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

描述（由申请人提供）：也许蛋白质质谱中最具影响力的概念之一是酶蛋白消化的概念，以提供适合常规串联质谱（碰撞激活，CAD）的肽集合毫无疑问，这种方法使全球蛋白质鉴定取得了重大进展；然而，许多研究者现在意识到这种方法有很大的局限性这一结论是基于以下观察得出的：首先，蛋白翻译后修饰（PTMs）在多结构域蛋白上以及蛋白-蛋白机器的组成部分之间协同工作；为了确定它们的生物学相关性，这些模式必须在彼此的背景下（在整个蛋白质中）进行检测其次，转录编辑过程在高等真核生物中普遍存在，即使基因组完全测序也难以预测。例如，3/4的人类蛋白质预计至少有1个剪接变异4-6，这些变异可能包含内含子序列。跳过密码子、移框、基因融合和单核苷酸多态性（snp）也会发生。因此，使用短肽作为基因的代理标记是不充分的，而且往往具有误导性。与CAD不同，电子转移解离（ETD）是由PI共同发明的一种新的碎片化技术，不需要短肽进行成功的序列分析（即胰蛋白酶消化）。ETD与肽长度或PTMs的存在无关，在允许与色谱偶联的时间尺度上进行，并且可以与其他离子/离子反应偶联。本提案旨在开发一套核心离子/离子反应工具，并在混合-

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-