Sample Preparation for MALDI-TOF/TOF >95% Success Protein Identification with Rates

Transcription

HUPO First World Congress, November 21–24, Versailles, France
12.6
13.1
Sample Preparation for MALDI-TOF/TOF
Protein Identification with >95% Success
Rates
Proteomic Map of the Human
Cholangiocarcinoma Cell Line
D. Suckau1, Peter Berndt2, Martin Schuerenberg1,
and H. Langen2
1
Bruker Daltonics, 28359 Bremen, Germany; and 2HoffmannLa Roche, 4070 Basel, Switzerland
The full characterization of human or bacterial proteomes is a task, which
is pursued in industrial proteomics today at increasing pace. As a rough
estimate, 50,000 MALDI fingerprint spectra of 2D gel spots are currently
required to obtain a basic insight into a proteome. Unfortunately only
about 60% of all spectra typically provide for protein identification. Therefore, there is dramatic demand for increased throughput AND success
rates of protein identification for industrial proteomics. Here we present
technologies, which provide significant improvements of these aspects.
Basically, advanced sample preparation protocols and MALDI-TOF/TOF
technology allow these performance increases. A novel sample preparation using “AnchorChips” i.e., MALDI sample plates with hydrophobic
profiles, was developed subsequently to the automatic digestion of protein spots from 2D gels. The digestion and sample preparation process
were fully automated for high throughput analysis. The process does not
involve any other purification step, such as microcolumns, etc. AnchorChips with a diameter of 600 ␮m of the hydrophilic sample patch were
used, which allow for multiple sample handling steps such as rinsing and
recrystallization on the target without increasing the size of the prepared
sample patch. Investigated samples came from gels from human embryonic kidney cell lysate. As a first step protein mass fingerprints were
recorded with a success rate of 90%. As a second step LIFT-TOF/TOF
MS/MS spectra were obtained on a novel MALDI-TOF/TOF mass spectrometer, which increase the rate of protein identification beyond 95%.
12.7
A Transgenic Model for Muscle Gene
Expression Profiling In Vivo
T. Crepaldi1, P. Accornero1, C. Prunotto1, R. Taulli1,
R. Chiarle2, and C. Ponzetto1
1
Dipartimento di Anatomia, Farmacologia e Medicina Legale,
Universita` di Torino, C.so M. D’Azeglio 52, 10126 Torino, Italy; and
2
Dipartimento di Scienze Biomediche e Oncologia, Universita` di
Torino, Via Santena 19, 10126 Torino, Italy
The HGF/SF ligand and its receptor (c-Met) promote migration of hypaxial
muscle precursors from the somites to their final destination into the limbs,
diaphragm and tip of the tongue. They also enhance proliferation of foetal
myoblasts and trigger the activation of satellite cells. Activation of Met
signaling in cultures of C2C12 cells results in expansion of cycling progenitors and block of myogenic differentiation. To verify the role of Met
receptor in muscle differentiation in vivo we produced double transgenic
mice where the oncogenic form of Met receptor, Tpr-Met, can be expressed in muscle under the control of tetO responsive promoter. The tTA
transactivator was controlled by the Muscle Creatine Kinase (MCK) promoter (Tet-Off system), which is expressed in mouse embryos from E14
dpc onwards, when secondary myogenesis and terminal differentiation
occur. In these mice EGFP was used as reporter gene.
Five different lines of Tpr-Met transgenics were crossed with homozygous MCK mice. Double transgenics (MCK-TprMet) from only two out of
five lines expressed EGFP in muscle. The line with stronger EGFP expression was lethal perinatally. A severe reduction in all skeletal muscles was
observed. Most fibres were disorganized and Z-lines were absent. In these
animals myotubes did not form and mononucleate cells accumulated. This
suggests that Met signaling interferes with muscle terminal differentiation.
Total RNA from muscle tissue of these mice will be used as target in
microarray experiments versus control RNA from normal muscles. This will
allow to delineate an in vivo expression profiling of genes involved in
skeletal muscle differentiation.
668
Molecular & Cellular Proteomics 1.9
C. Srisomsap1, P. Subhasitanont1, T. Panichakul1,
K. Lirdprapamongkol1, S. Keeratichamroen1,
P. Sawangareetrakul1, D. Chokchaichamnankit1,
J. Jai-nhuknan2, and S. Sirisinha1,3
1
Chulabhorn Research Institute, Bangkok 10210, Thailand; 2Bruker
South-East Asia, Bangkok, Thailand; and 3Mahidol University,
Bangkok 10400, Thailand
Cholangiocarcinoma (CCA), a malignant tumor derived from bile duct
epithelium, occurs with a higher incidence in tropical countries especially
in some areas of Southeast Asian countries such as Thailand. Twodimensional (2-D) protein map of human bile duct epithelial carcinoma cell
line (HuCCA-1) was studied in parallel with human breast epithelial cancer
cell lines (MCF-7) and hepatocellular carcinoma cell lines (HepG2 and
HCC-S102). The proteins from MCF-7 were identified by mass spectrometry (MALDI-TOF-MS and ESI/MS/MS) and protein sequencer. Our results
show that HuCCA-1 expressed a unique pattern of proteins. Twenty-eight
major proteins were identified by matching to the map of MCF-7. We
found higher expression of cytokeratin 18 (CK18) but lower expression of
glucose regulated protein 75 and heat shock protein 70 in HuCCA-1
compared to MCF-7. CK18 was also expressed at lower level in HepG2
and HCC-S102. There were 2 specific proteins expressed as a series of
isoelectric variants, which had MW/pI at 46.2/5.91 and 42.0/5.91 in
HuCCA-1, but showed MW/pI at 46.2/5.91 and 42.2/5.99 in HCC-S102.
These 2 spots were not expressed in MCF-7 and HepG2 and were
identified by ESI/MS/MS as Keratin, type II cytoskeletal 1 (Cytokeratin 1,
K1, CK 1, 67 kDa cytokeratin). This 67 kDa intermediate filament protein is
normally expressed only in the upper levels of the epidermis but has also
found in the serum of papillary thyroid carcinoma as a 35 kDa fragment
and in the tissue of esophageal cancer as 67 kDa CK1.
Supported by the Chulabhorn Research Institute.
13.2
13.3
Proteomics of Breast Cancer for Signal
Pathway Profiling and Therapeutic Target
Discovery
Comparative Proteome Analysis of Human
Colorectal Cancer Tissues
Ikram El Yazidi-Belkoura1, Eric Adriaenssens1,
Se´ verine Lottin1, Jean Jacques Curgy1,
Anne-Sophie Vercoutter-Edouart2, Je´ roˆ me Lemoine2,
and Hubert Hondermarck1
1
EA-1033, Universite´ des Sciences et Technologies de Lille, France;
and 2UMR8576, Universite´ des Sciences et Technologies de Lille,
France
Breast cancer is a major problem for public health, and the identification of
new markers as well as the definition of new therapeutic targets, are of
critical importance. Practical consequences for treatment derived from a
better understanding of the molecular basis of breast cancer cell growth
are now emerging, as evidenced by the development of therapeutic strategies based on the inhibition of tyrosine kinase receptors. In this context,
methods in functional proteomics have become a powerful approach for
deciphering the complex signaling circuitry involved in tumor growth. This
is well illustrated with intracellular signaling of fibroblast growth factor-2
(FGF-2) and nerve growth factor (NGF), two potent activators of breast
cancer cell survival, proliferation and metastasis (1). We have shown that
breast cancer cell transfection with 14-3-3 sigma, a molecular chaperone
that is down-regulated in breast cancer cells (2), resulted in a the inhibition
of FGF-2 and NGF intracellular signaling. After immunoprecipitation, 2Delectrophoresis and MALDI-TOF analysis, we have identified several targets of 14 –3-3 sigma. Interestingly, HSP90 was also found to interact with
PI-3-kinase and IKK and appears to be a crucial element involved in
growth signaling as well as a potential therapeutic target as its pharmacological inhibition resulted in breast cancer cell growth arrest and apoptosis. The complementarity between genomics and proteomics for identification of therapeutic targets is well illustrated with H19, an untranslated
mRNA, which is oncogenic for breast epithelial cells. H19 transfected
breast epithelial cells appear to grow faster in tissue culture, although no
molecular target has been assigned to H19 and its mechanism of action
remains unknown. Proteomic analysis of H19 transfected cells reveals
several modifications of protein synthesis and particularly a strong upregulation of thioredoxin, one of the major proteins regulating intracellular
redox metabolism, providing the first molecular target identified for the
oncogen H19 (3). Interestingly, this example point out that proteomics can
provide usefull data for the understanding of genes and mRNA function,
specially in a pathological context. Together with genomics, proteomics is
now opening a way to define molecular processes involved in breast
cancerogenesis and to identify new markers and therapeutic targets.
Technological innovations in large scale/high throughput analysis are now
ushering in new prospects.
1. Nurcombe V, Smart CE, Chipperfield H, Cool SM, Boilly B, Hondermarck H (2000) J. Biol. Chem. 275, 30009 –18.
2. Vercoutter-Edouart AS, Lemoine J, Le Bourhis X, Hornez L, Boilly B,
Nurcombe V, Revillion F, Peyrat JP, Hondermarck H (2001) Cancer Research 61, 76 – 80.
3. Lottin S, Vercoutter-Edouart AS, Adriaenssens E, Czeszak X, Lemoine J, Morad R, Coll J, Hondermarck H, Dugimont T, and Curgy JJ (2002)
Oncogene 21, 1625–1631.
Gyong-Sik Ha1, Hyung-Gon Koh1, Kwang-Ho Kim2,
Eung-Bum Park2, Wan-Je Park1, Jang-Yun Lee1,
and Young-Sun Sohn1
1
R&D Center of Pharmaceuticals, Cheiljedang Corporation, Ichon
467-810, Korea; and 2Mokdong Hospital, Ewha Womans University,
Seoul 158-710, Korea
Colorectal cancer is one of the most common malignancies in the world.
In an attempt to identify proteins that may specifically characterize colorectal cancer, comparative proteome analysis was performed with 5 human cancer tissues and their matching normal tissues, using two-dimensional gel electrophoresis coupled with matrix assisted laser desorption
ionization-time of flight (MALDI-TOF) mass spectrometry. It was found that
29 protein spots were differentially expressed in at least three tissues, 17
spots significantly increased and 12 spots decreased. Among those protein spots, twelve up-regulated and 2 down-regulated proteins were identified and classified based on their functions by searching SWISS-PROT
database. Proteins known to be related to protein folding; protein disulfide
isomerase A3 precursor and peptidyl-prolyl cis-trans isomerase A, formation of cytoskeleton; PDZ and LIM domain protein 1 and cofilin, metabolic
pathway; alpha enolase and triosephosphate isomerase, gene expression;
myc far upstream element-binding protein and heterogeneous nuclear
ribonucleoproteins A2/B1, and signal pathway; calgizzarin and putative
peroxisomal antioxidant enzyme were overexpressed in cancer tissues,
whereas the expression of transgelin related to formation of cytoskeleton
and transthyretin precursor to signal pathway decreased. It is expected
that these data would contribute to understanding the characteristics of
the celluar proteins in colorectal cancer tissues.
13.4
Parallel 2D-DIGE and Microarray
Expression Profiling of a Model Breast
Cancer Cell System
Severine Gharbi, Sarah White, Mariana Bertani,
Hong Lin Chan, Michael D. Waterfield, and John F. Timms
Ludwig Institute for Cancer Research, University College of London
Medical School, London, United Kingdom
We report here the use of 2D-Difference gel electrophoresis (DIGE) combined with microarray analysis for differential expression profiling of a
breast cancer cell model of ErbB-2 over-expression. The protein expression pattern of a normal human mammary luminal epithelial cell line (HB4a)
and a transfected derivative over-expressing ErbB-2 (C3.6) was analysed
by 2D-DIGE. Both cell lines were compared at various time points after
stimulation with a specific growth factor, heregulin b1 (Hrgb1). As well as
increased reproducibility by avoiding gel-to-gel variation, this approach
allowed quantitative and statistical evaluation of differential display. Decyder software was used for gel image processing and statistical analysis
revealed significant changes between both cell lines in response to Hrgb1.
Of 135 features displaying difference in abundance, 27 were identified by
in-gel tryptic digestion of spots followed by peptide mass mapping by
MALDI/MS. Protein identities were subsequently validated by 1D-western
blotting with specific antibodies. A parallel microarray experiment was
carried out to evaluate the mRNA levels of 9932 genes between the two
cell types under identical growth conditions. A comparison between expression levels of mRNA and the proteins identified herein revealed a good
correlation, and a linear regression of 0.66. This indicates post-transcriptional modulation of protein levels. This work is providing us with key
information on the regulation of particular proteins in an ErbB2 over
expressing cellular system.
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13.5
13.6
Proteomics-based Identification of PGP9.5
Protein Profiling of the Human Epidermis
from the Elderly Reveals Up Regulation of
a Signature of IFN-␥ Induced Polypeptides
That Includes MnSOD and PI3K
Myeong J. Nam, Cecilia E. Schmalbach, David E. Misek,
Hong Wang, Rork Kuick, and Samir M. Hanash
Department of Pediatrics, University of Michigan, Ann Arbor,
Michigan 48109
The detection of circulating tumor antigens or their related autoantibodies
provides a means for early cancer diagnosis, as well as providing leads for
therapy. We have implemented a proteomic approach to identify proteins
that commonly induce an antibody response in colon cancer. Aliquots of
proteins from a colon adenocarcinoma cell line (LOVO) were utilized for
two-dimensional polyacrylamide gel electrophoresis (2-DE), followed by
Western blot analysis in which individual sera were tested for autoantibodies. Sera from 25 newly diagnosed patients with colon cancer, 24
patients with lung adenocarcinoma and 45 non-cancer controls were
analyzed. Autoantibodies against a protein identified by Q-TOF mass
spectrometry as an isoform of PGP9.5 (ubiquitin C-terminal hydrolase)
were detected in 10 of 25 colon cancer patients. They were not detected
in 25 sera obtained from normal individuals and patients with colon adenomas. Sera from patients and with lung adenocarcinoma exhibited reactivity against some forms of PGP9.5 but not others. These findings were
confirmed by Western blot analysis of both colon tumors and the colon
tumor cell line, resolved by 2-DE, using an antibodies to PGP9.5. Expression analysis of microarray data obtained for a panel of 287 different
tumors and normal tissues allowed us to correlate serum reactivity with
expression patterns at the RNA level. Data based on microarray analysis
were confirmed by both RT-PCR and real time PCR. Thus, the findings of
anti-PGP9.5 autoantibodies in the serum of patients with colon cancer
suggest that this antigen may have utility in colon cancer screening and
diagnosis.
670
Pavel Gromov1, Gunhild Skovgaard2, Hildur Palsdottir3,
Irina Gromova1, Morten Ostergaard4, and Julio Celis1
1
Institute of Cancer Biology, The Danish Cancer Society and Danish
Centre for Molecular Gerontology, DK-2100 Copenhagen, Denmark;
2
Department of Dermatology, Bispebjerg Hospital, The University of
Copenhagen and Danish Centre for Molecular Gerontology,
DK-2400 Copenhagen NV, Denmark; 3Max-Planck Institute of
Biophysics, 60528 Frankfurt am Main, Germany; and 4Department
of Medical Biochemistry and Danish Centre for Molecular
Gerontology, The University of Aarhus, DK-8000 Aarhus C, Denmark
Aging of the human skin is a complex process that consists of chronological and extrinsic aging; the latter caused mainly by exposure to ultraviolet radiation (photoaging). We have used proteomic profiling technologies and 2D PAGE database resources to identify proteins whose
expression is deregulated in the epidermis of the elderly. One to two mm3
epidermal biopsies obtained from the forearm of young and old donors
were labeled with [35S]methionine for 18 h and subjected to 2D PAGE and
phosphorimage autoradiography. Proteins were identified by matching the
gels with the master 2D-gel image of human keratinocytes (http://proteomics.cancer.dk). Quantitative analysis of 172 well-focused and abundant polypeptides showed that the level of most proteins (148) remains
unaffected by the aging process. Twenty-two proteins were consistently
deregulated by a factor of 1.5 or more across the 20 sample pairs. Among
these we identified a group of six polypeptides (Mx-A, Manganese superoxide dismutase, tryptophanyl tRNA synthetase, phosphatidylinositol 3-kinase, and proteasomal proteins PA 28-␣ and SSP 0107), that is induced
by IFN-␥ in primary human keratinocytes, and that represents a specific
protein signature for the effect of this cytokine. Changes in the expression
of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60,
annexin I, and plasminogen activator inhibitor 2 were also observed.
Besides arguing for a role of IFN-␥ in the aging process, the biological
activities associated with the deregulated proteins support the contention
that aging is linked with increased oxidative stress that could lead to
apoptosis in vivo.
13.7
13.9
Two-dimensional Molecular Profiling of
Mantle Cell Lymphoma
Human Liver Tissue Two-dimensional Gel
Electrophoresis Map and Hepatocellular
Carcinoma-related Proteins
Francesca Antonucci1, Marco Chilosi2, Claudia Parolini2,
Mahmoud Hamdan3, Hubert Astner3,
and Pier Giorgio Righetti1
1
University of Verona, Department of Agricultural and Industrial
Biotechnologies, Verona, 37134, Italy; 2University of Verona,
Department of Pathology, Verona, 37134, Italy; and 3Computational,
Analytical & Structural Sciences, GlaxoSmithKline, Verona, 37135,
Italy
In present study we have compared 2-D maps of reactive lymph-nodes
and Mantle Cell Lymphoma tissue. Mass-spectrometry-compatible-colloidal Coomassie has revealed a total of ca. 750 spots in each maps.
Comparison of the 2-D maps by PDQuest established up- and downregulation of ca. 150 spots, with positive variations (up to 10 folds) and
negative variations (up to 13 folds). More than 20 proteins have been
identified by MALDI-TOF mass spectrometry, with an additional five spots
which could not be matched to any of the available databases. Some
proteins, such as the 78 kDa glucose-regulated protein precursor, appear
to be in common with other tumours. Others reflect changes in cellular
metabolism. T-cell leukemia/lymphoma protein 1A over-expression
agrees with the MCL microenvironment.
Jina Kim, Na-Young Ha, Jin Sook Ahn, Seung Uook Lee,
Yu Na Kim, Bok Im Cho, Sung-Jo Kang, Chang-Won Lee,
and Jae Won Kim
Division of Life Science, Research Institute of Life Sciences,
Gyeongsang National University, Jinju 660-701, South Korea
Hepatocellular carcinoma is a common malignancy worldwide and is a
leading cause of death. In recent years, a proteome analysis technology is
one of the most popular tools approaching cancer studies. The aim of this
work was to largely expand the currently available human liver tissue map
and to study the differential expression of proteins in tumor and normal
tissues. Proteins were separated in the first dimension by isoelectric
focusing on IPG strips and by 7.5–17.5% gradient SDS-PAGE gels in the
second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight mass spectrometry (DE-MALDI-TOF MS). Up to the
present, reference 2-DE maps of human liver tumor tissue include 212
protein spots (117 spots in pH 4 –7 map and 95 spots in pH 6 –9) corresponding to 127 different polypeptide chains. And we analyzed the differential protein expression of liver tumor samples. Proteins whose expression levels were different by more than three fold in at least 30% (four) of
the 11 patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, one
overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified.
13.8
13.10
Proteome Analysis of Gastric Cancer:
Overexpression of Thymidine
Phosphorylase in Gastric Cancer Tissue
Overexpression of Rho GDP Dissociation
Inhibitor 1 in Human Gastric Cancer
Tissues
Na-Young Ha, Jina Kim, Jin Sook Ahn, Bok Im Cho,
Yu Na Kim, Seung Uook Lee, Sung-Jo Kang, Jae Won Kim,
and Chang-Won Lee
Gastric cancer is the second most common cause of cancer-related
mortality worldwide and the 14th overall cause of death. Proteomic approach was undertaken for the analysis of differential expression of protein
between stomach cancer tissue and adjacent non-cancerous region. So it
is a powerful tool to find out candidate proteins for diagnostic markers and
therapeutic targets. Protein samples were prepared from cancer and
normal tissues of the same patients, and were subjected to isoelectric
focusing on immobilized pH 4 –7 gradient strips followed by 7.5–17.5%
gradient SDS-PAGE gels. Protein spots were detected by staining with
silver nitrate. Image analysis was carried out with PDQuest software.
Paired samples of seventy gastric cancer patients were obtained from
Gyeongsang National University Hospital and Inha University Hospital and
were analyzed by two-dimensional gel electrophoresis. Of the seventy
samples that gave readable gel images thirty five samples were positive
for overexpression (with a cutoff value of 3.0) of the spot NO.4528. The
spot was excised from preparative gels, digested by trypsin, and subjected to peptide mass fingerprinting. The spot was identified to be thymidine phosphorylase, with 16 matching peptides which corresponds to a
sequence coverage of 34%.
Yu Na Kim, Bok Im Cho, Na-Young Ha, Jina Kim,
Jin Sook Ahn, Seung Uook Lee, Sung-Jo Kang,
Jae Won Kim, and Chang-Won Lee
Gastric cancer remains a leading cause of cancer-related death worldwide. Advances in diagnostic and treatment technologies have enabled us
to offer excellent long-term survival results for early gastric cancer, but
prognosis of advanced gastric cancer still remains poor. The aim of this
work was to figure out the protein components that are differentially
expressed in stomach cancer tissues to develop the protein candidates for
tumor marker. In this study, we have analyzed the proteomes of 70
stomach cancer tissue samples and compared with those of noncancerous tissues of the same patients. Human stomach tissue samples were
prepared from resection materials of gastric cancer patients. The proteins
of stomach tissues were analyzed by two-dimensional electrophoresis,
stained with silver nitrate and the images were analyzed with the aid of
PDQuest. Of the 70 samples that gave readable gel image, 4 samples were
positive for overexpression (with a cutoff value of 3.0) of the spot no. 3211.
The spot was excised from preparative gels, digested by trypsin, and
subjected to peptide mass fingerprinting. The spot was identified to be
Rho GDP dissociation inhibitor 1 (Rho GDI 1), with the sequence coverage
of 28%. The Rho GDI forms a complex with the GDP-bound form of the
Rho family small G proteins and inhibits their activation. Rho GDI has been
implicated in tumor cell apoptosis, invasion and metastases. In the previous studies reported, overexpression of Rho GDI has been associated
with chemoresistance in several cancer cell lines.
671
13.11
13.13
Overexpression of Galectin-1 in Human
Gastric Cancer Tissue
Proteomic Analysis of Tyrosine
Phosphorylated Proteins in Human MCF10A Mammary Epithelial Cells Treated by
HGF/SF
Bok Im Cho, Yu Na Kim, Na-Young Ha, Jina Kim,
Jin Sook Ahn, Seung Uook Lee, Sung-Jo Kang,
Jae Won Kim, and Chang-Won Lee
Gyeongsang National Universit, Jinju 660-701, South Korea
Gastric cancer remains a great challenge for clinicians and scientists. It is
one of the most frequent cancers worldwide, and it is the second most
common cause of cancer-related deaths. Patients with gastric cancers
have a poor prognosis and low survival rates. The aim of this work was to
figure out of the protein components that are differentially expressed in
gastric cancer. In this study, we analyzed proteomes of gastric cancer
tissue samples and adjacent noncancerous tissues of same patients. The
protein of gastric cancer and normal tissue were separated two-dimensional gel electrophoresis, and stained with silver nitrate. The images of
stained gels were analyzed with aids software, PDQuest. Paired samples
were obtained from 70 gastric cancer patients in Gyeongsang National
University Hospital and Inha University Hospital in South Korea. Of the 70
samples that gave readable gel images, 8 samples were positive for
overexpression (with a cutoff value of 3.0) of the spot no. 3023. The spot
was excised from preparative gels, digested by trypsin, and subjected to
peptide mass fingerprinting. The spot was identified to be galectin-1, with
8 matching peptides which corresponds to a sequence coverage of 74%.
Galectin-1 with molecular masses of about 14.5 kilodaltons have been
found in a variety of normal and malignant cells and have been implicated
in the regulation of cell growth, cell adhesion, and metastasis.
13.12
A New Look at the Tumor Proteome by
Large-scale Analysis of MHC Peptides
Arie Admon1, Eilon Barnea2, Ilan Beer2, Lior Dassau1, and
Tamar Ziv1
1
Department of Biology, Technion-Israel Institute of Technology,
Haifa 32000, Israel; and 2IBM Research Laboratory, Haifa, Israel
Among the thousands of proteins expressed in cells, there are many that
do not accumulate to a significant level due to their rapid degradation and
therefore remain undetectable by standard proteomics approaches. Some
of the peptides resulting from these degradations end up being stabilized
and so accumulate while displayed within the Major Histocompatibility
Complex (MHC) molecules on the cell surface. The amounts of these
peptides correlate with the protein degradation rates and with their binding
affinity to the MHC molecules. In Addition to peptides derived from normal
cellular proteins, cancer cells display peptides originating from Tumor
Associated Antigens, which attract significant attention as candidates for
development of cancer vaccines.
We describe here the construction of large datasets of MHC peptides
presented by various human cancer cell types. The cells were transfected
with vectors for secreted, soluble MHC molecules (sMHC), the sMHC were
recovered in large amounts from the growth medium and the bound
peptides were analyzed by capillary ESI-LC-MS/MS. Peptides were identified from human lung, breast, ovarian, and prostate tumor cells, from the
MHC haplotypes: A2, B7 and Cw4. As expected, the majority of the
peptides were derived from housekeeping proteins and only a few originated from tumor antigens or from yet-uncharacterized proteins. A catalogue of these peptides is available online with relevant links. A new
bioinformatics tool, Pep-Miner, described in a separate poster, vastly
facilitated the analysis. The approach described here offers a staging point
for a ‘human cancer MHC-peptides project’, to follow cancer genomics
and proteomics analyses.
672
Julien Deheuninck, Sylvie Reveneau, David Tulasne,
Catherine Leroy, Bernard Vandenbunder, Yvan de Launoit,
and Ve´ ronique Fafeur
CNRS UMR8117, Institut de biologie de Lille, IPL, 1, rue du Pr
Calmette, 59021 Lille, France
Hepatocyte growth factor/scatter factor (HGF/SF) induces epithelial cell
proliferation, scattering and morphogenesis through activation of the MET
tyrosine kinase receptor. As deregulation of HGF/SF signalling is involved
in tumoral and metastatic progression, our goal is to improve the knowledge of molecular actors of cancer, in particular for breast cancer.
According to numerous studies in different cell types, HGF/SF regulates
by phosphorylation an extensive number of proteins. To clarify their relevance and to possibly discover novel targets of HGF/SF signalling, we
decided to investigate exhaustively tyrosine phosphorylated proteins in a
single human mammary epithelial cell line, in which HGF/SF is biologically
active. We first compared HGF/SF activity in human mammary epithelial
cell lines (MCF-7, MDA-MB231, MCF-10A). This led us to further investigate MCF-10A cells, which : 1) express mammary specific epithelial markers, 2) are sensitive to HGF/SF treatment, with efficient phosphorylation of
known targets such as MET, GAB1, ERK or AKT and 3) are biologically
responsive to HGF/SF, with efficient cell scattering at low cell density or in
wounding experiments. To identify phosphoproteins regulated by HGF/
SF, we first performed immunoprecipitation/immunoblotting experiments,
using anti-phosphotyrosine antibodies. Following separation by monodimensional electrophoresis, 4 bands were induced after HGF/SF treatment (of ⬃180, 165, 120 and 75 kDa). These proteins are not identified and
do not correspond to GAB1, CBL, nor SHC. Further experiments are
underway for their identification by mass spectrometry. Alternatively, we
are using two-dimensional electrophoresis of whole protein extract followed by immunoblotting using anti-phosphotyrosine antibodies and final
identification by mass spectrometry.
13.14
13.16
Database for Fluorescence 2D Difference
Gel Electrophoresis (2D-DIGE)
Protein Expression Fingerprints of Normal
Segments of the Colon
Yasuharu Mori, Tadashi Kondo, Tesshi Yamada,
and Setsuo Hirohashi
Irina Gromova, Pavel Gromov, and Julio Celis
Cancer Proteomics Project, National Cancer Center Research
Institute
Fluorescence 2D Difference Gel Electrophoresis (2D-DIGE) has been used
for several biological studies. In 2D-DIGE, protein lysates are labeled with
different fluorescence dyes and separated concomitantly in a same gel.
Because samples can be run with a common control sample in a same gel,
accurate spot matching can be performed with broad dynamic range of
fluorescence signals. We aimed to construct 2D database and to evaluate
a potential of 2D-DIGE for oncological studies. Protein extracted from a
human colon cancer cell line, DLD-1, was labeled with Cy5 and separated
according to their isoelectric point and molecular weight. Among 2,000
spots observed, approximately 500 spots were chosen randomly and 343
spots were identified using mass spectrometry and database search. They
were grouped based on their molecular function according to Panther
Categories in Celera Discovery System. Majorities of identified proteins
were a family of nucleic acid binding protein, oxidoreductases, cytoskeletal proteins and chaperons. Although one receptor and six proteins for
signal transduction were observed, proteins with lower amount, such as
transcription factors and cell cycle regulators, were not observed. These
results indicated that, 2D-DIGE has a limited potential to access mechanisms of cancer biology. Biopsied tissues of human colon cancer were
also subjected to 2D after they were labeled with Cy5. Most of identified
spots in 2D database of DLD-1 cells were observed in 2D images of
human colon cancer tissues. These results suggest that the database for
2D-DIGE using DLD-1 cells will be useful for rapid spot identification of
clinical samples.
Institute of Cancer Biology and Danish Center for Human Genome
Research
Colorectal cancer is one of the most common types of cancers in developed countries. Despite advances in treatment this disease remains life
threatening for a large number of people. Today, about 50% of all patients
with colorectal cancer are diagnosed with early stage cancer, and most of
these patients will be cured by surgery. However, approximately 30% of
these patients will experience recurrence over the next 5 years. Clearly,
survival can be improved if patients are diagnosed at early stages of the
disease.
In our laboratory we are using proteomic technologies to compare the
protein expression of normal and cancerous fresh colon biopsies with the
aim of revealing molecular signatures that may identify lesions at a very
early stage of the disease. One of the major challenges we face with these
studies relates to the fact that various segments of the normal colon
epithelia have different susceptibilities to neoplastic transformation and
may therefore express distinct proteome profiles. Here we present initial
studies intended to characterize the proteome profiles of normal colon
biopsies obtained from various regions of the colon.
13.15
13.17
Clustering Analysis of Human Cancer Cell
Lines Based on Quantitative 2D
Mass Spectrometry-based Analysis of
Tubulin Isoforms in Taxol-sensitive and
-resistant Human Cancer Cell Lines
Masahiro Seike, Tadashi Kondo, Tesshi Yamada, and
Setsuo Hirohashi
Institute
We aimed to perform clustering analysis of human cancer cell lines using
fluorescence 2D Difference Gel Electrophoresis (2D-DIGE). In 2D-DIGE,
different samples are labeled with different fluorescence dyes and coseparated in a same gel. Because samples are run with a common sample
in a same gel, accurate and rapid spot matching can be performed with
broad and quantitative dynamic range of fluorescence signals. To evaluate
a potential of 2D-DIGE for clustering analysis, two models of human
cancer cell lines were studied. First, cell lines of the same histological type
from different organs were examined. Adenocarcinoma cell lines derived
from lung, pancreas or colon cancer, were analysed by 2D-DIGE. Each
group involved 10 cell lines. A set of quantitative intensity of 180 spots was
subjected to clustering analysis. Clustering analysis resulted that most cell
lines used were grouped into their original organ. The proteins used to
segregate each group from the other ones included nucleoside diphosphate kinases. Second, cell lines of different histological types in a same
organ were examined. Thirty lung cancer cell lines of adenocarcinoma,
squamous cell and small cell cancer were subjected to clustering analysis
using quantitative data of 170 spots as above. The cell lines were grouped
into their original histological types using spots including stathmin and
keratin19. These results indicate that 2D-DIGE is a powerful tool for
clustering analysis of human cell lines and suggest possible applications
for clinical uses.
Pascal Verdier-Pinard, Fang Wang, Berta Burd,
Susan Horwitz, and George Orr
Albert Einstein College of Medicine, Bronx, New York 10461
Alterations to microtubule stability may be a crucial determinant in the
development of clinical resistance towards Taxol and other drugs with a
binding site on the microtubule polymer. Cancer cells may alter their
microtubule dynamics by differential expression of the six ␣-tubulin and
seven ␤-tubulin isotypes, by mutation of tubulin, or by differential posttranslational modifications of these tubulin isotypes. Most of the primary
sequence divergence in the various tubulin isotypes and all of the posttranslational modifications, except acetylation, occur within the C-terminal
20 amino acids. We have developed mass spectrometry-based methods
for the analysis of tubulin isoforms from human cancer cell lines. In the cell
lines examined, the major tubulin isotypes were K␣1 and ␤I, the next
abundant tubulin isotypes were ␣6 and ␤IVb, and the minor tubulin isotypes were ␣4 and ␤III. Only trace amounts of monoglutamylated tubulins
were detected and ␣ tubulins were almost totally tyrosinated. Additionally,
we detected and quantified the expression of mutant tubulin in Taxol- and
epothilone-resistant cell lines. These studies represent the first comprehensive analysis of tubulin isoform expression at the protein level in cancer
cells. We seek to adapt these approaches to normal and malignant tissues, and to design methodologies for absolute quantification of tubulin
isoforms in human samples.
673
13.18
13.20
Proteomic Studies on Hepatocellular
Carcinoma
Cell Surface Expression of Heat Shock
Proteins in Human Leukemia Cell Lines
Qi-chang Xia1, Rong Zeng2, Zhao-you Tang3,
and Hong-yang Wang4
Jun Ho Jang, Bong Kyung Shin, David E. Misek,
and Samir M. Hanash
1
Research Center for Proteome Analysis, Shanghai Inst.of
Biological Sciences, Chinese Academy of Sciences, 320 YueYang
Road, Shanghai, 200031, China; 2Research Center forProteome
Analysis, Shanghai Inst.of Biological Sciences, Chinese Academy of
Sciences, 320 YueYang Road, Shanghai, 200031, China; 3Shanghai
Zhongshan Hospital, Shanghai, 200032, China; and 4Eastern
Hepatobilliary Surgery Institute, Shanghai, 200438, China
Michigan 48109
We demonstrated the comparative proteomic analysis of human hepatocellular carcinoma (HCC). Firstly, two clones with high (MHCC97-H) and
low (MHCC97-L) were isolated from the parent cell line, which have the
same genetic background but have different metastatic behaviours. Then,
the proteomic profiles of the two cell clones were established and compared based on two dimensional electrophoresis. Several proteins with
different expression levels were further identified by MALDI-TOF-MS characterization, indicating their relation with the HCC metastatic mechanism.
Furthermore, immuno-blotting analysis on over 60 clinical tissues of hepatocellular carcinoma also confirmed the observations on cultured cells. In
addition, directed comparisons of clinical tumor and normal tissues were
carried out to find new markers for the carcinogenesis of HCC. Our
analysis gave insight into the carcinogenesis and metastatic mechanism
and helped us to find out clinical markers of hepatocellular carcinoma.
The plasma membrane is a sub-cellular compartment of substantial interest in various aspects of cancer, from molecular diagnosis to cancer cell
immune system avoidance. Profiling of the cell surface proteome in both
normal and cancer cells provides an effective approach for the identification of novel targets for diagnostics and therapeutics. We have implemented a biotinylation-based strategy for targeting plasma membranederived proteins to identify cell surface proteins of leukemia cell lines.
Following biotinylation of the Sup-B15 (human acute lymphoblastic leukemia B cell line) and the U 937 (human acute monoblastic leukemia cell
line, the labeled proteins were affinity-captured and purified on streptavidin columns, further resolved by 2-D PAGE, then transferred to PVDF
membranes. The biotinylated proteins were visualized by hybridization
with streptavidin/Horseradish peroxidase complex, then identified from
silver-stained gels using MALDI and tandem mass spectrometry. Remarkably, a large set of heat-shock proteins, including GRP78, GRP 75, heat
shock 70 kDa protein 2, heat shock 70 kDa protein 9B (mortilin-2), hsp72,
heat shock 60 kDa protein 1 and heat shock 27 kDa protein, all previously
considered to be associated with the endoplasmic reticulum were found to
be highly abundant on the cell surface. The findings were correlated with
gene expression at the RNA level by DNA microarray analysis. Further
investigation of heat shock proteins, thought to be chaperones in antigen
presentation, may help elucidate immune system avoidance mechanisms
of human leukemia cells.
13.19
13.21
New Differentially Expressed Stomach
Cancer Markers Identified through
Extended Proteomics Analysis on Highly
Selected Tumor Samples
Characterization of Proteome Analysis
Data and Comparison of Pre-processing
Methods for the Differential Expression
Analysis
M. L. Fogeron1* S. Lamer1*, S. Heim1, Ch. Ro¨ cken2,
M. Ebert2, and P. von Hoegen1
1
2
Europroteome, 16761 Henningsdorf/Berlin, Germany; and
Otto-von-Guericke University, 39120 Magdeburg, Germany
A case study was performed on stomach cancer tumor samples to demonstrate that the unique combination of a collection of highly purified, well
documented, clinical specimens with modern proteomics and transcriptomics technologies can lead to the identification of multiple new stomach
tumor markers with value for therapy and diagnostics.
These protein markers have been identified by leveraging EUROPROTEOME’s multidimensional system biology technology infrastructure (e.g.,
Proteomics, Transcriptomics and Bioinformatics) and using purified human tissue samples (applying the proprietary sample preparation method)
from the company’s extensive human tissue sample bank.
Applying 2D gels of normal and tumor tissues over 350 differentially
expressed marker spots could be identified. Many of these proteins have
been altered in several patients indicating the general relevance of them
for the disease. Many known changes in regard of de-differentiation and
increased proliferation could be confirmed and thereby validated our
approach. After extensive analysis nearly 50 markers could be identified as
yet unknown or as new stomach-cancer-associated markers. These provide a significant and proprietary list of new markers with potential for use
in diagnostic and therapy of cancer, an example being EP32.1.2. Many of
these markers are likely to be drug candidates (e.g., enzymes) making
them ideal candidates for therapeutic product development.
* Contributed equally.
674
Jae Won Kim, Sung Jin Ahn, Euy Hoon Suh, Jong Min Bae,
and Chang-Won Lee
Gyeongsang National University, Jinju 660-701, Korea
Gastric cancer is one of the leading causes of death worldwide. The
identification of new markers and therapeutic targets is of critical importance, and a proteomics project on gastric cancer has been launched
three years ago as an effort to attain the goal. Selection of differentially
expressed proteins in tumor and normal tissues would be the first rational
step toward finding markers and targets of the disease. Two-dimensional
gel electrophoresis was used as the main technological platform of proteome analysis, and proteome data for matched tissue samples from forty
gastric cancer patients have been generated by employing immobilized
pH gradient (pH 4 –7) isoelectric focusing in the first dimension and 7.5–
17.5% gradient SDS-PAGE in the second dimension. Protein visualization
was done by staining with silver nitrate. The images were transformed into
digital data by scanning and analyzed by PDQuest software for the automatic spot detection. As a prerequisite to the analysis of proteome data,
it is important to have a deep understanding the characteristics of the
data, which will enable to select the most appropriate analysis method(s)
that take the data characteristics best into consideration. Here, we have
characterized the proteome data obtained from the analysis of clinical
tissues, with respect to the spot numbers, spot intensities, and reproducibilities. Also, three different normalization methods were used to preprocess the raw data, and the t-test was performed to select the differentially expressed proteins. The results are compared and discussed.
13.22
13.24
Proteomics-based Microarray Technology
for the Identification of Tumor Antigens
Differential Expression of Sub-proteome
Between Human Liver Cancer HepG2 and
Hep3B Cell Lines
Juan Madoz-Gurpide, David E. Misek, Hong Wang, and
Samir M. Hanash
Michigan 48109
The identification of circulating tumor antigens that elicit a humoral response provides a means for early cancer diagnosis. We have developed
a novel proteomic method for probing the cell and tissue proteome by
combining liquid-phase protein separations with microarray technology.
The advantage of a liquid-based separation system is that proteins in
hundreds of individual fractions can be spotted onto microarrays directly
and used as targets for a variety of probes. These microarrays were
probed with the sera of cancer patients in order to detect disease-related
autoantibodies. The A549 lung adenocarcinoma cell line was resolved by
isoelectric focusing (Rotofor) in the first dimension. Each of twenty fractions was further separated by RP-HPLC, such that 88 fractions were
obtained for each first-dimension fraction. All 1760 fractions and controls
were arrayed in duplicate onto surface-modified glass slides. An antibody
against a previously identified cancer biomarker was used to detect 15
specific antigen-containing fractions (p ⬍ 0.01 and ratio ⬎ 2). The presence of the antigen in these fractions was confirmed by Q-TOF mass
spectrometry. The microarrays were individually hybridized with sera from
12 colon cancer patients, from 12 lung cancer patients as well as from 12
normal individuals. The reactivity of 15 selected fractions was analyzed.
8/12 colon patient sera and 10/12 lung cancer sera reacted positively,
whereas only 1/12 normal controls showed reactivity. The proteomic
approach we have developed has utility for the development of serumbased assays for cancer screening and diagnosis.
Chi-Yue Wu1,2, Szu-Pei Wu1,2, Chih-Lei Lee1,
Shiuan-Yi Huang2, Cheng-Liang Chen2, Ying-Ta Wu1,
Kay-Hooi Khoo2, Shui-Tein Chen2, and Andrew H.-J. Wang1
1
Core Facilities for Proteomic Research Academia Sinica, Taipei,
Taiwan; and 2Institute of Biological Chemistry Academia Sinica,
Taipei, Taiwan
In the search for new protein markers of human liver cancer, the method
of quantitative two-dimensional gel electrophoresis coupled with mass
spectrometry was applied to define the protein expression profiles of
human liver cancer HepG2 and Hep3B cell lines. Gel image matching and
statistic analysis showed that of the total 792 protein spots that were
matched, 537 spots were differentially expressed at a significant level. In
total, 111 protein spots were identified by peptide mass fingerprinting
combined with database searching. These identified proteins were classified into several functional groups, including chaperones and stress
response proteins, cytoskeletal proteins, enzymes involved in metabolism
and biosynthesis, and proteins with regulatory functions. Among these
differentially expressed proteins were several cancer-related proteins, i.e.,
annexin II, cathepsin B, dihydrodiol dehydrogenase II, heterogeneous
nuclear ribonucleoproteins (hnRNP A1, hnRNP A2/B1 and hnRNP C), heat
shock proteins (Hsp90 and Hsp70) and glucose-regulated proteins (Grp78
and Grp94). The strategy of rapid comparative proteomic studies of
HepG2 and Hep3B cells is demonstrated here to be effective in narrowing
down the range for screening novel cancer markers from the sea of whole
cell proteins. The experimentally derived model provides important leads
and several potential targets for the development of diagnostics and
therapeutics for liver cancer, a common cancer in Asia and Africa.
13.23
Comprehensive Profiling of Surface
Membrane Proteins of Cancer Cells
Bong Kyung Shin, Jun Ho Jang, David E. Misek,
Rong Zhao, Hong Wang, Rork Kuick, and Samir M. Hanash
Michigan 48109
Comprehensive profiling of surface membrane proteins of cancer cells is
highly relevant to cancer diagnostics and therapeutics and to our understanding of dysregulated pathways. Biotinylation of surface membrane
proteins of intact cells followed by selective capture of biotinylated proteins using avidin columns allowed us to undertake comprehensive profiling of surface membrane proteins of a variety of cancer cell lines and
freshly isolated tumor cells. Proteins were identified by MALDI and tandem
mass spectrometry analysis and finding were correlated with gene expression at the RNA level by DNA microarray analysis. A lung adenocarcinoma
cell line (A549), a colon adenocarcinoma cell line (Lovo), a neuroblastoma
cell line (Sy5y), a B-cell lymphoblastic leukemia cell line (Sup-B15), and
freshly isolated ovarian tumor cells were analyzed. Distinctive patterns of
expression were observed for each cell population based on patterns
analysis and the identification of over 100 surface membrane proteins. For
example, Annexin III which was present in A549, Lovo and ovarian tumor
cells but not in Sy5y or Sup-B15. Other proteins such as heat shock 70 kD
protein 2, HLA class I histocompatibility antigen, membrane associated
progesterone receptor component 1, and heat shock 27 kD protein 1,
showed significant quantitative difference in expression in different cell
types both in gel image analysis and RNA measurement. Comprehensive
analysis of surface membrane proteins of cancer cells provides an effective approach for the identification of cancer proteins of diagnostic and
therapeutic value.
675
13.25
13.26
Strategic Experimental Approach Towards
Establishing Model Hepatoma Cell Lines
for the Global Human Liver Proteome
Initiatives
Proteomic-based Identification of Tumor
Antigens in Pancreatic Cancer
Chih-Lei Lee1, Wei-Tzer Shyu1, Chin-Yi Lin1, He-Hsuan
Hsiao1, Cheng-Liang Chen2, Chi-Yue Wu1, Ying-Ta Wu2,
Kay-Hooi Khoo2, and Andrew H.-J. Wang1,2
Michigan 48109
1
Core Facilities for Proteomic Research, Academia Sinica, Taipei,
Taiwan; and 2Institute of Biological Chemistry, Academia Sinica,
Taipei, Taiwan
Liver cancer is a world-wide disease which has serious impact in Asia and
Africa and has become a leading cause of death in these areas. Recognizing the importance of liver diseases, the Asian and Oceanian Human
Proteome Organization (AOHUPO) has launched the human liver proteome project as one of its major initiatives. The well established human
hepatoma HepG2 (from hepatitis B surface antigen-negative patient) and
Hep3B (from a HBsAg-positive patient) cells are the designated in vitro
experimental model cell lines. In response to this initiative and to support
the nation-wide genomics and proteomics research programs, a National
Core Facilities for Proteomic Research was established in Taiwan a year
ago. A variety of proteomics related projects including in depth, comprehensive mapping of the hepatoma proteome were initiated which serve to
drive innovative technical developments in sample preparation, separation, identification, and further characterization. Both gel electrophoresisbased and liquid chromatography-based separation systems coupled with
integrated high-throughput mass spectrometer systems (including ESI-ion
trap, ESI-Q/TOF, MALDI-Q/TOF, and MALDI-TOF/TOF) are currently in
routine operation. At present, more than 1000 proteins from the human
HepG2 and Hep3B liver cell proteomes have been identified using either or
both approaches. The well recognized pitfalls in either methods alone
dictate that both platform technologies coupled with well designed fractionation and enrichment strategies are required if a true human proteome
database is to be established. Our data are compiled and presented here
to serve as an entre´ e for AOHUPO to set the standards and regulations in
the global human proteome initiatives.
Su-Hyung Hong, David E. Misek, Hong Wang, Rong Zhao,
Craig Logsdon, and Samir M. Hanash
Pancreatic cancer has a poor prognosis, in part due to lack of early
detection. There is substantial interest in the identification of human tumor
antigens and autoantibodies for early diagnosis and immunotherapy of
cancer. We have implemented a proteomic approach for the identification
of tumor proteins that elicit a humoral response in pancreatic cancer.
Proteins from two pancreatic cancer cell lines (Panc-1 and BxPC3) were
subjected to two-dimensional PAGE, followed by Western blot analysis in
which individual sera were tested for autoantibodies. Sera from 23 newly
diagnosed patients with pancreatic cancer, 14 patients with lung adenocarcinoma and 15 healthy subjects were analyzed. Autoantibodies against
a novel PDZ-Domain and LIM-Domain protein, identified by MALDI in the
BxPC3 cell line were detected in sera from 9 of 23 patients with pancreatic
cancer, in 1 of 14 patients with lung cancer but were not detected in sera
from normal individuals. In the Panc-1 cell line autoantibodies were detected against a calcium-binding protein (in 11 of 23 pancreatic cancer
sera) and against a phosphatase (in 8 of 23 pancreatic cancer sera), but
neither was detected in sera from 15 normal individuals. The phosphatase
was detected by 1 of 14 patients with lung cancer and the calcium-binding
protein was detected in sera from 8 of 14 patients. These tumor-associated antigens may have utility as a biomarker for pancreatic cancer
screening and diagnosis.
13.27
Proteome Mapping of Chronic
Lymphocytic Leukemia
C. A. Evans1, D. A. E Cochran1, D. Blinco1, J. Burthem1,
S. K. Stevenson2, S. J. Gaskell1, and A. D. Whetton1
1
Leukaemia Research Fund Proteomics Facility, UMIST,
Manchester, United Kindgom; and 2Southampton General Hospital,
Southampton, United Kingdom
B-Cell Chronic Lymphocytic Leukaemia (B-CLL) can be divided into two
groups depending on the mutational status of the VH locus. Ig VH status
correlates with prognosis. Cases with somatic mutation Ig VH CLL (M-CLL)
have a relatively less aggressive form of CLL compared to patients with
unmutated Ig VH CLL (UM-CLL). Despite this, the gene expression profiles
of UM-CLL and M-CLL Ig VH are extremely similar. We have undertaken a
proteomic analysis of these prognostic groups to identify proteins that
may have prognostic value in distinguishing between these clinical subtypes. Differential protein expression profiling was performed using 2D-gel
electrophoresis. Similar patterns of protein expression were obtained for
all patients. The gels were subjected to computerised image analysis
using the Progenesis/Progenera software. Four proteins were identified as
present/absent between UM-CLL and M-CLL. Principal Component Analysis confirmed that there were significant differences in the patterns of
protein expression analysed by 2-dimensional gel electrophoresis. Over
65 proteins were identified from gels using MALDI-ToF mass spectrometry. One of these was the apoptotic regulator, Smac/DIABLO. It was
expressed only in cases where Ig VH was mutated; the less aggressive
form of the disease. Proteomic analysis thus has value in determining new
prognostic and mechanistic detail in the leukaemias.
676
13.28
13.29
Identification of Metastasis-associated
Proteins by Proteomic Analysis and
Functional Exploration of IL-18 in
Metastasis
Using Antibody Microarray for Proteomic
Analysis of Esophageal Squamons Cell
Carcinoma
Daifeng Jiang1,2, Wantao Ying1, Yinglin Lu3, Jinghong Wan1,
Yun Zhai1, Wanli Liu1, Zongyin Qiu2, Xiaohong Qian1, and
Fuchu He1
1
Beijing Institute of Radiation Medicine, Beijing, 100850, People’s
Republic of China; 2Chongqing University of Medical Science,
Chongqing, 400000, People’s Republic of China; and 3Institute of
Basic Medical Sciences, Beijing, 100850, People’s Republic of
China
Protein phosphorylation is the most important reversible post-translational
modification that occurs in cells. Widespread metastasis is the major lethal
cause of cancer. So far, very little is known about its mechanisms. In the
present study, comparative proteomic analysis was used to find out
metastasis-associated proteome. Firstly, a pair of highly and lowly metastatic sublines (termed as PLA801D and PLA801C respectively), originated from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by
metastatic phenotypes analysis in vitro. Subsequently, their protein expression profiles were compared by a proteomic approach. Of them, 11
metastasis-associated proteins were identified and further validated by
1-D western blotting, northern blot and/or semi-quantitative RT-PCR analysis. Compared with that in lowly metastatic PLA801C subline, CK18,
TGLC, GDIR, TPMF, IL-18 and ANX1 were significantly up-regulated, while
ER60, CH60, PDX1, CLI1 and KCRB were significantly down-regulated in
highly metatsatic PLA801D subline. Intriguingly, all the candidate proteins
except CLI1 have been evidenced to be somehow associated with distinct
aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was only
present in highly metastatic PLA801D, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense or IL-18 antisense into PLA801C or PLA801D subline respectively. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro
and down-regulated E-cadherin expression of PLA801C transfectants,
and that IL-18 antisense remarkably decreased the invasion potency in
vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play role in metastasis by inhibiting
E-cadherin expression.
Xiaohang Zhao, Yu Liu, Yousheng Mao, Huixin Wang,
Xiaoguang Ni, Fang Liu, and Min Wu
Cancer Institute of Chinese Academy of Medical Sciences, Beijing
100021, People’s Republic of China
Esophageal squamons cell carcinoma (ESCC) has very poor prognosis.
The early detection of ESCC is very depends on the discovery of some
specific and sensitive molecular biomarkers. We have studied the proteomic alterations of ESCC using antibody microarrays in a comparative
fluorescence assay to measure the abundance of many specific cellular
proteins simultaneously. Two groups samples, one based on the individual
patient’s tissue for comparative quantitation, the other based on the
pooled samples for subtraction of the individual differences, were labeled
by covalent attachment of spectrally resolvable fluorescent dyes. Specific
antibody-antigen interactions localized specific components in the array,
where the relative intensity of the fluorescent signal representing the
experimental sample and the reference standard provided a measure of
each protein’s abundance. Several kinds of proteins which low- or highexpressed in both groups involving in different signaling pathways and cell
cycle control, such as apoptosis pathway and cell cycle progression.
These results suggest that protein microarrays can provide a practical
approach to characterize patterns of variation in hundreds of thousands of
different proteins in proteomic analysis.
This work was supported by grants from the special funds for Major
State Basic Research (G19980512), State 863 High-tech R&D
(2001AA227091) and the NNSF (39990570 and 30171049) of China.
13.30
Metabolic Proteome Analysis of
Hepatocellular Carcinoma
Kang-Sik Park1, Hoguen Kim2, and Young-Ki Paik1,3
1
Yonsei Proteome Research Center, Yonsei University; 2Department
of Pathology, Yonsei University School of Medicine, Yonsei
University; and 3Department of Biochemistry, Yonsei University
To understand functional roles of proteins in hepatocelluar carcinoma
(HCC) at the protein level, 19 cases of liver tumor tissues were analyzed by
proteomic tools. Results were compared with those of paired adjacent
nontumorous tissues. We were able to identify more than 150 protein
spots out of 1000 spots in HCC in which total 32 types of protein spots
representing 24 protein families were induced while 34 types of protein
spots representing 27 protein families decreased. These differentially expressed proteins were confirmed by Western blot analysis. To our surprise, most of the induced proteins were closely related to metabolic
pathway of ATP synthesis and amino acid transformation. Interestingly,
the decreased proteins are liver-specific proteins involved in detoxification. This result is consistent with recent report on the inhibition of ATP
synthesis as a novel therapy for HCC (Geschwind et al. (2002) Cancer Res.
62, 3909 –3913). Taken together, our results suggest that HCC is closely
related to liver-specific metabolic process in which mitochondiral events
become more evident in hepatocellular carcinogenesis.
677
13.31
13.32
Complementary Proteome Analysis of a
Human Cancer Cell Line by MALDI-MS
and MALDI-MS/MS
Human 20S Proteasome: Reference Map,
Post-translational Modifications, and
Comparative Cancer Cell Lines Analyses
E. Claude1, A. Wallace1, D. Gostick1, A. Alaiya2, G. Auer2,
and J. Langridge1
Sandrine Uttenweiler-Joseph, Odile Burlet-Schiltz,
Ste´ phane Claverol, Elisabeth Neuhauser, Bo Xu,
Jean Edouard Gairin, and Bernard Monsarrat
1
Waters Corporation, Floats Road, Wythenshawe, Manchester,
United Kingdom; and 2Unit of Cancer Proteomics, Department of
Oncology and Pathology, Cancer Center Karolinska, Karolinska
Hospital, Stockholm, Sweden
Colorectal cancer represents an ideal model system to study development
and progression of human tumors. This is because the epithelial cells of
the colon follow a systematic process of cellular proliferation, differentiation and adenoma-carcinoma transformation. The p53 gene is actively
involved during the different stages of colo-rectal carcinogenesis. In this
study, we have examined the role of p53 in a human colorectal cell line
(HCT 116) a diploid cell line deficient in DNA mismatch repair mechanism.
To understand the role of p53 in the development of colon cancer, we
have studied the protein expression profiles in HCT 116 cells with two wild
type p53 alleles (p53⫹/⫹) and their p53 knockout counterparts (p53⫺/⫺).
Representative 2D gel images were derived from the two samples and
were analysed to identify proteins that were significantly up or down
regulated. Spots were automatically excised from the gels and then processed on an automated protein digestion robot, used to carry out all the
necessary de-staining, alkylation, reduction and protein digestion steps.
Automated MALDI-MS was used to rapidly identify many proteins from the
digest samples. Some of these proteins however remained unidentified,
henceforth a MALDI Q-Tof mass spectrometer was used to provide
MS/MS information the extra specificity required to achieve unambiguous
protein identification by databank searching.Using this approach it was
possible to identify many proteins that were potentially important in the
process of colo-rectal carcinogenesis and to localise their positions on
specific chromosomes.
678
Institut de Pharmacologie et de Biologie Structurale, CNRS, 205
Route de Narbonne, 31077 Toulouse Cedex, France
The proteasome is an ubiquitous multicatalytic complex, present in the
cytoplasm and the nucleus of all eukaryotic cells, which represents the
main protein degradation machinery in the cell. Evidence accumulates to
show that the proteasome is involved in crucial cellular processes, including cell cycle, apoptosis, morphogenesis, stress response, regulation of
intracellular proteins content, removal of abnormal proteins, and major
histocompatibility complex (MHC) class I antigen processing.
The catalytic core of the proteasome is a barrel-shaped complex called
20S proteasome, which is composed of 28 subunits assembled into two
outer ␣ rings and two inner ␤ rings containing 7 subunits each. The outer
a subunits regulate the entrance of denatured proteins into the catalytic
cavity formed by the ␤ subunits. Three ␤ subunits (␤1, ␤2, and ␤5) per ring
possess a catalytic active site and are responsible for three major peptidase activities (trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide hydrolyzing). The catalytic activities of 20S proteasome may be altered by the cellular origin and/or environment of the proteasome. It is
known for example that, in the presence of the cytokine ␥-interferon, the
three catalytic ␤ subunits of mammalian 20S proteasomes are replaced by
three other catalytic subunits, leading to modified peptidase activities.
Moreover, post-translational modifications of proteasome subunits, such
as phosphorylations, have been shown to affect proteasomal catalytic
activities.
The aim of our work is to understand the effects of cellular environment
on 20S proteasome structure/activity relationships and their consequences on MHC class I tumor antigen processing by proteasome. Here
we present the study of the subunit composition of human 20S proteasomes of various cellular origins by proteomics. Post-translational modifications have been characterized using mass spectrometric based
strategies.
The separation of 20S proteasome subunits by 2D gel electrophoresis
and their identification by MALDI-TOF mass spectrometry analyses combined with database searches, provided a reference map of human 20S
proteasome subunit composition. This study was performed with 20S
proteasome purified from human erythrocytes and allowed us to identify
all expected a and b subunits. Moreover, these analyses revealed the
presence of numerous isoforms associated to most subunits, including the
catalytic subunits. Post-translational modifications, including phosphorylation and N-acetylation, were identified by a combination of chromatographic techniques and mass spectrometry (MALDI-TOF and nanospray
MS/MS) for the various isoforms of one a subunit. Comparison of subunit
composition maps obtained with human 20S proteasomes purified from
various cellular origins (normal versus cancer cell lines) showed quantitative differences for the catalytic subunits and for a number of isoforms.
13.33
13.35
Differential Cell Surface Protein
Expression on Normal and Neoplastic
Humal Prostate Cells and Their
Regulation by Interferons
Proteomic-based Approach for the
Identification of Early Tumor Markers
Associated with Hepatocellular Carcinoma
Soren Naaby-Hansen, Claire Hastie, Akunna Akpan,
Malcolm Saxton, Rainer Cramer, Kohji Nagano,
and Jonn R. Masters
Ludwig Institute For Cancer Research, Royal Free and University
College Medical School, London, United Kingdom
The cell surface protein expression of a normal prostate epithelial cell line
and a prostate cancer cell line derived from the patient were investigated
by a combination of vectorial labeling with sulfo-biotin derivatives, 2D gel
electrophoresis, avidin blotting, affinity chromatography, and mass spectrometry analysis. Several proteins sere solely detected on the surface of
either the cancer or the normal cell line, and many others were found to be
expressed at different levers between two cell line. The repertoire of
proteins available for biotin labeling changed both qualitatively and quantitatively when the cells were grown in the presence of either or both the
therapeutic agents INF-␣ and INF-␥. Certain cell surface proteins including
gp96 and Annexin II, were differentially regulated in normal and cancer cell
lines by these interferons. Surface exposed Annexin II levels sere specifically down-regulated by INF-␥, while the total cellular levels were unaffected by cytokine treatment. INF-␥ mediated down-regulation of surface
exposed Annexin II, which captures and creates a reservoir of surface
proteases such as plasmin and procathepsin B, significantly reduced the
invasive potential of prostate epithelial cells in cell invasion assays. Our
data thus provides a potential explanation for the reduction of metastatic
potential observed in response to treatment with this cytokine.
Kapil Dua1, Franc¸ ois Le Naour1, Jorge A. Marrero2,
David E. Misek3, Christian Bre´ chot 4, Samir M. Hanash3,
and Laura Beretta1
1
Department of Microbiology and Immunology, University of
Michigan, Ann Arbor, Michigan, 48109-0666; 2Department of Internal
Medicine, University of Michigan, Ann Arbor, Michigan, 48109-0666;
3
Michigan, 48109-0666; and 4INSERM U.370, Necker Hospital, 75015
Paris, France
The incidence of hepatocellular carcinoma (HCC), the major histological
form of primary liver cancer, has substantially increased over the past two
decades. Chronic infections with hepatitis B (HBV) and hepatitis C (HCV)
viruses are major risk factors for HCC. We have utilized a proteomic
approach to determine whether a distinct repertoire of autoantibodies can
be identified in HCC. We recently reported the identification of 8 proteins
including calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A and F1-ATP synthase beta subunit, for each of which, autoantibodies were detected in sera from more than 10% of patients with HCC
but not in sera from healthy individuals (p ⬍ 0.05) (Le Naour et al. (2002)
Mol Cell Proteomics 1, 197–203). The common occurrence of autoantibody formation to HCC-related proteins may also have value in HCC
screening. We are currently investigating the utility of identified autoantibodies for the early diagnosis of HCC among high-risk subjects with
chronic HCV infection alone or in combination with cirrhosis. To that
effect, two-dimensional gel electrophoresis followed by 2-D Western blotting are used for simultaneous identification of proteins eliciting a humoral
response in 36 HCV patients with different degrees of liver fibrosis and
cirrhosis. Further prospective patient follow-up will allow validation of
these markers for the early diagnosis of HCC.
13.34
Identification of Tumor Antigens That
Elicit a Humoral Response to Dendritic
Cell-based Immunotherapy in Patients
with Melanoma
Kapil Dua1, Hong Wang2, Franc¸ ois Le Naour1, Jim Mule3,
Samir M. Hanash2, and Laura Beretta1
1
Michigan, Ann Arbor, Michigan, 48109-0666; 2Department of
Pediatrics, University of Michigan, Ann Arbor, Michigan,
48109-0666; 3Department of Surgery, University of Michigan, Ann
Arbor, Michigan, 48109-0666; and 4University of Michigan, Ann
Arbor, Michigan, 48109-0666
We have utilized a proteomic approach to determine whether the humoral
response to tumor antigens observed in melanoma patients was modified
following dendritic-cell based immunotherapy. Two-dimensional gel electrophoresis was used for simultaneous separation of individual proteins
from tumor tissue or cell lines. Proteins eliciting a humoral response in
melanoma patients were identified by 2-D Western blotting using sera
obtained before and after immunotherapy treatment, followed by mass
spectrometry analysis. We identified proteins, for each of which, autoantibodies were absent in sera from healthy individuals but detected in
melanoma patients, in different amounts before and after immunotherapy.
The identification of these tumor antigens may have utility in melanoma
diagnosis or in establishing an appropriate dendritic-cell based vaccine.
679
13.36
13.37
Proteomic Characterization of the HCV
NS5A Natural Mutants Isolated from
Interferon Responder Versus
Non-responder Patients with HCV
Proteome Differential Display of MCF-7
Cell Lines with High Capability for Active
Dissociation of Hoechst 33342 and Wild
Type
Alison Elafros1, Christian Bre´ chot2, Samir M. Hanash3, and
Laura Beretta1
Olga V. Tikhonova1 and Vera V. Levina2
1
Michigan, Ann Arbor, Michigan, 48109-0666; 2INSERM U.370,
Necker Hospital, 75015 Paris, France; and 3Department of
Pediatrics, University of Michigan, Ann Arbor, Michigan, 48109-0666
Hepatitis C virus (HCV) is a positive-stranded RNA virus, which exhibits
marked viral heterogeneity. Therapy using interferon alpha has been
shown to be effective in chronic hepatitis C infection, but only 30% to
40% of treated patients show a sustained virologic response. The
polyprotein precursor is co- and post-translationally processed by both
cellular and viral proteases to yield mature, structural and nonstructural
proteins. We have shown that the HCV non-structural NS5A protein
inhibits the antiviral activity of interferon alpha (Podevin et al. (2001)
Hepatology 33, 1503–1511; Girard et al. (2002) Virology 295, 272–283).
We isolated full-length NS5A sequences, from patients with or without
response to interferon therapy and combined transient and stable expression of these NS5A mutants in a human hepatocytic cell line (Huh7).
We analyzed and compared the phosphorylation and stability of the
various NS5A mutants. Several NS5A isoforms were identified by twodimensional gel electrophoresis. Interestingly, the NS5A proteins isolated from non responder patients, migrated into two major spots with
pI values of 5.57 and 5.43 respectively, whereas the NS5A proteins
isolated from responder patients presented two additional spots with pI
values of 5.29 and 5.16 respectively. Using pretreatment of Huh7 protein extracts with calf intestinal phosphatase, we demonstrated that the
two additional, more acidic, spots correspond to hyper-phosphorylated
isoforms of NS5A. This result demonstrates distinct phosphorylation
patterns among the NS5A mutants.
1
Institute of Biomedical Chemistry RAMS, Moscow, Russia; and 2St.
Petersburg Institute of Nuclear Physics, St. Petersburg, Russia
Living cells are capable to remove noncovalently binding molecules from
DNA. This process of active dissociation or ’DNA clearing’ from noncovalently bound agents in living mammalian cells was studied on the model
of the vital fluorescent bisbenzimidazole dye Hoechst 33342 and cytofluorimetric technique. It was showed that DNA clearing is one of the mechanisms of multidrug resistance in cancer cells. The method of stepwise
selection with increasing concentrations of Hoechst 33342 was used to
develop a set of Hoechst 33342 resistant MCF7 human breast adenocarcinoma cell lines. The cell lines with enhanced level of DNA clearing were
found to be 300 fold more resistant to toxic action of Hoechst 33342 and
netropsin and 20 fold more resistant to etoposide.
To recognise the proteins involved in DNA clearing we have studied
differences between proteomes of parent MCF7 line, mutant cell line
MCF7 HoeR-3 in early stages of selection and strong resistant MCF7
HoeR-7 mutant cell line using the conventional 2D-EF technique.
A comparison of corresponding 2D gels shows differences at several
protein spots, being more expressed in strongly resistant mutant or vice
versa. Some of differential proteins were identified by MALDI-MS.
13.38
BD PowerBlotTM Western Array Analysis
of Protein Expression in Differentiated
SH-SY5Y Neuroblastomas
M. T. Wilson1, J. K. Stevenson1, E. B. Heywood1,
A. M. Siemers1, A. L. Claxon1, B. J. Nelson1, C. H. Keith2,
and R. Campos-Gonzalez1
1
BD Transduction Laboratories, Lexington, Kentucky; 2Cellular
Biology, University of Georgia, Athens, Georgia; 3PNNL, Richland,
Washington 99352; and 4Institute of Biological Chemistry, Academia
Sinica, Taipei, Taiwan
A variety of in vitro models of neuronal differentiation and survival have
been developed for studying the protein expression changes that occur
during these processes. Few studies have compared the global changes
in protein expression that occur as a result of using different differentiation
factors, such as staurosporine vs NGF. In this study, we used BD Transduction Laboratories Western Array Screening Service (BD PowerBlotTM)
to identify protein expression changes for more than 800 proteins in
SH-SY5Y cells exposed to NGF for 24 hrs and staurosporine for 16 hrs.
Our results show that NGF exposure caused a modest increase in the
number of neurite-bearing cells, which correlates with alterations in the
expression of some proteins. Staurosporine caused more robust differentiation of SY5Y cells, and this correlates with changes in the expression of
many protein families. However, staurosporine also caused detachment of
a subpopulation of cells from the substratum, and led to staurosporinespecific decreases in the expression of essential enzymes, cytoskeletal
components, and calcium signaling proteins. Many of the proteins expressed after staurosporine-induced differentiation are also expressed
during development of the rat cerebrum, however some unique protein
expression changes found in staurosporine-differentiated cells do not
occur in the rat cerebrum. Thus, BD PowerBlotTM screening analysis is a
valuable tool for examining protein expression profiles for pathways involved in neuronal differentiation and death.
680
13.39
13.41
Two-dimensional Gel Electrophoresis Map
for Human Stomach Tissue
Application of Fluorescence Dyes in a
Linkage of Laser Microdissection and
Two-dimensional Gel Electrophoresis: As
a Tool for Cancer Proteomic Study
Seung Uook Lee, Na-Young Ha, Jina Kim, Jin Sook Ahn,
Yu Na Kim, Bok Im Cho, Sung-Jo Kang, Jae Won Kim, and
Chang Won Lee
Stomach cancer is one of the most common cancers in Korea both in men
and women. Also, stomach is one of the leading causes of cancer death
in the world. We recently reported the proteome analysis of human stomach tissue (Electrophoresis (2002) 23, 2513–2524), using two dimensional
gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF). We have successfully
identified 243 protein spots (168 spots acidic map and 75 spot in basic
map) from the 2-DE gel of human stomach tissue. Here, we update
previously published in addition to urea has been found to further improve
solubilization. With these result, we could be detected many low-abundance proteins and high molecular proteins. Second, to improve success
rate of protein identification in this study, we performed protein identification using reversed-phase nano column. The use of reversed-phase
nano column improved the concentration of extracted peptides and the
purification of peptide mixture. Using this approach, 2-DE map have been
established for soluble protein of human stomach tissue. 174 protein
spots (104 spots acidic map and 71 spot in basic map) were identified
newly. The 2-DE maps resulting from this study will server important
resources in studies on the differential protein expressions of human
stomach cancer and also other human stomach pathologies.
Tadashi Kondo, Masahiro Seike, Yasuharu Mori,
Kazuyasu Fujii, Tesshi Yamada, and Setsuo Hirohashi
Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
The combination of laser microdissection and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used for proteome analysis on specific populations of cells in cancer tissues. However, because of
low sensitivity of spot detection method, silver staining, the microdissection to obtain enough amount of protein for 2D-PAGE is too laborious and
only restricted numbers of protein spots were visualized. As a consequence, this technology looks impractical for direct clinical applications
and had a limited impact on cancer study. To solve these problems, we
developed new application in which sensitive fluorescent dyes label the
proteins extracted from microdissected tissues prior to 2D-PAGE separation. In this application, small amount of proteins, less than 6.6 ␮g, was
enough to generate a 2D profile with approximately 1,500 protein spots.
For accurate quantification of spot intensity, different samples were labeled with different fluorescence dyes and run in a single 2D gel. This
technique was applied to compare the proteome of normal intestinal
epithelium and adenoma tissues of Min mice. Among 1,500 spots, 37
spots changed their intensities reproducibly: 27 spots showed increased
intensity and 10 spots decreased in adenoma tissues compared with their
normal counterparts. Mass spectrometric analysis and subsequent database search successfully identified 4 of them, which included prohibitin,
14 –3-3␨, tropomyosin 3 and Hsp84. These results indicate that the fluorescence labeling of proteins from microdissected tissues prior to 2DPAGE is a powerful tool in cancer proteomic study.
13.40
Overexpression of Nucleoside
Diphosphate Kinase A in Human Gastric
Cancer Tissue
Sung-Jo Kang, Na-Young Ha, Jina Kim, Jin Sook Ahn,
Yu Na Kim, Bok Im Cho, Seung Uook Lee, Jae Won Kim,
and Chang Won Lee
Gastric cancer is the most common cancer in Korea and Asian countries.
Although incidence rates in western world are much lower than Asia,
gastric carcinoma is still a significant worldwide health burden, second
only to lung tumors as a leading cause of cancer deaths. In this study, in
order to develop novel markers for gastric cancer, 70 cases of gastric
cancer tissue were analyzed by two-dimensional electrophoresis, and
stained with silver nitrate. The images of stained gels were analyzed with
aid of software, PDQuest. Of the 70 samples, 16 samples (22.86%)
showed higher expressions of the spot 6117 in cancer tissue. The spot
was excised from preparative gels, digested by trypsin, and subjected to
peptide mass fingerprinting. The spot was identified to be nucleoside
diphosphate kinase A, with 10 matching peptides which corresponds to a
sequence coverage of 53%. Nucleoside diphosphate kinase A is a ubiquitous enzyme responsible for the synthesis of most cellular non ATP
nucleoside triphosphates. Beside their role in nucleotide synthesis, these
enzymes present additional functions, possibly independent of catalysis,
in processes such as differentiation, cell growth, tumor progression, metastasis and development.
681
13.42
13.43
Proteomics of Renal Disorders: Urinary
Proteome Analysis by Two-dimensional
Electrophoresis and MALDI-TOF Mass
Spectrometry
Isolation of Rheumatoid Arthritis-specific
Proteins Using Proteomic Analysis
Yadunanda Kumar1, Nageshwar Rao Venkata Uppuluri1,
Kishore Babu2, Kishore Phadke3, Prasanna Kumar2,
Sudarshan Ballal4, and Utpal Tatu1
1
Department of Biochemistry, Indian Institute of Science,
Bangalore 560 012, India; 2Department of Endocrinology, M.S.
Ramaiah Medical College Hospital, Bangalore 560 054, India;
3
Department of Pediatrics, St. John’s Medical College Hospital,
Bangalore 560 034, India; and 4Manipal Institute of Nephrology and
Urology, Manipal Hospital, Bangalore 560 017, India
The proteomes of urinary samples from patients with different renal conditions were analyzed by 2-DE and MALDI-TOF technology. Samples from
three different renal conditions namely kidney failure, nephrotic syndrome
and microalbuminuria, were included in the analysis. Apart from the presence of albumin, the profiles of protein spots found in these urine samples
were quite distinct. While kidney failure patients showed predominantly
low molecular weight proteins, the nephrotic syndrome patients showed
an abundance of relatively high molecular weight proteins clustering in the
acidic range of the 2-D gels. Two different protein spots from kidney failure
patients, four from nephritic syndrome and three from microalbuminuria
patients were identified by in-gel protease digestions and analysis of
resulting peptides by MALDI-TOF. The proteins identified were albumin,
␣-1-antitrypsin, ␣-1-acid glycoprotein 2, Zn-␣-2-glycoprotein and ␣-1microglobulin. Among these only one was common between the proteomes of renal failure and nephrotic syndrome patients. Among the
limited proteins found in microalbuminuria patients, three were common
with the proteome of nephrotic syndrome. Overall profiles were, however,
quite different. In all, our study showed that urinary proteomes of different
renal conditions were different and emphasized the potential of urinary
proteome analysis to augment existing tools in the diagnosis of renal
disorders.
Deok Ryong Kim1, Young-Sool Hah1, Eun-Hye Cho1,
Yun Jong Lee2, and Choong Won Kim1
1
College of Medicine, Gyeongsang National University,
92 Chilam-dong, JinJu, KyeongNam, South Korea 660-751; and
2
Department of Biochemistry, Department of internal medicine,
Gyeongsang National University, 92 Chilam-dong, JinJu,
KyeongNam, South Korea 660-751
Rheumatoid Arthritis (RA), an autoimmune disease with chronic inflammation and destruction of cartilage and bone, has a prevalence of 1% over
the world. The immunological and molecular mechanism in RA pathogenesis has not been elucidated clearly, although many efforts were involved
in the research of RA. Cytokines (IL-10, TNF␣, etc.) that regulate immune
cell proliferation have been thought the key controller in RA pathogenesis.
In fact, blocking the function of TNF␣ using antagonist antibodies prohibits
the progression of RA. However, the role of such cytokines in RA pathogenesis is not clearly demonstrated. The immune response to the self
antigens in RA is triggered by the activation of potential self-reacting B or
T cells with mimicry antigens that might be derived from viral or bacterial
infection. Here we are trying to isolate RA-specific proteins from comparative proteomic analyses of proteins obtained from Korean RA, Osteoarthritis, or other arthritis patients. We separated total cell proteins (about
2,000) or subcellular (soluble or insoluble) proteins extracted from patients
in two-dimensional gel electrophoresis and analyzed the pattern of protein
expression using PDQuest software. We found that some proteins were
uniquely expressed in RA patients and expression of many proteins either
increased or decreased. Isolation of RA-specific proteins and new autoantigens will provide some insights into understanding the molecular mechanism in RA pathogenesis and separating target proteins for new drug
development.
13.44
Discovery, Purification, and Identification
of Biomarkers from Complex Samples by
Combining ProteinChip姞 Array Analysis
with Micropurification Strategies
Rosa I. Viner, Siyu Fu, Ning Tang, and Scot R. Weinberger
Ciphergen Biosystems, Inc., Fremont, California 94555
In this study we present a novel approach to biomarker identification via
Retentate Chromatography™-Mass Spectrometry (RC-MS) on the SELDI
ProteinChip platform. The work combines RC-MS protein profiling and
spin column micropurification followed by gel electrophoresis using limiting amounts of starting material. This strategy consists of four main
components: biomarker discovery by SELDI; biomarker enrichment by
combining micropurification with electrophoresis; recovering purified proteins using passive gel elution; and confirmation of protein identity utilizing
on-spot proteolysis followed by tandem ms analysis.
We have used Q-HyperD姞F anion exchange micro-spin columns for
pre-fractionation and RC-MS to monitor human Parathyroid Hormone
(PTH) spiked into Cerebral Spinal Fluid (CSF). RC-MS analysis has shown
the presence of this protein in an organic fraction which strongly bound to
reversed phase arrays, even after 50% acetonitrile wash. The protein was
then purified by 4 –20% Tris-Glycine SDS PAGE and detected by a reverse
negative staining procedure. The PTH band was excised from the gel,
destained and the protein was eluted as described in (1) with minor
modifications. The purified protein has shown the same molecular weight
and chromatographic properties on the H50 array as PTH and its identity
was confirmed with high confidence after AspN on-spot digestion through
a combination of peptide mass fingerprinting by SELDI-TOF MS and
peptide sequencing by SELDI-QqTOF MS/MS. We have successfully applied this technique to identify potential biomarkers from other complex
biological systems.
1. Cohen, S. L., and Chait, B. T. (1997) Anal. Biochem. 247, 257–267
682
13.45
14.1
Post-translational Modification of Serum
Proteins Underlies the Mass Spectral
Fingerprinting of Disease
Proteomic Analysis of Acholeplasma
laidlawii Mutants Resistant to Tetracycline
and Fluoroquinolone
John Marshall, Peter Kupchak, Weimin Zhu, Jason Yantha,
Tammy Vrees, Shirley Furesz, Kelly Jacks, Chris Smith,
Inga Kireeva, Rulin Zhang, Miyoko Takahashi, Eric Stanton,
and George Jackowski
Sergei Moshkovskii, Vadim Govorun, and Eugene Goufman
Synx Pharma, 1 Marmac Drive, Toronto, Ontario, M9W 1E7 Canada
The mass distribution and intensity of poly-peptides from human sera
obtained using mass spectrometry has served as a fingerprint used to
diagnose disease. However, the mechanism underlying mass spectral
diagnosis has not been demonstrated. We extended the method to obtain
a serum fingerprint of myocardial infarction patients which forms the basis
of a powerful diagnostic tool that rapidly and conclusively confirms the
presence of myocardial infarction. We show that in general mass spectral
diagnosis works on the principle of detecting post-translational modifications of major serum proteins effected by disease associated activities in
the blood. In the case of myocardial infarction, the MALDI-TOF spectra of
peptides collected by C18 reversed-phase chromatography form a diagnostic pattern resulting from the post-translational modification of complement C3 ␣ chain to release the C3f fragment and cleavage of fibrinogen
to release the alpha peptide. We used time course and PMSF studies to
demonstrate that the peptides that form diagnostic patterns in the serum
result from polypeptides that were continually being generated by serinecentered endo-proteinases. However, we also show that the peptides
cleaved from serum proteins by endo-proteinases are themselves in turn
degraded by N-terminal exopeptidase(s), i.e., aminopeptidase. Thus the
mass spectral patterns that form the basis of diagnosis reflect a balance
of the proteinase and aminopeptidase specificity and activity in the sera.
On this basis we conclude that MALDI-TOF, or other mass spectral
fingerprinting, of sera may reflect the interaction of disease-associated
molecules with the proteins of the blood.
13.46
Analysis of Complex Autoantiboy
Repertoires by SELDI-TOF
V.N. Orekhovich Institute of Biomedical Chemistry, Moscow, Russia
Acholeplasma laidlawii was firstly investigated as a good experimental
model for membrane transport studies of antimicrobial drugs like fluoroquinolones, tetracycline and similar compounds. However, the role of
mycoplasma plasma membrane transport systems in the resistance phenomena has not yet been studied. The 2D electrophoretic (2DE) maps of
Acholeplasma laidlawii cell and membrane proteins were obtained for the
comparative analysis of 2DE maps of mutants resistant to tetracycline and
fluoroquinolones and wild type.
For the solubilization and separation of Acholeplasma laidlawii plasma
membrane proteins in the first dimension (IPG-strips pH 3–10, Bio-Rad)
we used detergents, such as CHAPS (ICN), Triton X-100 (Bio-Rad), Noctylglucoside (ICN), NP-40 (ICN) and Octylpol (Alexis, USA). For the
second dimension 5–18% gradient SDS-PAGEs were used. Silver staining
procedure was performed for the visualization of separated proteins.
Analysis of the 2DE gels was carried out using Melanie II software. Several
2DE of Acholeplasma laidlawii were obtained using different detergents.
NP-40 was the most effective detergent for the solubilization and separation of Acholeplasma laidlawii cell proteins. In contrast, Octylpol has been
chosen as optimal detergent to solubilize membrane proteins. There were
670 individual membrane protein spots on gel after silver staining. Analysis
of Acholeplasma laidlawii mutants resistant to tetracycline and fluoroquinolones 2D-PAGE maps revealed increasing expression of several
proteins which were suggested to be involved in the resistance mechanism. Both mutants resistant to tetracycline and fluoroquinolones had 4
protein spots of molecular weight about 9 –14 kDa with increased intensity. Using MALDI-MS of tripsin digests of these spots it has been found
that three of them occur derived from the same gene and one protein was
different.
Selected fragments of these two individual proteins were investigated
using ESI-MS-MS in order to determine amino acid sequence. Basing this
information PCR primers were constructed and corresponding genes were
cloned and sequenced using conventional technique known in the art.
F. H. Grus, S. C. Joachim, and N. Pfeiffer
Department of Ophthalmology, University of Mainz, Mainz, Germany
Normal sera contain a large number of naturally occurring auto-antibodies
which can mask important disease-associated ones. In previous studies
we could show the diagnostic potential of the analysis of auto-antibodies
in autoimmune diseases by means of multivariate statistics and artificial
neural networks. However, conventional Western blotting procedures remain very time-consuming limited in sensitivity.
Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDITOF (surface-enhanced laser desorption/ionisation in time-of-flight mass
spectrometry) technology (Ciphergen, Fremont, California) for capturing,
detection, and analysis of proteins without labelling or without the need of
chemical modification. The micro-scale design of the arrays allows the
analysis of very small quantities of proteins. In the present study, we used
arrays with biological activated surfaces that permit antibody capture
studies.
In the present study, we could show complex on-chip antibody-antigen
reactions. At higher molecular weights (⬎30 kDa) the detection sensitivity
of this on-chip method was comparable to conventional Western blotting.
At lower molecular weights, the Western blot technique is easily exceeded
by the on-chip method.
Considering that this on-chip procedure is quite easy to use, is much
less time consuming than Western blotting, and is much more sensitive at
least in the low molecular weight range, the SELDI-TOF technology is a
very promising approach for the screening of auto-antibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow
the large-scale screening for complex auto-antibody distributions for diagnostic purposes and early detection of autoimmune diseases could be
made possible.
683

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Sample Preparation for MALDI-TOF/TOF &gt;95% Success Protein Identification with Rates

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Sample Preparation for MALDI-TOF/TOF >95% Success Protein Identification with Rates