title: N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry.
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.
authors: Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences , Dalian 116023, China.
authors: Liwei
authors: L
authors: Cao
authors: Long
authors: L
authors: Yu
authors: Zhimou
authors: Z
authors: Guo
authors: Aijin
authors: A
authors: Shen
authors: Yunü
authors: Y
authors: Guo
authors: Xinmiao
authors: X
authors: Liang
mesh_terms: D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry
chemical_list: D006023:Glycoproteins; D010455:Peptides; D011134:Polysaccharides; D029701:Saccharomyces cerevisiae Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase