Characterization of Cell Surface Glycoproteins Using Enzymatic Treatment and Mass Spectrometry

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