Characterization\nof Cell Surface Glycoproteins Using\nEnzymatic Treatment and Mass Spectrometry

Almost all proteins on the cell surface are modified\nby glycosylation.\nCell surface glycoproteins participate in various cellular pathways,\nsuch as cell adhesion, cell–cell communication, and immune\nresponse. Due to their functional importance, glycoproteins on the\ncell surface often serve as potential therapeutic targets. Recent\nadvancements in mass spectrometry (MS) have facilitated the characterization\nof glycoproteins that are generally localized on the cell surface,\nsecreted to the extracellular environment, or found in intracellular\norganelles such as the endoplasmic reticulum, Golgi apparatus, and\nperoxisome. However, the selective characterization of glycoproteins\non the cell surface remains challenging. In this study, we applied\nenzymatic treatment to live cells, followed by MS-based glycoproteomics\nanalysis, to assess changes in protein glycosylation at different\ntreatment time points as a method to identify cell surface glycoproteins.\nTo demonstrate this approach, a renal cell carcinoma cell line, A498,\nwas treated with glycosidases, sialidase and PNGase F, over two treatment\ntime intervals, 2 and 24 h. Glycoproteins were identified as cell\nsurface glycoproteins from A498 cells when enzyme treatment altered\nthe glycosylation of the glycoproteins. The results revealed the effectiveness\nof integrating enzymatic treatment with MS-based glycoproteomics for\nanalyzing cell surface glycoproteins. Our established method has demonstrated\nthe potential applications for assessing accessibility of therapeutic\ntargets on the cell surface over time and supporting the development\nof new targeted therapies.