Supporting discovery in the next steps for biology

Supporting
discovery in the
next steps for
biology
The Centre for Genomics and Proteomics
As biology has advanced so has the need to define key cellular processes in molecular
detail. In responding to this, The University of Auckland Centre for Genomics and
Proteomics (CGP) has become a recognised hub for its unique cluster of high-tech
equipment and leading research and teaching. It has established expertise in all aspects
of genomics, proteomics, metabolomics and bioinformatics. It has also built a strong
track record in helping scientists from other research centres and biotechnology
companies to advance their own discovery programmes by drawing on its expertise.
The Centre undertakes DNA sequencing, genotyping and gene expression analysis,
proteomics and protein characterisation, as well as metabolite identification and
metabolomics analysis. In addition to extensive in-house research, it also regularly
provides specialist resources via collaborations or contract services for a full range of
proteomic and mass spectrometric services. These include 2-D electrophoresis, HPLC,
small molecule elucidation, peptide mass fingerprinting, bottom-up and top-down
sequencing and quantitative proteomics.
CGP Genetics Research Resources
• LIQUID-HANDLING ROBOTIC WORKSTATIONS
Applications include:
- Library management – clones, oligonucleotides, compounds, etc
- Dilutions, replications, sub-libraries, etc
- High-density replication (eg. yeast colonies)
- High-throughput extractions (DNA, RNA, protein)
- Assay plate set-up (PCR, qPCR, ELISA, kinase, etc)
• NUCLEIC ACID QC
Genomic applications require high quality RNA/DNA. The Centre operates a
Nanodrop Spectrophotometer and an Agilent Bioanalyzer to quantify and QC RNA
and DNA.
• REAL-TIME PCR
An ABI 7900HT Fast Real-Time PCR System, with automated plate handling and
barcode reader, is used for qPCR and automated SNP genotyping.
• DNA SEQUENCING AND GENOTYPING
The Centre uses a 3130XL capillary sequencer (Applied Biosystems) and BigDye
version 3.1 Dye terminator chemistry. Genotyping and sequencing reactions can
be seamlessly entered into the production flow.
• MICROARRAY - GENE EXPRESSION AND GENOTYPING
The Centre uses an Affymetrix Genechip Workstation, capable of high throughput
whole genome expression analysis, and whole-genome genotyping and copy
number variation.
CGP Proteomics Facility
Our Proteomics Facility combines in-depth expertise with state-of-the-art equipment for
the identification and characterisation of proteins expressed in cells, tissues and body
fluids under different conditions, such as health and disease states. Our area of work
includes the large scale purification of proteins from recombinant or biofluidic sources,
the analytical fractionation of biochemical compounds and peptides, the analysis and
comparison of proteomes, identification of proteins in protein spots/bands by mass
spectrometry, analysis of expressed proteins for quantitative studies and analysis of
proteins for post-translational modifications.
Resources available include:
Mass Spectrometry Instrumentation:
• LTQ-FT
The LTQ-FT is a hybrid Linear Ion Trap Fourier Transform Ion Cyclotron Resonance
mass spectrometer. This instrument provides a powerful combination of a fastscanning linear ion trap that is capable of up to eight levels of MSn, with a high
resolution FT cell (up to 500,000 FWHM) which can provide sub-ppm mass
accuracies. Multiple ionisation sources (ESI, APCI, APPI, nano-ESI and atmospheric
pressure MALDI) and fragmentation methods (CID and ECD) are available, and with
the attached Surveyor HPLC system, the instrument is a highly functional platform
for both proteomic and metabolomic studies. Data can be processed using ProSight
PC, ProMass ID 2.5, MetWorks 1.1, Peaks 4.5 and Mass Frontier 5 packages.
• QSTAR XL
This Quadrupole-Time-of-Flight MS/MS instrument coupled to a capillary HPLC
system is a very capable workhorse for proteomic applications including precise
molecular weight determinations, protein identifications, characterisation of
modifications and quantitative studies using the iTRAQ labelling system. Software
packages available in-house include ProteinPilot, Mascot and PEAKS, providing
comprehensive cover for protein identifications, relative quantitation and de novo
sequencing. The high resolution mass measurement (15,000 FWHM) is also useful
for small molecule work, and the ability to perform Precursor Ion Scans provides
a powerful option for more specialised analyses.
• Voyager DE-Pro
Our MALDI-TOF instrument provides an alternative ionisation source for molecular
analyses. The use of various matrices permits the mass measurement of peptides,
intact proteins, polymers and some metabolites. Protein identifications from digests
are possible in conjunction with Mascot software.
• GC-MSD
Combining the high resolution separation of small molecular weight compounds by
capillary gas chromatography with the determination of their EI mass spectra, this
GC-MS offers us a highly informative platform for low molecular weight analyses.
A CTC PAL autosampler provides unattended automation of injection, while a Flame
Ionisation Detector is also installed to allow for more traditional detection.
Other instrumentation:
• System Gold HPLC
This integrated HPLC-UV system can be used for the separation of analytes for either
quantitative analysis or for purification with fraction collection. The autosampler
tray has a Peltier cooler to maintain sample integrity, while a column oven permits
temperature control during separation. Data is analysed using 32Karat software.
• Multiphor II flatbeds and PharosFX Plus scanner
We have four flatbeds that can run large format 2D gels for proteomic studies. Our
PharosFX Plus scanner with additional 488nm external laser is capable of scanning
both colorimetric and multi-colour fluorescent stained samples on a 35 x 43cm
platen. Resolutions down to 50 microns are possible, with a linear dynamic range
of almost five orders of magnitude.
DNA Sequencing Facility:
Kristine Boxen
Phone: +64 9 373 7599 x87293
Email: [email protected].
Affymetrix Facility:
Liam Williams
Phone: +64 9 373 7599 x87653
Email: [email protected].
Proteomics Facility:
Martin Middleditch
Phone: +64 9 373 7599 x87540
Email: [email protected].