Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.     

Comparative proteomic analysis of matched primary and metastatic melanoma cell lines

Identification of the biochemical pathways involved in the transformation from primary to metastatic melanoma is an area under intense investigation. A 2DE proteomics approach has been applied herein to the matched patient primary and metastatic melanoma cell lines WM-115 and WM-266-4, respectively, to better understand the processes that underlie tumor progression. Image analysis between samples aligned 470 common gel spots. Quantitative gel analysis indicated 115 gel spots of greater intensity in the metastatic line compared with the primary one, leading to the identification of 131 proteins via database searching of nano-LC-ESI-Q-TOF-MS/MS data. This more than tripled the number of proteins previously shown to be of higher abundance during melanoma progression. Also observed were 22 gel spots to be of lesser intensity in the metastatic line with respect to the primary one. Of these gel spots 15 proteins could be identified. Numerous proteins from both groups had not been reported previously to participate in melanoma progression. Further analysis of one protein, cyclophilin A, confirmed that this protein is expressed at higher levels in metastatic melanoma compared with primary melanoma and normal fibroblasts. Overall, this study expands our knowledge of protein modulation during melanoma stages, and suggests new targets for inhibitor development.     

A systems biology analysis of metastatic melanoma using in-depth three-dimensional protein profiling

Melanoma is an excellent model to study molecular mechanisms of tumor progression because melanoma usually develops through a series of architecturally and phenotypically distinct stages that are progressively more aggressive, culminating in highly metastatic cells. In this study, we used an in-depth, 3-D protein level, comparative proteome analysis of two genetically, very closely related melanoma cell lines with low- and high-metastatic potentials to identify proteins and key pathways involved in tumor progression. This proteome comparison utilized fluorescent tagging of cell lysates followed by microscale solution IEF prefractionation and subsequent analysis of each fraction on narrow-range 2-D gels. LC-MS/MS analysis of gel spots exhibiting significant abundance changes identified 110 unique proteins. The majority of observed abundance changes closely correlate with biological processes central to cancer progression, such as cell death and growth and tumorigenesis. In addition, the vast majority of protein changes mapped to six cellular networks, which included known oncogenes (JNK, c-myc, and N-myc) and tumor suppressor genes (p53 and transforming growth factor-β) as critical components. These six networks showed substantial connectivity, and most of the major biological functions associated with these pathways are involved in tumor progression. These results provide novel insights into cellular pathways implicated in melanoma metastasis.     

Proteomic analysis of infiltrating ductal carcinoma tissues by coupled 2-D DIGE/MS/MS analysis

There is a growing interest in protein expression profiling aiming to identify novel diagnostic markers in breast cancer. Proteomic approaches such as two-dimensional differential gel electrophoresis coupled with tandem mass spectrometry analysis (2-D DIGE/MS/MS) have been used successfully for the identification of candidate biomarkers for screening, diagnosis, prognosis and monitoring of treatment response in various types of cancer. Identifying previously unknown proteins of potential clinical relevance will ultimately help in reaching effective ways to manage the disease. We analyzed breast cancer tissues from five tumor and five normal tissue samples from ten breast cancer subjects with infiltrating ductal carcinoma (IDC) by 2-D DIGE using two types of immobilized pH gradient (IPG) strips: pH 3-10 and pH 4-7. From all the spots detected, differentially expressed (p < 0.05 and ratio > 2) were 50 spots. Of these, 39 proteins were successfully identified by MS, representing 29 different proteins. Ten proteins were overexpressed in the tumor samples. The 2-D DIGE/MS/MS analysis revealed an increase in the expression levels in tumor samples of several proteins not previously associated with breast cancer, such as: macrophage-capping protein (CAPG), phosphomannomutase 2 (PMM2), ATPase ASN1, methylthioribose-1-phosphate isomerase (MRI1), peptidyl-prolyl cis-trans isomerase FKBP4, cellular retinoic acid-binding protein 2 (CRABP2), lamin B1 and keratin, type II cytoskeletal 8 (KRT8). Ingenuity Pathway Analysis (IPA) revealed highly significant (p = 10(-26)) interactions between the identified proteins and their association with cancer. These proteins are involved in many diverse pathways and have established roles in cellular metabolism. It remains the goal of future work to test the suitability of the identified proteins in samples of larger and independent patient groups.     

Proteomic analysis in clear cell renal cell carcinoma: identification of differentially expressed protein by 2-D DIGE

Renal cell carcinoma (RCC), the most common neoplasm affecting the adult kidney, is characterised by heterogeneity of histological subtypes, drug resistance, and absence of molecular markers. Two-dimensional difference gel electrophoresis (2-D DIGE) technology in combination with mass spectrometry (MS) was applied to detect differentially expressed proteins in 20 pairs of RCC tissues and matched adjacent normal kidney cortex (ANK), in order to search for RCC markers. After gel analysis by DeCyder 6.5 and EDA software, differentially expressed protein spots were excised from Deep Purple stained preparative 2DE gel. A total of 100 proteins were identified by MS out of 2500 spots, 23 and 77 of these were, respectively, over- and down-expressed in RCC. The Principal Component Analysis applied to gels and protein spots exactly separated the two sample classes in two groups: RCC and ANK. Moreover, some spots, including ANXA2, PPIA, FABP7 and LEG1, resulted highly differential. The DIGE data were also confirmed by immunoblotting analysis for these proteins. In conclusion, we suggest that applying 2-D DIGE to RCC may provide the basis for a better molecular characterization and for the discovery of candidate biomarkers.     

Drosophila melanogaster larval hemolymph protein mapping

With the completion of the genome sequence of Drosophila melanogaster the importance of constructing a proteome map is to be considered. Therefore, with the application of recent advances in proteomic analysis approaches, a protein map of D. melanogaster larvae hemolymph proteins was obtained using 2-DE in the range of pH 3-10. After Coomassie colloidal detection of 289 spots, a total of 105 were excised from the gel and digested with trypsin. Identification was done based on a combination of MALDI-TOF/TOF MS and MS/MS spectra. The 99 proteins identified using this approach include a large number of metabolic enzymes, translational apparatus components, and structural proteins. Among these we emphasize the identification of proteins with molecular chaperone properties (heat shock proteins and PPIases) and protein spots involved in defense responses such as antioxidant and immunological defense mechanisms (thioredoxin, prophenoloxidase, and serine proteases), as well as in signal transduction pathways.     

The Encysted Dormant Embryo Proteome of <Emphasis Type="Italic">Artemia sinica</Emphasis>

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.     

Proteomics in Drosophila melanogaster: first 2D database of larval hemolymph proteins

A proteomic approach was used for the identification of larval hemolymph proteins of Drosophila melanogaster. We report the initial establishment of a two-dimensional gel electrophoresis reference map for hemolymph proteins of third instar larvae of D. melanogaster. We used immobilized pH gradients of pH 4-7 (linear) and a 12-14% linear gradient polyacrylamide gel. The protein spots were silver-stained and analyzed by nanoLC-Q-Tof MS/MS (on-line nanoscale liquid chromatography quadrupole time of flight tandem mass spectrometry) or by Matrix assisted laser desorption time of flight MS (MALDI-TOF MS). Querying the SWISSPROT database with the mass spectrometric data yielded the identity of the proteins in the spots. The presented proteome map lists those protein spots identified to date. This map will be updated continuously and will serve as a reference database for investigators, studying changes at the protein level in different physiological conditions.     

Serum proteomics of lung adenocarcinomas induced by targeted overexpression of c-raf in alveolar epithelium identifies candidate biomarkers

We previously reported a proteome map of lung adenocarcinomas in serine-threonine kinase of the Raf family (c-raf) transgenic mice. We now extend our initial studies to serum proteins at early stage (1 month) and advanced stages of tumorigenesis (12 months). Notably, serum proteins from wild-type and tumor bearing mice were extracted with a lysis buffer containing 5 mol/L urea, 2 mol/L thiourea, 40 mmol/L Tris, 4% CHAPS, 100 mmol/L DTT, 0.5% BioLyte 3-10, separated by 2-DE and studied by image analysis. On average 400 protein spots per gel were excised and analyzed by MALDI-TOF MS. We identified 45 common and 5 uniquely expressed proteins in wild-type and tumor bearing mice. Apart from uniquely identified proteins we observed for n = 9 proteins differential expression when wild-type and tumor bearing mice were compared. This included serpins and other protease inhibitors, lipocalins, transthyretins, globins, and Igs. Notably, we demonstrate significant regulation of alpha-1-antitrypsin, alpha-2-macroglobulin, hemoglobin subunit alpha, vitamin D-binding protein, major urinary proteins, and transthyretin (up to eight-fold) in serum of lung tumor bearing mice. Disease association of these proteins in human malignancies has been reported. Thus, an identification of regulated serum proteins in this lung cancer disease model provides excellent opportunities for the search of novel biomarkers.     

Proteome profiling of human epithelial ovarian cancer cell line TOV-112D

A proteome profiling of the epithelial ovarian cancer cell line TOV-112D was initiated as a protein expression reference in the study of ovarian cancer. Two complementary proteomic approaches were used in order to maximise protein identification: two-dimensional gel electrophoresis (2DE) protein separation coupled to matrix assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and one-dimensional gel electrophoresis (1DE) coupled to liquid-chromatography tandem mass spectrometry (LC MS/MS). One hundred and seventy-two proteins have been identified among 288 spots selected on two-dimensional gels and a total of 579 proteins were identified with the 1DE LC MS/MS approach. This proteome profiling covers a wide range of protein expression and identifies several proteins known for their oncogenic properties. Bioinformatics tools were used to mine databases in order to determine whether the identified proteins have previously been implicated in pathways associated with carcinogenesis or cell proliferation. Indeed, several of the proteins have been reported to be specific ovarian cancer markers while others are common to many tumorigenic tissues or proliferating cells. The diversity of proteins found and their association with known oncogenic pathways validate this proteomic approach. The proteome 2D map of the TOV-112D cell line will provide a valuable resource in studies on differential protein expression of human ovarian carcinomas while the 1DE LC MS/MS approach gives a picture of the actual protein profile of the TOV-112D cell line. This work represents one of the most complete ovarian protein expression analysis reports to date and the first comparative study of gene expression profiling and proteomic patterns in ovarian cancer.     

Proteomic analysis of human epithelial ovarian cancer xenografts in immunodeficient mice exposed to chronic psychological stress

This work aims at comparing alterations in the proteomes of human epithelial ovarian cancer xenografts between stressed and non-stressed immunodeficient mice as well as exploring the molecular mechanisms linking chronic psychological stress to ovarian cancer oncogenesis and progression. SK-OV-3 cells were injected subcutaneously into nude mice. The stress group was subjected to a chronic physical restraint protocol for 6 h on 35 consecutive days, while the control group was unrestrained. All mice were sacrificed on day 36 after SK-OV-3 cell injection, and tumors were excised. Tumor tissues were processed for 2D gel electrophoresis, mass spectrometry (nanoUPLC-ESI-MS/MS) and Western blotting. The expression of 20 proteins was found to be significantly altered between the stress and control groups, of which 14 were up-regulated, five were down-regulated, and one protein was found only in the stress group. All proteins were identified by UPLC-ESI-MS/MS, and Western blotting results were consistent with those of proteomic methods. The present results provide new evidence relating to the molecular mechanism underlying the relationship between psychological stress and tumor progression.     

Comparative proteome analysis of breast cancer and normal breast

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p<0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.     

Comparative proteomic analysis provides new insights into the fiber elongating process in cotton

A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers. The temporal changes of global proteomes at five representative development stages (5-25 days post-anthesis [dpa]) were examined using 2-D electrophoresis. Among approximately 1800 stained protein spots reproducibly detected on each gel, 235 spots were differentially expressed with significant dynamics in elongating fibers. Of these, 120 spots showed a more than 2-fold change in at least one stage point, and 21 spots appeared to be specific to developmental stages. Furthermore, 106 differentially expressed proteins were identified from mass spectrometry to match 66 unique protein species. These proteins involve different cellular and metabolic processes with obvious functional tendencies toward energy/carbohydrate metabolism, protein turnover, cytoskeleton dynamics, cellular responses and redox homeostasis, indicating a good correlation between development-dependent proteins and fiber biochemical processes, as well as morphogenesis. Newly identified proteins such as phospholipase D alpha, vf14-3-3 protein, small ras-related protein, and GDP dissociation inhibitor will advance our knowledge of the complicated regulatory network. Identification of these proteins, combined with their changes in abundance, provides a global view of the development-dependent protein changes in cotton fibers, and offers a framework for further functional research of target proteins associated with fiber development.     

Proteomic analysis of methyl parathion-responsive proteins in Sparus latus liver

The contamination of marine ecosystems by organophosphate pesticides is of great concern. The use of protein expression profiles may provide a good method to help us understand the methyl parathion (MP) toxicity to aquatic organisms. In this study, Sparus latus, was selected as the target organism. The toxicological effects of MP were investigated after 24 h exposure using proteomics to analyze their liver tissues. Certain enzyme activity parameters of the liver extracts were also examined, including CAT. After analyzing the proteomic profile of the liver using 2D gel electrophoresis, we found that the protein expression levels of 25 spots increased or decreased significantly in the exposed groups. Sixteen of the 25 protein spots were successfully identified using MALDI-TOF MS/MS. These proteins were roughly categorized into diverse functional classes such as cell redox homeostasis, metabolic processes and cytoskeleton system. These data demonstrated that proteomics was a powerful tool to provide valuable insights into the possible mechanisms of toxicity of MP contaminants in aquatic species. Additionally, these data may provide novel biomarkers for evaluation of MP contamination in the environment.     

Proteomic analysis of the hydrophobic fraction of mesenchymal stem cells derived from human umbilical cord blood

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering, but their application has been impeded by lack of knowledge of their core biological properties. In order to identify MSC-specific proteins, the hydrophobic protein fraction was individually prepared from two different umbilical cord blood (UCB)-derived MSC populations; these were then subjected to two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). Although the 2D gel patterns differed somewhat between the two samples, computer-assisted image analysis identified shared protein spots. 35 spots were reliably identified corresponding to 32 different proteins, many of which were chaperones. Based on their primary sub-cellular locations the proteins could be grouped into 6 categories: extracellular, cell surface, endoplasmic reticular, mitochondrial, cytoplasmic and cytoskeletal proteins. This map of the water-insoluble proteome may provide valuable insights into the biology of the cell surface and other compartments of human MSCs.     

Proteomic analysis of tumor establishment and growth in the B16-F10 mouse melanoma model

The B16-F10 mouse model of melanoma is a widely used model to study many aspects of cancer biology and therapeutics in a solid tumor. Melanomas aggressively progress within a dynamic microenvironment containing in addition to tumor cells, stroma cells and components such as fibroblasts, immune cells, vascular cells, extracellular matrix (ECM) and extracellular molecules. The goal of this study was to elucidate the processes of tumor progression by identifying differentially expressed proteins in the tumor mass during specific stages of tumor growth. A comparative proteome analysis was performed on B16-F10 derived tumors in C57BL/6 mice at days 3, 5, 7, and 10. Statistical approaches were used to determine quantitative differential protein expression at each tumor time stage. Hierarchical clustering of 44 protein spots (p < 0.01) revealed a progressive change in the tumor mass when all 4 time stages were classified together, but there was a clear switch in expression of these proteins between the day 5 and the day 7 tumors. A trend analysis showed 53 protein spots (p < 0.001) following 6 predominant kinetic paths of expression as the tumor progressed. The protein spots were then identified using MALDI-TOF mass spectrometry. Proteins involved in glycolysis, inflammation, wounding, superoxide metabolism, and chemotaxis increased during tumorigenesis. From day 3 to day 7 VEGF and active cathepsin D were induced 7-fold and 4-fold, respectively. Proteins involved in electron transport, protein folding, blood coagulation, and transport decreased during tumorigenesis. This work illustrates changes in the biology of the B16-F10 tumor mass during tumor progression.     

Proteome analysis on an early transformed human bronchial epithelial cell line,BEP2D,after alpha-particle irradiation

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.     

Global expression study in colorectal cancer on proteins with alkaline isoelectric point by two-dimensional difference gel electrophoresis

Colorectal cancer is one of the leading causes of cancer death worldwide. To identify candidates for biomarkers and therapeutic targets, we investigated the proteome of colorectal cancer tissues. Using 2D-DIGE in combination with our original large format electrophoresis apparatus, we compared surgically resected normal and tumor tissues from 53 patients with colorectal cancer. We focused on proteins with an alkaline pI using IPG gels for the alkaline range. We observed 1687 protein spots, and found 100 spots with statistical (p<0.01) and significant (>2-fold) differences between the normal and the tumor tissue groups. Among these 100 protein spots, five showed a different intensity between tumor tissues from the stage-II and the stage-III patients. MS experiments revealed that these 100 protein spots corresponded to 58 unique proteins. These included six proteins which had not been previously reported to be associated with colorectal cancer. Among these proteins, five were not reported in any type of malignancy. IEF/western blotting confirmed the differences in protein expression between the normal and the tumor tissues. These results may provide an insight for biomarker development and drug target discovery in colorectal cancer.     

Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.