Sequential ion/ion reactions for large peptide and whole protein characterization

描述(申请人提供)：蛋白质质谱学中最有影响力的概念之一可能是酶促蛋白质消化的概念，以提供适合传统串联质谱学(碰撞激活，CAD)的大小的多肽集合。1毫无疑问，这种方法在全球蛋白质鉴定方面取得了重大进展；然而，许多研究人员现在认识到这种方法有很大的局限性。2这一结论基于以下观察：第一，蛋白质翻译后修饰(PTM)在多结构域蛋白质上以及蛋白质-蛋白质机器的组件之间协同工作；为了确定它们的生物学相关性，必须在彼此(整个蛋白质)的背景下检测这些模式。3其次，转录编辑过程在高等真核生物中普遍存在，即使在完全测序的基因组中也很难预测。例如，人类所有蛋白质中的3/4预计至少有1个剪接变种，4-6个变种可能包含内含子序列。还会发生密码子跳过、移码、基因融合和单核苷酸多态(SNPs)。因此，使用短肽作为基因的代理标记是不够的，而且往往具有误导性。与CAD不同的是，电子转移解离(ETD)是PI共同发明的一种新的碎裂技术，它不需要短肽就能成功进行序列分析(即胰酶消化)。ETD与多肽长度或PTMS的存在无关，在允许与层析耦合的时间尺度上进行，并可与其他离子/离子反应耦合。这项提议旨在开发一套核心离子/离子反应工具，并使它们在混合动力中的使用自动化-

DESCRIPTION (provided by applicant): Perhaps one of the most influential concepts in protein mass spectrometry has been the notion of enzymatic protein digestion to render a collection of peptides of suitable size for conventional tandem mass spectrometry (collisional-activation, CAD).1 Doubtless this methodology has enabled significant progress for global protein identification; however, many investigators now realize this approach has significant limitations.2 This conclusion is based upon the following observations: First, protein posttranslational modifications (PTMs) on multi-domain proteins, and among components of protein-protein machines, work in concert; to determine their biological relevance, these patterns must be detected within the context of one another (across the whole protein).3 Second, transcriptional editing processes are pervasive in higher eukaryotes and difficult to predict, even with a completely sequenced genome. For example, 3/4 of all human proteins are expected to have at least 1 splice variant4-6 - variants that could contain intronic sequences. Skipped codons, frameshifting, gene fusion, and single nucleotide polymorphisms (SNPs) also occur. Thus, the use of short peptides as proxy markers for genes is inadequate and often misleading. Unlike CAD, electron transfer dissociation (ETD), a new fragmentation technique co-invented by the PI, does not require short peptides for successful sequence analysis (i.e., trypsin digestion). ETD is indifferent to peptide length or the presence of PTMs, is performed on a time-scale that permits coupling with chromatography, and can be coupled to other ion/ion reactions. This proposal aims to develop a suite of core ion/ion reaction tools, and automate their use in a hybrid-