title: Rapid characterization of N-linked glycosylation site using non-specific protease digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry.
abstract: Rapid identification of glycosylation sites of glycoproteins is urgently needed in glycoproteomics study. In the present work, a rapid and simple method based on non-specific digestion of gel-separated glycoproteins and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry was described, which can efficiently identify the N-linked glycosylation sites. One-step in-gel digestion of Ribonuclease B (RNase B) by proteinase K was employed to generate glycopeptides with short and discrepant peptide composition. When compared with glycopeptides prepared by two-step in gel-digestion using trypsin-proteinase K or trypsin-pronase, the  direct proteinase K treatment showed obvious superiority in both glycopeptide recovery and preparation simplicity. Most importantly, it helps to generate greater variety of glycopeptide series with rich information for glycosylation site identification. In addition, binary matrices 5-chloro-2-mercaptobenzothiazole (CMBT) /2,5-dihydroxybenzoic acid (DHB) were found to form homogeneous microcrystal on the target with the purified glycopeptides, leading to improved detection sensitivity. Thus, the present work provides an optimized solution to speed up the characterization of N-linked glycosylation sites in glycoproteins.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Tingting
authors.initials: T
authors.lastname: Yu
authors.forename: Yuanming
authors.initials: Y
authors.lastname: Luo
authors.forename: Keqian
authors.initials: K
authors.lastname: Yang
mesh_terms: D001665:Binding Sites; D004789:Enzyme Activation; D005782:Gels / Q000737:chemistry*; D006031:Glycosylation; D009584:Nitrogen / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D005782:Gels; D010447:Peptide Hydrolases; D009584:Nitrogen