Gas-phase Bio-conjugation in the Tandem Mass Spectrometry of Peptides Proteins

这项研究倡议的总体主旨是为结构设计开发新的和改进的方法 与生物医学相关的离子的特征，特别是由多肽形成的离子 从酶或化学消化、整个蛋白质和蛋白质复合体中提取。我们的目标是 开发串联质谱学的新方向，使一系列新的策略能够处理 存在生物分子鉴定/表征问题。绝大多数串联质谱学 实验依赖于碎片作为关键的结构信息反应。在这项工作中，我们试图增加 在MSN的背景下对气体离子进行选择性共价或非共价衍生的能力 做实验。特定的气相化学反应的发展从根本上能够实现 具有非常广泛的新颖应用。就像溶液中的生物共轭化学已经成为 分子生物学研究界在广泛的测量策略中广泛使用 实现气相生物共轭的能力将使许多新的结构表征方法成为可能。 气相法的有趣特征之一是它速度快(即，几十毫秒的时间刻度) 而且它完全避免了样本处理问题。这项工作将涉及小说的发展 选择性反应，其中许多将受到丰富的生物偶联文献的启发，发展 基于多肽离子的应用，以及基于整体蛋白质/蛋白质络合物离子的应用的开发， 具体目标体现为： 具体目标1：发展基于选择性类酶的自上而下的蛋白质表征方法 早些年发现的官能团特定的离子/离子反应导致的碎裂 项目。 具体目标2：开发选择性离子/离子反应在多肽和MSN中的应用 蛋白质离子用于鉴定/表征两种基因的亚化学计量比翻译后修饰 自下而上和自上而下的蛋白质组学场景。 具体目标3：开发利用离子/离子表征蛋白质和蛋白质复合体的新方法 电荷转移策略。这些方法将应用于蛋白质复合体和大型生物 复合体(如病毒)。

The overall thrust of this research initiative is to develop new and improved means for the structural characterization of ions of biomedical relevance, with particular emphasis on those formed from peptides derived from enzymatic or chemical digestion, whole proteins, and protein complexes. The objective is to develop a new direction in tandem mass spectrometry that will enable a wide range of novel strategies to deal with biomolecule identification/characterization problems. The vast majority of tandem mass spectrometry experiments rely on fragmentation as the key structurally informative reaction. In this work, we seek to add the capability for selective covalent or non-covalent derivatization of gaseous ions within the context of an MSn experiment. The development of specific gas-phase chemistries fundamentally enables the implementation of a very wide range of novel applications. Just as bio-conjugation chemistries in solution have become broadly used by the molecular biology research community in a wide range of measurement strategies, the ability to effect gas-phase bio-conjugation will enable many novel approaches to structural characterization. Among the interesting features of the gas-phase approach are that it is fast (i.e., tens of milliseconds timescale) and it completely avoids sample manipulation issues. The work will involve the development of novel selective reactions, many of which will be inspired by the rich bio-conjugation literature, the development of peptide ion-based applications, and the development of whole protein/protein complex ion-based applications, as reflected in the specific aims: Specific Aim 1: Develop top-down protein characterization methods based on selective "enzyme-like" fragmentation enabled by the functional group specific ion/ion reactions discovered in the early years of this project. Specific Aim 2: Develop applications for the use of selective ion/ion reactions in the Msn of peptide and protein ions for the identification/characterization of sub-stoichiometric post-translational modifications in both bottom-up and top-down proteomics scenarios. Specific Aim 3: Develop novel approaches for protein and protein complex characterization using ion/ion charge transfer strategies. These approaches will be applied to protein complexes and large biological complexes (e.g., viruses).