Glycoproteomic Analysis of Malignant Ovarian Cancer Ascites Fluid Identifies Unusual Glycopeptides

Abstract

Ovarian cancer is a major cause of cancer mortality among women, largely due to late diagnosis of advanced metastatic disease. More extensive molecular analysis of metastatic ovarian cancer is needed to identify post-translational modifications of proteins, especially glycosylation that is particularly associated with metastatic disease to better understand the metastatic process and identify potential therapeutic targets. Glycoproteins in ascites fluid were enriched by affinity binding to lectins (ConA or WGA) and other affinity matrices. Separate glycomic, proteomic, and glycopeptide analyses were performed. Relative abundances of different N-glycan groups and proteins were identified from ascites fluids and a serum control. Levels of biomarkers CA125, MUC1, and fibronectin were also monitored in OC ascites samples by Western blot analysis. N-Glycan analysis of ascites fluids showed the presence of large, highly fucosylated and sialylated complex and hybrid glycans, some of which were not observed in normal serum. OC ascites glycoproteins, haptoglobin, fibronectin, lumican, fibulin, hemopexin, ceruloplasmin, alpha-1-antitrypsin, and alpha-1-antichymotrypsin were more abundant in OC ascites or not present in serum control samples. Further glycopeptide analysis of OC ascites identified N-and O-glycans in clusterin, hemopexin, and fibulin glycopeptides, some of which are unusual and may be important in OC metastasis.

Multiple Reaction Monitoring for the Quantitation of Serum Protein Glycosylation Profiles: Application to Ovarian Cancer

“…Furthermore, since IgG and IgA are among the most abundant glycosylated proteins in ascites fluid [23], N-glycans attached to these proteins are likely a source of the most abundant N-glycans being measured in total N-glycan analysis of OC ascites fluid as previously reported [24] . These results demonstrate our ability to identify N-glycans attached to some of the major sources of N-glycosylation (IgG and IgA) in OC ascites.…”

“…Trypsin digestion was performed as previously described [12]. After trypsin digestion and clean-up (ZipTip, Omix C18 100 ml tips, Agilent, Santa Clara CA), peptides were lyophilized.…”

Ovarian Cancer Dissemination—A Cell Biologist’s Perspective

“…EOC dissemination rarely follows an invasion-metastasis cascade where single cells or collective cell populations break through the basal lamina, penetrate surrounding tissues, and intravasate into the vasculature [26,27]. EOC can form loosely attached outgrowths that extend the apical boundary of the tissue mucosa [28].…”

“…One of the characteristics of ascites-derived tumors is their high degree of cell-to-cell adhesion, cell surface deposition of ECM, and integrin activation [40,59,135]. Ascites contain soluble ECM [136,137], and EOC deposits ECM on cell surfaces [27,39,[59][60][61], suggesting that, in the suspended environment of a peritoneal cavity, EOC cells engage integrins to organize ECM adhesion and, thus, suppress cell death associated with anchorage deprivation (anoikis). ECM adhesion is required for growth factor-mediated signaling [138].…”

Ascitic Fluid in Ovarian Carcinoma – From Pathophysiology to the Treatment

“…In malignant ascites also are an abundance of other biochemical components, including N, haptoglobin, fibronectin, lumican, fibulin, hemopexin, ceruloplasmin, alpha-1-antitrypsin, alpha-1-antichymotrypsin, and clusterin, hemopexin, and fibulin glycopeptides [89].…”