pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3.
pGlyco: a pipeline for the identification of intact N-glycopeptides by using HCD- and CID-MS/MS and MS3.
复制标题
pGlyco：使用 HCD-和 CID-MS/MS 和 MS3 鉴定完整 N-糖肽的管道。
DOI：
10.1038/srep25102
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发表时间：
2016-05-03
影响因子：
4.6
通讯作者：
Yang P
中科院分区：
文献类型：
--
作者：
Zeng WF;Liu MQ;Zhang Y;Wu JQ;Fang P;Peng C;Nie A;Yan G;Cao W;Liu C;Chi H;Sun RX;Wong CC;He SM;Yang P
通过鉴定完整的糖肽来确定蛋白质糖基化的微观异质性仍然是糖蛋白组学最艰难的分析挑战之一。最近提出的基于质谱（MS）的方法仍然存在一些缺陷，例如缺乏用于聚糖鉴定的错误发现率（FDR）分析和缺乏用于肽鉴定的足够的片段化信息。本文提出了一种新的利用互补质谱技术鉴定完整糖肽的方法pGlyco：1）利用HCD-MS/MS和产物依赖性CID-MS/MS提供互补片段来鉴定糖肽，并提出了一种新的靶-诱饵方法来估计糖肽鉴定的错误发现率; 2）对HCD-MS/MS的一些最强峰的MS 3的数据依赖性采集用于提供片段以鉴定肽骨架。通过HCD-MS/MS、CID-MS/MS和MS 3的整合，可以可靠地鉴定完整的糖肽。使用pGlyco，在Orbitrap Fusion中分析标准糖蛋白混合物，并鉴定了309种非冗余完整糖肽，具有聚糖和肽的详细光谱信息。
Confident characterization of the microheterogeneity of protein glycosylation through identification of intact glycopeptides remains one of the toughest analytical challenges for glycoproteomics. Recently proposed mass spectrometry (MS)-based methods still have some defects such as lack of the false discovery rate (FDR) analysis for the glycan identification and lack of sufficient fragmentation information for the peptide identification. Here we proposed pGlyco, a novel pipeline for the identification of intact glycopeptides by using complementary MS techniques: 1) HCD-MS/MS followed by product-dependent CID-MS/MS was used to provide complementary fragments to identify the glycans, and a novel target-decoy method was developed to estimate the false discovery rate of the glycan identification; 2) data-dependent acquisition of MS3 for some most intense peaks of HCD-MS/MS was used to provide fragments to identify the peptide backbones. By integrating HCD-MS/MS, CID-MS/MS and MS3, intact glycopeptides could be confidently identified. With pGlyco, a standard glycoprotein mixture was analyzed in the Orbitrap Fusion, and 309 non-redundant intact glycopeptides were identified with detailed spectral information of both glycans and peptides.