Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS

Proteomics
Ying XuBenjamin L Schulz

Abstract

Asparagine-linked glycosylation is a common post-translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site-specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH-MS to enable automated measurement of site-specific occupancy at many glycosylation sites. Deglycosylation with peptide-endoglycosidase H, leaving a remnant N-acetylglucosamine on asparagines previously carrying high-mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide-N-glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site-specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase....Continue Reading

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Citations

Jan 6, 2016·Mass Spectrometry Reviews·Han HuJoseph Zaia
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Feb 27, 2019·ACS Chemical Neuroscience·Charmaine EnculescuBenjamin L Schulz
Sep 1, 2021·Bioanalysis·Shulei Liu, Benjamin L Schulz

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