N-Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid Chromatography-Based Enrichment, Parallel Deglycosylation, and Mass Spectrometry
N-Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid Chromatography-Based Enrichment, Parallel Deglycosylation, and Mass Spectrometry
复制标题
使用亲水相互作用液相色谱富集、平行去糖基化和质谱法对酿酒酵母蛋白质进行 N-糖基化位点分析
DOI：
10.1021/pr401049e
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发表时间：
2014-03-01
影响因子：
4.4
通讯作者：
Liang, Xinmiao
中科院分区：
文献类型：
--
作者：
Cao, Liwei;Yu, Long;Liang, Xinmiao
对酿酒酵母N-糖基化位点的分析对阐明人类先天性糖基化紊乱的分子机制具有重要意义。在这里，我们提出了一种基于质谱学(MS)的工作流程，用于分析酿酒酵母蛋白质中的N-糖基化位点。在这个工作流程中，通过使用一种名为Click TE-Cys的亲水性材料来丰富蛋白质水解性糖肽，以提高糖肽的选择性和覆盖率。为了提高鉴定结果的可靠性，在LC-MS/MS分析之前，分别使用两种内切糖苷酶(即PNGase F和Endo H-f)对富含糖肽的糖肽进行平行脱糖。在工作流程的基础上，共鉴定了135个N-糖基化位点，其中包括6个已知位点、93个潜在位点和36个新位点，并将其定位于79个蛋白质。在新类型的位点中，通过PNGase F和Endo H-f脱糖基化同时鉴定的8个蛋白质中的9个位点被认为具有高度的置信度。所建立的工作流程，以及N-糖基化位点的描述，将有助于改进酿酒酵母模型以揭示CDG的发病机制。
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 H-f), 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 H-f 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.