation. For the measurements capillaries with 200 mm
length, and 50 μm I.D., coated with non-crosslinked acrylamide to eliminate electroendozmosis were used. The buffer containing 18 mM
Tris, 18 mM boric acid, 0.3 mM EDTA (pH 8.4)3 was used as background electrolyte. Run voltage was10 kV. IEF experiments were made
with the sequential injection setup4 in uncoated capillary applying various pH range ampholytes. The different transferrin samples were
analyzed with capillary electrophoresis. The electropherograms show the differences between iron-free-, and iron-complexed molecular
forms of human serum transferrin. The iron-complexed transferrin sample containing the different anions as synergistic complexing
agents were characterized by different electrophoretic parameters. The differences in the binding constants and in the electrophoretic
parameters might give information about the bioactivity of the transferrin-iron-anion complexes.
1 P. Aisen, R. Aasa, B. G. Malström, T. Vänngård, The Journal of Biological Chemistry 242, 10, 2484-2490 (1967)
2 P. T. Gomme, K. B. McCann., Reviews-Drug Discovery Today, 10, 4, 267-273 (2005)
3 F. Kilár, S. Hjertén, Electrophoresis 10, 23-29 (1989)
4 F. Kilár, Á. Végvári, A. Mód, Journal of Chromatography A, 813, 349-360 (1998)
181
Poster Abstracts
P241:
Development of new UHPLC-MS/MS method for the determination of antibiotics in environmental waters
Marcela Seifrtová1, Jana Aufartová1, Angelina Pena3, Lucie Nováková1, Petr Solich1
1 Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Hradec Králové, Czech Republic
3 Group of Bromatology, Centre of Pharmaceutical Studies, University of Coimbra, Coimbra, Portugal
Antibiotics are a class of environmental pollutants of an increased concern in last years. They were detected in wide range of environmental waters, including surface waters, ground waters and wastewaters. Their presence and persistence in the aquatic environment
can cause important problems for the public health. The main problem of antibiotic residues in aquatic environment is potency for the
development of antibiotic resistance of sensitive microorganisms.
Ultra high performance liquid chromatography (UHPLC) is nowadays new trend for the separation in analytical chemistry. UHPLC offers
the great advantages over conventional HPLC due to ultrahigh back pressures of up to 15 000 psi (100 MPa) and using the sub-2-μm
particle sorbents yielding high separation efficiency. New automatic development approach specially arranged for UHPLC in connection
with mass spectrometry offers an efficient tool in development of high-throughput selective and sensitive analytical methods. This
approach used changing 4 columns (Acquity BEH C18, Acquity BEH Phenyl, Acquity BEH Shield and Acquity HSS C18), two modificators
(acetonitrile or methanol) and two buffers pH 3 and pH 9 in gradient mode of elution. This automatical approach generates 14 different
chromatograms in about 5 hours, including all variables (column, buffer, modificator).
A new UHPLC method for the determination of five commonly used fluoroquinolone antibiotics (ofloxacin, norfloxacin, ciprofloxacin, pefloxacin and enrofloxacin) in wastewaters using both tandem mass spectrometry and fluorescence detection was developed. Systematic
method development approach was compared to a conventional one. The best separation of fluoroquinolones was obtained on BEH
Phenyl analytical column using ammonium acetate solution pH 10.5, which is not in agreement to all published results. The sensitivity
and selectivity of fluorescence (FD) and mass spectrometry detection was compared. FD at basic pH 10.5 demonstrated lower sensitivity than conventionally performed analyses at pH 3.0. UHPLC-MS/MS was found about two orders of magnitude more sensitive. Both
methods were validated and compared in terms of linearity, accuracy, precision and limits of detection and quantification. UHPLC-MS/
MS method was used for the analysis of water samples.
The project was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (KJB 601100901).
P242:
On-plate concentration and analysis of low-molecular compounds using hydrophobic surfaces in MALDI MS
Maria Borissova, Merike Vaher
TUT Institute of Chemistry
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been extensively used for the analysis of large and
medium-size molecules as proteins and peptides. The detection of small molecules with common matrices is usually limited due to the
abundance of interfering matrix ions in the low-mass range of spectra. Moreover, the main reason for the problems encountered with
method sensitivity and reproducibility is the non-homogeneous distribution of the analyte using conventional target plates. Nowadays
this problem can be resolved by developing of the new methods for the formation of various hydrophobic supports and introduction of
new types of matrixes based on novel reagents. These supports were successfully applied for rapid analysis of complex low-molecular
analyte mixtures with excellent reproducibility and low limits of detection.
Present research introduces the combination of the two above-mentioned strategies for the improvement of the mass spectrum quality.
The potential application of this merging was demonstrated with several examples. Firstly, on-plate oxidation process of glutathion
followed by MALDI - MS was investigated involving minimal sample handling and eliminating sample loss. Secondly, biological fingerprinting of Taxus extracts was applied to screen the traces of multiple bioactive compounds (alkaloids) in natural products by selective
concentration of the sample droplets into microspots. And finally, the effective method for quantitative analysis of free fatty acids
using mixture of powdered carbon aerogels and Teflon was developed and evaluated by application of internal standards and improved
properties of target surface.
182
Autor´s Index
Autor´s Index
Abele S.
P028
Beyer J.
P234
Abraham B.
P240
Bezuidenhout L. W.
P150
Adam T.
P015, P105, P106, P108, P109,
P111
Bhagat A. A. S.
P129
Bilkova Z.
P004, P013, P082, P135, P192,
P210, P216
Adriaenssens L.
P047
Ahmed A.
P197
Blaga I.
L61, P041
Ahn J.
P200
Blaha M.
P227
Aldini G.
L5
Blomberg L. G.
L8
Alley W. R.
P077
Boardman C.
P031, P167
Alley W. R. Jr.
P195
Bocek P.
P068
Allmaier G.
P117
Bodor R.
L38, P088, P095, P124
Al-Othman Z.
P176
Bogdan G.
P041
Anderson G. J.
L27
Bohlin E. M.
L8
Andrieux K. K.
P149
Bohoyo D.
P013
Anik N.
L36
Bomastyk B.
P124, P172
Ansley D. M.
P103, P180
Borissova M.
P242
Aona K.
P119
Boros B.
P238, P239
Aparicio E. E.
L9
Boschetti E.
PL3, L22
Archakov A. I.
L3, L41, L50
Böttger J.
P130
Arima T.
P131
Bouajila J.
P169
Arriaga E. A.
L35
Boutonnet A.
P161
Asahi T.
P225
Poinsot V.
P169
Aturki Z.
L4
Boven K. H.
L63
Aufartova J.
P241
Boyd K.
P006
Avagyan A. P.
P053
Brabazon D.
P021
Baba Y.
P131, P151
Brabcova I.
P217
Babula P.
P145
Brambilla D. D.
P149
Badjah-Hadj-Ahmed A.
P176
Brandmüller A.
P197
Bachi A.
L22
Breadmore M. C.
L18, L54, P070
Bai Y.
L32
Brett M. J.
P150
Baldock S. J.
P051
Brezovsky J.
P193
Balog C. I. A.
L58
Britz-McKibbin P.
P001, P183
Balonova L.
P082, P216
Brüggermann O.
P019
Barbas C.
P003
Brunnert H.
P084
Barron A. E.
PL8, P034, P036, P042
Bruns S.
P022
Bednar P.
P061
Bucinska L.
P195
Bek F.
L54, P118
Bui A.
P237
Bekarek V.
P108, P109
Bukharina N. S.
L41
Belcinski R.
P041
Bunz S.
L6
Bellouard Y.
P115
Buscaino J.
P041
Belsanova B.
L65
Busnel J. M.
L10, P134, P199
Benesova L.
L65
Buszewski B.
L39
Benoit F.
P087, P175
Butini S.
P184
Beres T.
P178
Butler P.
L61
Berezovski M. V.
P008
Buzas E.
P209
184
Autor´s Index
Campiani G.
P184
Du F.
P056
Canova E.
P060
Dubsky P.
P048
Canovi M. M.
P149
Dupejova J.
P195
Carrow J.
P041
Dusa F.
P094
Caslavska J
P182
Dvorak P.
P193
Caslavsky J.
P006, P189
Dziubakiewicz E.
L39
Causse E.
P161, P231
E H.
P181
Cedazo-Minguez A.
P113
Ebata T.
P138
Cifuentes A.
L19, P040, P055, P113, P207
Eberhardt D.
L61
Cisar P.
P072
Eckhart A.
P208
Cohen S. A.
L28
Eckslager T.
P005
Cologna S. M.
P206
Edigo J.
P003
Colombo R.
L5, P184
Eeltink S.
L68, P024, P074
Cottet H.
L36
Eggertson M.
P200
Couderc F.
P161, P169, P231
Ehala S.
P069
Couvreui P. P.
P149
Echene G.
P231
Cretier G.
L55
Eickenberg B.
P234
Cretich M.
P211
Ek P.
P232
Crhova M.
P202
El-Sissi O.
P041
Crommen J.
P044, P046, P049
Emmer Å.
P234
Crooks R. M.
L43, P212
Emmerlich P.
P130
Cui Y. R.
P146
Emmerova T.
P010
D´Orazio G.
L4
Emon H.
P156
Damborsky J.
P193
Engel H.
P039
Damin F.
P211
Erwin G. S.
P140
Danckwardt N. Z.
L29
Esaka Y.
P089
Davich N. J.
L42
Esselmann H.
P004
Davis M.
L45
Fadgen K.
P200, P203
De Jong G. J.
L24
Fakhari A. R.
P045
De Lorenzi E.
L5, P184
Falmagne J. B.
P087, P167, P175
Deelder A. M.
L24, L25, L58, L59, L60, L67, P079
Fanali S.
L4
Dejaegher B.
P162, P165
Fang N.
P128
Deng Q. P.
P086, P146
Fasoli E.
L22
Deng X.
P073, P197
Faure A. Ch.
L52
Derks R.
L25
Faure K.
L55
Descroix S.
L52
Felinger A.
P238, P239
Desmarais S. M.
P042
Felts K.
P223
Dewald J. A.
P031, P167
Fenclova Z.
P014
Di Carlo G.
P211
Feurer B.
P161
Dickerson J. A.
L42
Fielden P. R.
P051
Díez-Masa J. C.
P003
Fillet M.
P044, P046, P049
Dolezal K.
P178
Filuszova M.
P012
Dolhain R.
L67
Flieger M.
P226
Dolman B.
P074
Florence X.
P044
Dolnik V.
L31
Fodor R.
P240
Doneanu A.
L28
Dörnyer A.
P237, P238, P239
Foret F.
L54, P028, P101, P118, P152, P155,
P158, P159, P207
Dráb T.
P196
Forster R. E.
P034
Droumaguet B. B.
P149
Fouad M.
P151
185
Autor´s Index
Franklin H.
P041
Greiner M.
P084, P110
Franssila S.
P142
Grieve B. D.
P051
Franzen U.
L47, P171
Grishina A. A.
P069
Franzreb M.
L29
Grubhoffer L.
P195
Freudigmann Ch.
P130
Grym J.
P101
Friedecky D.
P015, P105, P111
Guarino E.
P184
Frutos M.
P003
Guber A.
L29
Frydlova J.
P007
Guijt R. M.
L54
Fu X.
L32
Gumulec J.
P145
Fuchsberger K.
L63
Gupta R.
P051
Fujii S.
P236
Gurske W. A.
L31
Fukahori O.
P138
Guttman A.
L54, L57, P002, P009, P083, P118
Fung Y.
P056
Haan B.
L68, P024, P074
Fung Y.
P168, P213, P214
Hagmeyer B.
L63, P130
Fung Y. S.
P136
Halasiova M.
P088
Gabrielyan E. S.
P053
Halewyck H.
P162, P165
Galicza J.
P240
Halim N. H. A.
L63
Garcia-Cañas V.
L19, P040
Han J.
P129
Gareil P.
L48, L52, P044
Hankmeier T.
PL9
Garelly L.
P188
Hanzlikova E.
P196
Garrigues C.
L61
Harano A.
P138
Gas B.
L17, L65, P048, P220, P221
Harms Z. D.
L49
Gassmen M.
L65
Hartmanova L.
P099
Gassner A. L.
P143
Hase T.
P131
Gebauer P.
P068
Hasegawa Y.
P131
Gebhardt R.
P130
Hashiba K.
L66
Gebler J. C.
P190
Hauser P. C
P172
Gelder E.
L61
Havlicek V.
P099
Gemma S.
P184
Hayes M. A
P066
Gemrotova M.
P178
Heegaard N. H. H.
L8
Gerardin C.
L36
Hegrova B.
P096
Ghowsi H.
P075
Hennig R.
L67
Ghowsi K.
P075
Hensbergen P. J.
L58
Gibb S.
P006
Hernychova L.
P082, P210, P216, P233
Gilar M.
P081, P190
Herrero M.
L19
Gillece-Castro B.
P081
Herwig E.
P117
Gillotin F.
P049
Heyden Y. V.
P162, P165
Ginterova P.
P104
Hezinova V.
P158, P207
Girault
P134
Hiu Y.
P180
Girault H. H.
P143, P199
Hjertér S.
L70
Glatz Z.
P107, P166, P187
Hlushkou D.
L43, P022, P212
Glennon J. D.
P085
Hohnova B.
P062
Glovinova E.
P071
Hokke C. H.
L58
Gobbi M. M.
P149
Holcapek M.
L23, P060, P065
Goetz J. A.
P080
Holopainen J. M.
P191
Gong M.
L27
Höltzel A.
P017
Goodall D. M.
P188
Holzner F.
P130
Goor T. A.
P223
Honegr J.
P215
Greibrokk T.
P027
Hong H. X.
P146
186
Autor´s Index
Hoogmartens
P147, P187
Chomoutska J.
P145
Horak D.
P010, P030, P135
Chu I. Ch.
P205
Horka M.
P173, P186
Chudoba R.
L65
Horky J.
P173, P186
Chung D. S.
P176
Horn J.
L61
Ibañez C.
P113
Horvath G.
P238
Ibañez E.
P055
Hoshiba K.
P122
Iguchi W.
P133
Hotta A.
P038
Inaba M.
P157
Hou H. W.
P129
Inagaki K.
P236
House M. G.
P080
Ishibashi R.
P157
Hrabec R.
P145
Ishihara K.
P126
Hrouzek P.
P076
Isida Y.
P089
Hruska V.
P221
Ito K.
P224, P225
Hrynkiweicz K.
L39
Ivanov Y. D.
L41, L50
Hsieh Y. T.
L20
Jacksen J.
P234
Hsieh Y. Z.
P185
Jacobson S. C.
L49, P121, P140
Hu J. B.
P050
Jagr M.
P208
Huang Ch. Ch.
P144
Jakabova S.
P238
Hubalek J.
P145
Janca J.
L34
Hudson J. C.
L15
Jandera P.
L21, P029, P057
Huhn C.
L67, P079
Jang K.
P126, P139
Hui Y.
P103
Jankovicova B.
P004, P013, P135, P210
Hung H. Y.
P185
Janosova V.
P095
Hurak Z.
P115
Janson N. L.
P001
Hustoft H.
P027
Jensen H.
P163
Hynek R.
P202
Jiang X.
L7
Cha S.
L2
Jim S. R.
P150
Chaloupkova R.
P193
Jiruskova M.
P222
Chambers R.
P141
Jolivet P.
P059
Chang H. T.
L20
Josserand J.
P143
Chang H. T.
P033, P097, P144, P153, P154
Jovanovich S. B.
L61, P041
Chankvetadze B.
P046
Jungova P.
L37, P102
Chapman J.
L15, P037, P038, P052
Juskova P.
P155
Chardot T.
P059
Kacer P.
P014
Chauhan V.
P184
Kádas J.
P009
Cheglakov I.
L3
Kahle V.
P093, P094
Chen D. D. Y.
L16, P103, P128, P180
Kaji N.
P131, P151
Chen Q.
P214
Kamenik Z.
P226, P229
Chen R.
L7, P011
Kaniansky D.
L38, P088, P095, P100, P124, P179
Chen S. J.
P144
Kanicky V.
L37, P102
Chen T.
L1
Karenga S.
L69
Chen W. T.
P097, P154
Karger B. L.
PL1, L2, L57
Chian M.
P211
Kasicka V.
L10, P047, P055, P069
Chiang CH. K.
P097
Kasparova M.
P194
Chiap P.
P044, P049
Kato M.
P157
Chiba K.
P236
Kaupilla T.
P142
Chiu T. Ch.
L20
Choi K.
P176
Cho W.
PL7
Keebaugh M. W.
P066
187
Autor´s Index
Kennedy R. T.
L27
Krylov S. N.
P008
Kerby M. B.
P036
Kubesova A.
P173, P186
Kerékgyárto M.
P002
Kubickova R.
L21
Khachvankyan G. U.
P053
Kubon M.
L63
Kilar A.
P235, P237
Kubosek L.
P007
Kilar F.
L13, P090, P235, P237, P238, P239, P240
Kucerova J.
P004
Kim I. W.
P018
Kucerova Z.
P007, P010,P012
Kim J.
P176
Kuckova S.
P202
Kitagawa F.
L66, P114, P122, P123, P137
Kuchtickova S.
P145
Kitamori T.
L33, L56, P032, P125, P126, P138,
P139, P156, P157, P218
Kumar A. P.
P018
Kupcova R.
P104
Kitamura Y.
P218
Kurbatov L.
L3
Kizek R.
P005, P145
Kurucz I.
P009
Klafka H.
P013
Kuss H. M.
P124
Klejdus B.
P063, P064
Kutter J. P.
P127
Kleparnik K.
P152, P158, P159, P207
Kuzma M.
P014
Klodzinska E.
L39
Kvasnicka F.
P222
Klvana M.
P193
Kwak J. Y.
P230
Knittle J.
P041
Kwan H. Y.
L11
Kobayashi H.
P025
l´Escaille F.
P087, P167, P175
Kocsis B.
P235, P237
Labeau M. P.
L36
Kodicek M.
P059, P202
Lacinova K.
P208
Koeleman C.
L59
Lacroix-Desmazes P.
L36
Kohler I.
P098
Ladner Y.
L55
Kojima N.
P025
Laine J.
P191
Kondo M.
P131
Lam M. P. Y.
P205
König M.
P039
Lan G. Y.
P153
Konno T.
P126
Langridge J.
P203
Kopecky J.
P064, P076, P229
Lannfelt L.
P232
Kopylov A.
L3
Lanyi S.
P240
Korecka L.
P192, P210
Lau E.
P205
Kort B. J.
L44
Laurel T.
PL6
Kostal V.
L35
Laws D. R.
L43, P212
Kostiainen R.
P142
Le Potier I.
P013
Kotiaho T.
P142
Lebedova J.
P014
Kovács A.
P009
Lecoeur M.
L48
Koval D.
L10, P047
Lee B.
P052
Kovalczuk T.
P106
Lee H. S.
P230
Kovarik M. L.
P140
Lee R.
L26
Kowalska M.
P178
Lee Y. M.
P230
Kracmerova J.
P196
Lee Yvonne M.
P223
Kral V.
P148
Lee Y. P.
P185
Krasensky P.
L37
Lehmann D.
P039
Krätschmerova H.
P015
Lechmann M.
P008
Krcmova L.
P194
Leitner T. K.
P042
Kren V.
P061
Lemr K.
P061, P099
Krizkova S.
P005
Leon C.
P055
Kroupa T.
P210
Li A.
P035
Krizkova S.
P005
Li H. Y.
P086
188
Autor´s Index
Li Ch.
P164
Marchetti-Deschmamm M.
P117
Li Q.
L1
Martin-Alvarez P. J.
P003
Li Y.
L61
Martin-Ventura J. L.
P003
Liao J.
P164
Martirosyan S. S.
P053
Liberda J.
P196
Masar M.
L38, P124
Lies M.
P078
Masarik M.
P005, P145
Ligon S. C.
P160
Masuda M.
P224, P225
Lim CH. T.
P129
Matouskova H.
P173
Lin B.
L53
Matsumoto H.
P131
Lin H. Ch.
P033
Lin Y. Ch.
P033, P116
Mawatari K.
L56, P032, P125, P126, P138, P156,
P157, P218
Lin Y. W.
P097, P154
Maxwell E. J.
L14, L16, P103, P128
Link M.
P082, P219
Mayboroda O. A.
L24, L25
Lion N.
P134
Mayboroda O. M.
L58
Lisa M.
P060, P065
Mayumi K.
P224, P225
Liskova M.
P158, P159
Mazanec K.
P173
Liu H.
L32, P164
McDonnell L. A.
L60
Liu Ch.
L16, P128
McDowall M. A.
P198
Liu S.
L64
McIntosh R.
P041
Liu X.
L32
McKibbin P. B.
L26
Lojkova L.
P063, P064
Megova M.
P104
Lokajova J.
P191, P220
Meighan M. M.
P066
Lokmer N.
P039
Mevissen M.
L11, P112
Luc M.
P124
Micova K.
P105
Ludewig R.
P023, P026
Michienzi J.
L28
Lundanes E.
P027
Miklossy I.
P240
Luo Y.
P128
Miksik I.
P201, P208
Lyapkalo I. M.
P069
Milanova D.
P141
Lyubarskaya Y.
L46
Minamikawa H.
P224, P225
Ma S.
L45
Minarik M.
L65
Maan B. F.
P080
Misjak P.
P209
Macka M.
L54, P021, P028, P118, P177
Mittermayr S.
P002, P083
Macova D.
P006
Mizutani M.
P089
Madr A.
P166
Moeller A.
L63
Madren A. M.
P121
Moeller E. H.
L47
Mai T. D.
P172
Mogensen K. B.
P127
Maier V.
P099, P104, P178, P179
Moharami A.
P174
Mak J. L. L.
P214
Mohrlok R.
L63
Mark L.
P239
Mokaddem M.
L48
Mak T. W.
P008
Mondella M.
P040
Makszin L.
P235
Morandini J.
P143
Mala Z.
P068
Moravcova D.
P093, P094, P186
Malkova K.
P228
Morlock G. E.
P150
Mann B. F.
P082, P195, P233
Morschhauser T.
P239
Mantim T.
P172
Mortensen N. A.
P127
Manz A.
PL5
Mosher R. A.
P070
Marak J.
P100, P179
Moschallski M.
L63
Maramai S.
P184
Moskalyova N.
L3
Marhol P.
P061
Mravcova L.
P189
189
Autor´s Index
Mueller M. B.
L51
Østergaard J.
L47, P163, P171
Muench S.
L63
Otsuka K.
L66, P114, P122, P123, P137
Müllerova L.
P220
Otto M. M.
L9
Munro A.
L41
Oukacine F.
P188
Murphy J.
P120
Ozohanics O.
P209
Murphy J. P.
L28
Pagano S.
P041
Musheev M. U.
P008
Pager C.
L13, P240, P090
Musilova J.
P107
Pallandre A. A.
L9
Myjavcova R.
P061
Palmblad M.
L60
Naccache P.
P161, P169
Papouskova B.
P061
Nagai H.
P132, P133
Park J. H.
P018
Nahlik T.
P072
Park N. Y.
P230
Nan L.
P052
Park Y. K.
P176
Naruishi N.
P132, P133
Parshintsev J.
P191
Nasab K. H.
P058
Pataridis S.
P208
Nashabeh W.
L45
Pauwels J.
P147, P187
Naumova O. V.
L50
Pelclova D.
P014
Navratilova J.
L37, P102
Peltre G.
L10
Navratilova K.
P148
Pena A.
P241
Nesterenko P. N.
L51
Perdue R. K.
L43, P212
Neusuess Ch.
L6
Perry J. M.
L49
Nevedomskaya E. V.
L25
Petr J.
L52
Nicolas J. J.
P149
Petru K.
P215
Nicolas V. V.
L9
Phillips S. M.
L26
Nicolas V. V.
P149
Piasecki T.
P021, P028
Niedringhaus T. P.
P034
Pirrote B.
P044
Nielsen W.
L61, P041
Plaza de Moral M.
P063
Nilsson M.
L56, P032, P218
Plhackova K.
P229
Nischang I.
P017, P019, P067
Plichta Z.
P135
Niwa D.
P133
Pluijm I.
L25
Nobilis M.
L23
Plumb R. S.
P120
Nogier M. B.
P231
Poirier F.
P013
Nojavan S.
P045, P058, P174
Poitevin M.
L10
Novakova L.
P227, P241
Pol J.
P142
Novotna L.
P010
Polasek M.
P215
Novotny M. V.
PL2, P077, P080, P082, P195,
P216, P233
Polovka M.
P062
Polynkova A.
P105
Nuhn H.
P036
Popov V. P.
L50
Nuchtavorn
P177
Pormsila W.
P172
Nunes P. S.
P127
Portmann S.
L11, P112
O W.
P168
Pospichal J.
P071
Oefner P. J.
PL4
Pospisilova M.
P215
Oguchi K.
P133
Pourhaghighi M. R.
P199
Oita I.
P162, P165
Preisler J.
L37, P096, P102
Okamoto Y.
P131, P151
Prikryl J.
P152, 158
Olsovska J.
P226, P229
Proczek G.
P134
Olszowy P.
L39
Prokop Z.
P193
Oohashi T.
P138
Puerta A.
P003
Oostingh G. J.
L40
Purkrtova Z.
P059
190
Autor´s Index
Qui S.
P146
Salmanowicz B. P.
P054
Raedschelders K.
P103, P180
Salmona M. M.
P149
Rainville P. D.
L28, P120
Salplachta J.
P186
Ramautar R.
L24, L25
Salvayre R.
P231
Rampal S.
P078
Samcova E.
P228
Ramsay L. M.
L42
Sandor V.
P240
Ranc V.
P099, P104, P179
Sandritter M.
L63
Rapp E.
L67
Santiago J.
P141
Rassi Z. E.
L69
Santrucek J.
P059
Rebout J.
L36
Satinsky D.
P217
Redweik S.
P073, P197
Sato Kae
L33, L56, P032, P218
Reeuwijk B.
PL9
Sato Kiichi
P119, P218
Regan F.
L12
Sawaga H.
PL9
Regazzoni L.
L5
Sazelova P.
P047, P055
Regnier F. E.
PL7
Sciabica K.
P037, P038
Rehulka P.
P233
Scriba G. K. E.
P023, P026
Rehulkova H.
P233
Sedlakova P.
P208
Rejtar T.
L2, L57
Seifrtova M.
P241
Reminek R.
P187
Selman M. H. J.
L60
Rezanka P.
P148
Sénéchal H.
P134
Ribes D.
P231
Senitkova I.
P219
Riesova M.
P220, P221
Seno M.
P114
Righetti P. G.
PL3, L22
Servais A. C.
P044, P046, P049
Riviere C.
P013
Sevcik J.
P104, P179
Rocco A.
L4
Severa L.
P047
Roeraade J.
P232
Shakalisava Y.
L12
Rogeberg M.
P027
Shang F.
P085
Röhnert P.
P130
Shanmuganathan M.
P183
Rombaut B.
P162, P165
Shesheney A. E.
P069
Romestand B.
P188
Shi N.
L62, P043
Rossier J. S.
P134
Shieh Ch.
L1
Rostaing L.
P231
Shimizu T.
P224, P225
Roth M.
P062
Shu S.
P025
Roth U.
P039
Schappler J.
P098, P204
Rousseau A.
P044, P046, P049
Schepdael A. V.
P147, P187
Rovny A.
P145
Schmitz A.
L11, P112
Row K. H.
P016
Schröder S.
P197
Rozing G.
PL9, P084
Schuette J.
P130
Rudaz S.
P098, P204
Schurek J.
P106
Rudolf K.
P239
Siaugue J. M.
L52
Rüfer A.
P117
Simanek V.
P061
Ruhaak L. R.
L67, P079
Simo C.
L19, P113, P207
Russell D. H.
P206
Siroka J.
P215
Russell W. K.
P206
Sisperova E.
P071
Ryvolova M.
L54, P118, P177
Siu S. O.
P205
Saha M.
P052
Skerikova V.
L21, P057
Saile V.
L29
Slais K.
P091, P092, P093, P094, P173, P186
Sakai Y.
P224
Slovakova M.
P013
Salek P.
P030
Smarda J.
L37, P102
191
Autor´s Index
Smejkal P.
L54, P028, P118
Szumski M.
L39
Smirnova A.
P125
Taborsky P.
P159
Smith N. W.
P120
Tachi T.
P131
Sneekes E. J.
L68, P024, P074
Takács L.
P009
Snoblova M.
P063
Takacsi-Nagy A.
L13
So H. Y.
P230
Takatsu A.
P236
Sobotka L.
P194
Tallarek U
L43, P017, P022, P067, P212
Soga T.
P110
Tanaka R.
L66, P122
Sola L.
P211
Tanaka Y.
L33, L56, P032, P132, P133, P139
Solich P.
P194, P217, P227, P241
Tanaka Yuki
L56
Solichova D.
P194
Tanigawa K.
P123
Solinova V.
L10
Tarkowski P.
P178
Somsen G. W.
L24, L44
Taschuk M. T.
P150
Song Ch.
L7
Taverna M. M.
L9, P013, P149
Soukuo J.
P029
Tazawa H.
P138
Sperling E.
P009
Tempels A.
PL9
Spidlova P.
P210, P219, P233
Teply F.
P047
Squier A. H.
P006
Tesarova E.
P048
Stanova A.
P100, P179
Teste B.
L52
Stapels M.
P200, P203
Thakur D.
L2
Stastna M.
P091
Theurillat R.
L11, P112
Staub A.
P204
Thormann W.
L11, L13, L18, P070, P112, P182
Stavikova L.
P062
Thys B.
P162, P165
Stelzl M.
L63
Tie C.
P170
Stelzle M.
P130
Ticha I.
P196
Stem S.
L61
Ticha M.
P007, P010, P012, P196
Sterba J.
P195
Tichy A.
P233
Sterbova D.
P063
Tikhonova O.
L3
Stojkoviæ M.
P172
Tokeshi M.
P131, P151
Strnad M.
P178
Tomany M.
L28, P120
Stulik J.
P082, P216, P219, P233
Trounstine M.
L61
Stutz H.
L40
Tsukahara T.
P156
Stys D.
P072, P076
Tu P. Y.
P116
Su H. L.
P185
Tuma P.
P228
Su P.
P181
Tuñon J.
P003
Su Y. T.
P153
Turiak L.
P209
Sueyoshi K.
L66, P114, P122, P123, P137
Tylova T.
P226
Sumitomo K.
P089, P224, P225
Ugaz V. M.
L62, P035, P043
Suntornsuk L.
P177
Ulanova D.
P229
Svec F.
L30
Ulrichova J.
P061
Svidrnoch M.
P099, P104
Umemura T.
P025
Svobodova I.
P158, P159
Uno B.
P089
Svobodová J.
P048
Urba J.
P072
Svobodova Z.
P004, P135
Urban J.
L21, P076
Swart R.
L68, P024, P074
Vaculovic T.
L37, P102
Sykora D.
P148
Vaher M.
P242
Syslova K.
P014
Vancova M.
P195
Szabo Z.
L57, P237
Vanek J.
P076
Szekrényes Á.
P002
Váradi C.
P002
192
Autor´s Index
Varenne A.
L48, L52, P044, P090, P240
Wuhrer M.
L58, L59, L60, L67, P079
Vasquez J.
P066
Xi H.
P032
Vavrova M.
P189
Xie H.
P081, P190
Vekey K.
P209
Xing X.
L1
Velinska H.
P065
Xu J. X.
P086
Ventura K.
P192
Xu L.
P146
Vermachova M.
P059
Xu Y.
P126, P139
Vermehren Ch.
P171
Yamaguchi Y.
P089, P224, P225
Verpillot R. R.
L9, P149
Yamashita T.
L33
Veuthey J. L.
P098, P204
Yang E.
L47
Vigh G.
P206
Yang Y.
P164, P181
Villemin D.
P044
Yavorska O.
P028
Vistoli G.
L5
Ye M.
L7, P011
Viteckova H.
P189
Yeh CH. H.
P116
Vlcek J.
P064
Yeung E. S.
P096
Vlckova H.
P194, P227
Yeung W. S. B.
P136
Vlckova S.
P014
Yim Y. H.
P230
Vuong Ch.
L36
Yin H.
P223
Vydrova L.
P189
Yokoyama H.
P224
Vykydalova M.
P093, P094
Yoshimura E.
P119
Wakabayashi J.
L56
You T.
P213
Wakida S.
P132
Yowanto H.
P037, P052
Wakita Y.
P025
Yu C.
P213
Wallace G. G.
L51
Yuan B.
P213
Wang D.
L2
Zakovicova S.
P100
Wang F.
P020
Zapletalova E.
P186
Wang S.
P181
Zauner G.
L59
Wang S. P.
P050
Zavareh A. R. F.
P058, P174
Wang W.
P146
Zeisbergerova M.
P166
Wang X.
L64
Zemanek J.
P115
Wätzig H.
P073, P197
Zeng R.
L1
Weinbauer M.
L40
Zgoda V.
L3
Wenz Ch.
P084, P117
Zhang D. W.
P086
Werner M. K. S.
P130
Zhang X. X.
P086, P146, P170
West D.
L26
Zhang Z.
P020
Wheat T. E.
P081
Zhao W.
P168
Wiberg H.
P232
Zhong X.
L16, P180
Wiedmer S. K.
P191
Zhou K.
L49
Wilmer F.
P039
Zhou L.
P085, P086
Wilson S. R.
P027
Zhu T.
P016
Wiltfang J.
P013
Zidkova L.
P108, P111
Wojtowicz P.
P106, P108, P109, P111
Zmatlikova Z.
P201, P208
Wolinsky S. M.
P042
Znaleziona J.
P104, P179
Wolken G. G.
L35
Zondag G.
L25
Woltjen K.
P038
Zou H.
L7, P011, P020, P023, P026
Wu M.
P020
Zrostlikova J.
P106
Wu R.
P020
Zvatora P.
P148
Wu Ruige
P136
Wu Y.
P052
193
Notes
194
Notes
Notes
196

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