Activated-Ion Negative Electron Transfer Dissociation For High-Throughput Glycoproteomics

 描述（由申请人提供）：蛋白质糖基化是一种普遍且重要的超类翻译后修饰（PTM），参与调节多种细胞内和细胞间功能。糖基化占蛋白质组多样性最大的任何其他PTM，因为大约一半的所有表达的蛋白质经历这种修饰;表征糖蛋白质组的复杂结构和功能是推进我们对细胞生物学和疾病的理解不可或缺的。也就是说，人们对哪些聚糖修饰哪些蛋白质以及在哪里修饰蛋白质知之甚少，这表明这一关键PTM存在技术差距。今天的技术依赖于质谱（MS），这是一种高通量和灵敏的技术，能够将修饰定位于单个氨基酸，从而在PTM分析中发挥巨大作用-例如，蛋白质磷酸化的全球研究通常在一个实验中包含数万个位点。然而，检测O-糖基化的方法落后于其他PTM，这是由于几个固有的分析挑战，包括：阳离子电离差、广泛的结构异质性、没有已知的共有序列基序以及缺乏同时进行肽和聚糖表征的方法。我们得出结论，改善O-糖肽分析的最佳方法是从根本上改变我们的分析方法，以更好地与分析物的化学特性配对。具体而言，我们将开发一种负离子模式MS方法，该方法将包含糖肽的酸性，以实现优先电离，而不是歧视性电离。由于缺乏与肽阴离子相容的解离方法，迄今为止这一直是具有挑战性的。然而，在过去的几年里，我们已经开发了ETD的负离子类似物-称为负ETD（NETD）。在这里，我们提出了全球糖蛋白组学分析的NETD的第一个实现。在NETD中，糖肽阴离子被自由基试剂阳离子氧化，促进电子重排，切割C-Ca骨架键以产生a-和x-型产物离子，使聚糖修饰完整用于位点特异性定位。为了提供同时的聚糖结构阐明，我们将NETD与光活化偶联（同时，即，活化离子NETD，AI-NETD），以在单串联MS分析中鉴定完整的糖肽（聚糖和肽）。

 DESCRIPTION (provided by applicant): Protein glycosylation is a prevalent and important super-class of post-translational modifications (PTMs) involved in regulation of a wide array of intra- and inter-cellular functions. Glycosylation accounts for the greatest proteome diversity over any other PTM, as approximately half of all expressed proteins undergo this modification; characterizing the complex structures and functions of the glycoproteome is integral to advancing our understanding of cell biology and disease. That said, strikingly little is known about exactly which glycans modify which proteins and where, indicating a technology gap exists for this key PTM. Today's technology relies upon mass spectrometry (MS), a high-throughput and sensitive technique with the ability to localize modifications to a single amino acid, lending it great power in PTM analysis - global studies of protein phosphorylation, for example, routinely harbor tens of thousands of sites in a single experiment. Methods of detecting O-glycosylation, however, lag behind other PTMs due to several inherent analytical challenges, including: poor ionization as cations, broad structural heterogeneity, no known consensus sequence motif, and a lack of methods for concurrent peptide and glycan characterization. We conclude that the best way to improve O-glycopeptide analysis is to fundamentally change our analytical methods to better pair with the chemical characteristic of the analytes. Specifically, we will develop a negative ion mode MS method that will embrace the acidity of the glycopeptide to achieve preferential, rather than discriminatory, ionization. This has been heretofore challenging, due to a lack of dissociation methods compatible with peptide anions. Over the last several years, however, we have developed the negative ion analog of ETD - termed negative ETD (NETD). Here, we propose the first implementation of NETD for global glycoproteomic analyses. In NETD, glycopeptide anions are oxidized by radical reagent cations, promoting electron rearrangements that cleave the C-Ca backbone bond to produce a¿- and x-type product ions, leaving the glycan modification intact for site-specific localization To provide concurrent glycan structure elucidation we shall couple NETD with photo-activation (simultaneously, i.e., activated-ion NETD, AI-NETD) to enable intact glycopeptide (glycan and peptide) identification in single tandem MS analysis.