Proteomics Analysis of O-GaINAc Glycosylation in Human Serum by an Integrated Strategy
Proteomics Analysis of O-GaINAc Glycosylation in Human Serum by an Integrated Strategy
复制标题
通过综合策略对人血清中 O-GalNAc 糖基化进行蛋白质组学分析。
DOI：
10.1021/acs.analchem.6b02887
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发表时间：
2017-02-07
影响因子：
7.4
通讯作者：
Zou, Hanfa
中科院分区：
文献类型：
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作者：
Qin, Hongqiang;Cheng, Kai;Zou, Hanfa
O-连接聚糖结构的多样性由于其重要的生物学作用而引起越来越多的关注。然而，具有不同聚糖的完整O-糖肽通常不能使用当前方法很好地阐明。在这项工作中，开发了一种集成的策略，通过结合亲水相互作用色谱（HILIC）尖端富集，梁型碰撞诱导分解（beam-CID）检测，和在电脑去糖基化方法光谱解释的O-GalNAc糖基化的综合分析。在该策略中，选择性富集完整的O-GalNAc糖肽，并使用计算机去糖基化方法对通过飞行时间（TOF）-CID获得的原始光谱进行预处理，从而能够直接搜索而无需设置多个糖基化修饰，这可以显著减少搜索空间。应用该策略分析了人血清中O-GalNAc糖蛋白组，从93种糖蛋白中鉴定出407种完整的O-GalNAc糖肽。约81%的糖肽含有至少一个唾液酸，这可能揭示了O-GalNAc糖基化的微观异质性。到目前为止，这是在蛋白质组水平上来自复杂生物样品的完整O-GalNAc糖型的最大数据集。此外，该方法也适用于其他复杂生物系统中O-糖型异质性的研究。
The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.