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

Xu, Ying, Bailey, Ulla-Maja and Schulz, Benjamin L. (2015) Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS. Proteomics, 15 13: 2177-2186. doi:10.1002/pmic.201400465


Author Xu, Ying
Bailey, Ulla-Maja
Schulz, Benjamin L.
Title Automated measurement of site-specific N-glycosylation occupancy with SWATH-MS
Journal name Proteomics   Check publisher's open access policy
ISSN 1615-9853
1615-9861
Publication date 2015-07-01
Year available 2015
Sub-type Article (original research)
DOI 10.1002/pmic.201400465
Open Access Status Not yet assessed
Volume 15
Issue 13
Start page 2177
End page 2186
Total pages 10
Place of publication Weinheim, Germany
Publisher Wiley - V C H
Language eng
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. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site-specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.
Keyword Glycoproteomics
Glycosylation
Macroheterogeneity
Mass spectrometry
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 631615
APP1031542
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
School of Chemistry and Molecular Biosciences
 
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Created: Fri, 06 Mar 2015, 21:32:23 EST by Mrs Louise Nimwegen on behalf of School of Chemistry & Molecular Biosciences