School of Pharmacy, University of Wisconsin, Madison, WI, 53705, USA.
Department of Surgery, Wisconsin Institutes for Medical Research, Madison, WI, 53705, USA.
J Am Soc Mass Spectrom. 2017 Sep;28(9):1751-1764. doi: 10.1007/s13361-017-1701-4. Epub 2017 Jul 10.
Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization. Graphical Abstract ᅟ.
蛋白质糖基化是最具异质性的翻译后修饰之一，它在细胞信号转导和疾病进展中起着重要作用。传统的基于质谱（MS）的大规模糖蛋白测序研究严重依赖于分别鉴定酶切释放的聚糖及其原始肽骨架，因为没有有效的碎片化方法可以在单个光谱中同时产生无偏的糖肽产物离子，并且可以方便地应用于高通量糖蛋白质组学的特征分析，特别是对于 N-糖肽，其糖侧链分支比相对较少的复杂 O-连接糖更多。在这项研究中，重新定义的电子转移/更高能量碰撞解离（EThcD）碎片化方案被应用于将聚糖和肽片段合并在一个单一的光谱中，从而能够收集完整的信息并揭示出更多的微观异质性细节。胎球蛋白首先被用来证明该方法的适用性，共鉴定了 19 个糖肽和相应的五个糖基化位点。随后的实验测试了它在人血浆 N-糖蛋白中的应用。大规模研究在血管再狭窄进展过程中对大鼠颈动脉的 N-糖蛋白质组学进行了研究，以探讨糖基化的潜在作用。这种集成的碎片化方案为分析完整的 N-糖肽和 N-糖蛋白质组学提供了一个强大的工具。我们还预计这种方法可以很容易地应用于大规模的 O-糖蛋白质组学特征分析。
