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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Carbohydrate Sequence
  • Chromatography, Liquid / methods
  • Glycoproteins / analysis*
  • Glycoproteins / chemistry
  • Glycosylation
  • Hydrolysis
  • Hydrophobic and Hydrophilic Interactions
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / chemistry
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / chemistry
  • Peptides / analysis*
  • Polysaccharides / analysis*
  • Polysaccharides / chemistry
  • Protein Processing, Post-Translational*
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / analysis*
  • Saccharomyces cerevisiae Proteins / chemistry

Substances

  • Glycoproteins
  • Peptides
  • Polysaccharides
  • Saccharomyces cerevisiae Proteins
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase