Author(s): Haun RS, Fan CY, Mackintosh SG, Zhao H, Tackett AJ
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Abstract BACKGROUND: The development of novel targeted cancer therapies and/or diagnostic tools is dependent upon an understanding of the differential expression of molecular targets between normal tissues and tumors. Many of these potential targets are cell-surface receptors; however, our knowledge of the cell-surface proteins upregulated in pancreatic tumors is limited, thus impeding the development of targeted therapies for pancreatic cancer. To develop new diagnostic and therapeutic tools to specifically target pancreatic tumors, we sought to identify cell-surface proteins that may serve as potential tumor-specfic targets. METHODS: Membrane glycoproteins on the pancreatic cancer cell lines BxPC-3 were labeled with the bifunctional linker biocytin hydrazide. Following proteolytic digestion, biotinylated glycopeptides were captured with streptavidin-coupled beads then released by PNGaseF-mediated endoglycosidase cleavage and identified by liquid chromatography-tandem mass spectrometry (MS). A protein identified by the cell-surface glycoprotein capture procedure, CD109, was evaluated by western analysis of lysates of pancreatic cancer cell lines and by immunohistochemistry in sections of pancreatic ductal adenocarcinoma and non- neoplastic pancreatic tissues. RESULTS: MS/MS analysis of glycopeptides captured from BxPC-3 cells revealed 18 proteins predicted or known to be associated with the plasma membrane, including CD109, which has not been reported in pancreatic cancer. Western analysis of CD109 in lysates prepared from pancreatic cancer cell lines revealed it was expressed in 6 of 8 cell lines, with a high level of expression in BxPC-3, MIAPaCa-2, and Panc-1 cells. Immunohistochemical analyses of human pancreatic tissues indicate CD109 is significantly overexpressed in pancreatic tumors compared to normal pancreas. CONCLUSIONS: The selective capture of glycopeptides from the surface of pancreatic cancer cell lines can reveal novel cell-surface glycoproteins expressed in pancreatic ductal adenocarcinomas.
This article was published in J Proteomics Bioinform
and referenced in Journal of Proteomics & Bioinformatics