SWATH-MS glycoproteomics reveals consequences of defects in the glycosylation machinery

Zacchi, Lucia F. and Schulz, Benjamin L. (2016) SWATH-MS glycoproteomics reveals consequences of defects in the glycosylation machinery. Molecular and Cellular Proteomics, 15 7: 2435-2447. doi:10.1074/mcp.M115.056366

Author Zacchi, Lucia F.
Schulz, Benjamin L.
Title SWATH-MS glycoproteomics reveals consequences of defects in the glycosylation machinery
Journal name Molecular and Cellular Proteomics   Check publisher's open access policy
ISSN 1535-9476
Publication date 2016-04-19
Sub-type Article (original research)
DOI 10.1074/mcp.M115.056366
Open Access Status Not Open Access
Volume 15
Issue 7
Start page 2435
End page 2447
Total pages 39
Place of publication Rockville, MD, United States
Publisher American Society for Biochemistry and Molecular Biology
Language eng
Subject 1602 Analytical Chemistry
1303 Biochemistry
1312 Molecular Biology
Abstract Glycan macro- and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro- and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared to wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. While ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3Δ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation.
Keyword Glycoprotein Pathways
Label-free quantification
Q-Index Code C1
Q-Index Status Provisional Code
Grant ID DP160102766
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: HERDC Pre-Audit
School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 13 times in Thomson Reuters Web of Science Article | Citations
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Created: Mon, 25 Apr 2016, 22:59:14 EST by Lucia Zacchi on behalf of School of Chemistry & Molecular Biosciences