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J Proteome Res. 2014 Mar 7;13(3):1485-93. doi: 10.1021/pr401049e. Epub 2014 Feb 21.

N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry.

Author information

1
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.

Abstract

N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.

PMID:
24527708
DOI:
10.1021/pr401049e
[Indexed for MEDLINE]

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