Glycosylation is the process by which oligosaccharides, termed glycans, are appended onto membrane and secreted proteins and lipids. It is the most common and complex form of post-translational modification, with ϳ50% of all eukaryotic proteins glycosylated (1, 2). A majority of glycans are found on the cell surface, where they are optimally poised to be the first cellular components encountered by approaching cells, pathogens, antibodies, or other molecules, as well as advertise information about the internal state and homeostasis of the cell (3, 4). Therefore, glycans play an essential role in many biological processes, including cell development and differentiation, cell-cell or cell-matrix communication, and pathogen-host recognition (3,(5)(6)(7). In fact, differences in glycan profiles between healthy and diseased states are utilized for clinical diagnosis (7), providing targets for many novel classes of therapeutics including cancer chemotherapy, diabetes treatment, and antibiotic and anti-viral medicine (5, 8). Glycans are highly heterogeneous in nature, varying in the composition of individual monosaccharide building blocks, the positions with which these building blocks link to each other, and the stereochemical disposition of the linkages (␣ or ␤). This complexity has presented a significant challenge for obtaining structural information about glycans at the molecular From the

Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.

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