ABSTRACT
BACKGROUND: We used intensive modern proteomics approaches to identify predictive proteins in ovary cancer. We identify up-regulated proteins in both serum and peritoneal fluid. To evaluate the overall performance of the approach we track the behavior of 20 validated markers across these experiments. METHODOLOGY: Mass spectrometry based quantitative proteomics following extensive protein fractionation was used to compare serum of women with serous ovarian cancer to healthy women and women with benign ovarian tumors. Quantitation was achieved by isotopically labeling cysteine amino acids. Label-free mass spectrometry was used to compare peritoneal fluid taken from women with serous ovarian cancer and those with benign tumors. All data were integrated and annotated based on whether the proteins have been previously validated using antibody-based assays. FINDINGS: We selected 54 quantified serum proteins and 358 peritoneal fluid proteins whose case-control differences exceeded a predefined threshold. Seventeen proteins were quantified in both materials and 14 are extracellular. Of 19 validated markers that were identified all were found in cancer peritoneal fluid and a subset of 7 were quantified in serum, with one of these proteins, IGFBP1, newly validated here. CONCLUSION: Proteome profiling applied to symptomatic ovarian cancer cases identifies a large number of up-regulated serum proteins, many of which are or have been confirmed by immunoassays. The number of currently known validated markers is highest in peritoneal fluid, but they make up a higher percentage of the proteins observed in both serum and peritoneal fluid, suggesting that the 10 additional markers in this group may be high quality candidates.
Subject(s)
Ascitic Fluid/metabolism , Blood Proteins/metabolism , Ovarian Neoplasms/blood , Proteomics , Up-Regulation , Enzyme-Linked Immunosorbent Assay , Female , Humans , Insulin-Like Growth Factor Binding Protein 1/metabolism , Mass Spectrometry , Ovarian Neoplasms/metabolismABSTRACT
BACKGROUND: Strategies to discover circulating protein markers of ovarian cancer are urgently needed. We developed a novel technology that permits us to isolate recombinant antibodies directed against the potential serum biomarkers, to facilitate the further development of affinity reagents necessary to construct diagnostic tests. METHODS: This study presents a novel discovery approach based on serum immunoprecipitation with cancer-specific in vivo biotinylated recombinant antibodies (biobodies) derived from differentially selected yeast-display scFv, and analysis of the eluted serum proteins by electrophoresis and/or mass spectrometry. RESULTS: Using this strategy we identified catabolic fragments of complement factors, EMILIN2, Von Willebrand factor and phosphatidylethanolamine-binding protein 1 (PEBP1 or RKIP) in patient sera. To our knowledge, this is the first report of a soluble form of PEBP1 in human. Independent evidence for ovarian cancer-specific expression of PEBP1 in patient sera was found by ELISA assays and antibody arrays with anti-PEBP1 antibodies. PEBP1 was detected in 29 out of 30 ascites samples and discriminated ovarian cancer sera from controls (p = 0.02). Finally, we confirmed by western blots the presence of a 21-23 kDa fragment corresponding to the expected size of PEBP1 but we also showed additional bands of 38 kDa and 50-52 kDa in various tissues and cell lines. CONCLUSION: We conclude that the novel strategy described here allows the identification of candidate biomarkers that can be variants of normally expressed proteins or that display cancer-specific post-translational modifications.
Subject(s)
Antibodies/immunology , Biomarkers, Tumor/blood , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/genetics , Yeasts , Antibodies/genetics , Biotinylation , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique, Indirect , Glycoproteins/blood , Humans , Neoplasm Staging , Ovarian Neoplasms/pathology , Phosphatidylethanolamine Binding Protein/blood , Phosphatidylethanolamine Binding Protein/genetics , Recombinant Proteins/immunology , Reproducibility of Results , Sensitivity and Specificity , Solubility , von Willebrand Factor/immunologyABSTRACT
Preventing peritoneal implantation of carcinoma cells could prolong ovarian cancer patient remission and survival. Peritoneal cells constitutively express mesothelin, a ligand for CA125 that is expressed by tumor cells. Thus blocking CA125/mesothelin-dependent cell attachment may prevent or delay peritoneal metastatic recurrence. We developed a high-throughput screening system for reagents able to block CA125/mesothelin-dependent cell attachment with a sensitive quantitative readout. Using a novel yeast-expression system to produce secreted, in vivo biotinylated proteins and biobodies (Bbs), we generated anti-mesothelin Bbs. Anti-mesothelin Bbs derived from high affinity yeast-display scFv detected both membrane-bound and soluble mesothelins and inhibited CA125/mesothelin-dependent cell attachment.
