Comprehensive Analysis of Protein N‑Glycosylation Sites by Combining Chemical Deglycosylation with LC–MS

Glycosylation
is one of the most important protein modifications
in biological systems. It plays a critical role in protein folding,
trafficking, and stability as well as cellular events such as immune
response and cell-to-cell communication. Aberrant protein glycosylation
is correlated with several diseases including diabetes, cancer, and
infectious diseases. The heterogeneity of glycans makes comprehensive
identification of protein glycosylation sites very difficult by MS
because it is challenging to match mass spectra to peptides that contain
different types of unknown glycans. We combined a chemical deglycosylation
method with LC–MS-based proteomics techniques to comprehensively
identify protein N-glycosylation sites in yeast. On the basis of the
differences in chemical properties between the amide bond of the N-linkage
and the glycosidic bond of the O-linkage of sugars, O-linked sugars
were removed and only the innermost N-linked GlcNAc remained, which
served as a mass tag for MS analysis. This chemical deglycosylation
method allowed for the identification of 555 protein N-glycosylation
sites in yeast by LC–MS, which is 46% more than those obtained
from the parallel experiments using the Endo H cleavage method. A
total of 250 glycoproteins were identified, including 184 membrane
proteins. This method can be extensively used for other biological
samples.