GPQuest: A Spectral Library Matching Algorithm for Site-Specific Assignment of Tandem Mass Spectra to Intact N-glycopeptides.
GPQuest: A Spectral Library Matching Algorithm for Site-Specific Assignment of Tandem Mass Spectra to Intact N-glycopeptides.
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DOI：
10.1021/acs.analchem.5b00024
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发表时间：
2015
影响因子：
7.4
通讯作者：
Zhang H
中科院分区：
文献类型：
--
作者：
Toghi Eshghi S;Shah P;Yang W;Li X;Zhang H
在生物学或病理学过程中，糖蛋白的变化不仅发生在蛋白质丰度上，而且发生在每个糖基化位点被不同糖型占据的情况下。质谱仪器和技术的最新进展通过允许用户生成具有连接到每个特定糖基化位点的聚糖的完整糖肽的光谱，促进了复杂生物样品中完整糖肽的分析。然而，分配这些光谱，导致识别的糖肽，是具有挑战性的。在这里，我们报告了一个算法，命名为GPQuest，用于位点特异性识别完整的糖肽，使用更高的能量碰撞解离（HCD）碎片的复杂样品。在该算法中，通过使用HCD LC-MS/MS分析分离的含糖肽，建立样品中含糖肽的光谱库。通过使用聚糖氧鎓离子作为糖肽光谱的特征离子，选择完整糖肽的光谱。然后将这些含氧鎓离子的光谱与含糖基肽产生的光谱库进行比较，从而将每个完整糖肽MS/MS光谱分配给特定的含糖基肽。通过将完整糖肽的前体离子与含糖位点的肽之间的质量差与聚糖数据库匹配来确定占据每个糖位点的聚糖。使用GPQuest，我们分析了前列腺肿瘤LNCaP细胞蛋白提取物的LC-MS/MS光谱。在没有从整体胰蛋白酶肽富集糖肽的情况下，在1%的错误发现率下，1008个含聚糖的MS/MS谱被分配给769个独特的完整N-连接糖肽，代表344个N-连接糖位点和57个不同的N-聚糖。使用GPQuest的光谱库匹配以自动化和高通量方式分配HCD LC-MS/MS生成的完整糖肽光谱。此外，光谱库匹配为用户提供了在预定聚糖数据库中可能缺失的特定糖位点上识别新型或修饰聚糖的可能性。
Glycoprotein changes occur in not only protein abundance but also the occupancy of each glycosylation site by different glycoforms during biological or pathological processes. Recent advances in mass spectrometry instrumentation and techniques have facilitated analysis of intact glycopeptides in complex biological samples by allowing the users to generate spectra of intact glycopeptides with glycans attached to each specific glycosylation site. However, assigning these spectra, leading to identification of the glycopeptides, is challenging. Here, we report an algorithm, named GPQuest, for site-specific identification of intact glycopeptides using higher-energy collisional dissociation (HCD) fragmentation of complex samples. In this algorithm, a spectral library of glycosite-containing peptides in the sample was built by analyzing the isolated glycosite-containing peptides using HCD LC-MS/MS. Spectra of intact glycopeptides were selected by using glycan oxonium ions as signature ions for glycopeptide spectra. These oxonium-ion-containing spectra were then compared with the spectral library generated from glycosite-containing peptides, resulting in assignment of each intact glycopeptide MS/MS spectrum to a specific glycosite-containing peptide. The glycan occupying each glycosite was determined by matching the mass difference between the precursor ion of intact glycopeptide and the glycosite-containing peptide to a glycan database. Using GPQuest, we analyzed LC-MS/MS spectra of protein extracts from prostate tumor LNCaP cells. Without enrichment of glycopeptides from global tryptic peptides and at a false discovery rate of 1%, 1008 glycan-containing MS/MS spectra were assigned to 769 unique intact N-linked glycopeptides, representing 344 N-linked glycosites with 57 different N-glycans. Spectral library matching using GPQuest assigns the HCD LC-MS/MS generated spectra of intact glycopeptides in an automated and high-throughput manner. Additionally, spectral library matching gives the user the possibility of identifying novel or modified glycans on specific glycosites that might be missing from the predetermined glycan databases.