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

描述（由申请人提供）：本研究计划的总体目标是开发用于多肽离子结构表征的新的和改进的方法，包括由酶或化学消化产生的肽、完整蛋白质和蛋白质复合物形成的多肽离子。近期的目标是开发一个新的方向，串联质谱，这将使广泛的新的策略来处理蛋白质鉴定/表征问题。绝大多数串联质谱实验依赖于片段化作为关键的结构信息反应。在这项工作中，我们试图添加选择性共价或非共价衍生的气体离子的MSn实验的背景下的能力。特定气相化学的发展从根本上实现了非常广泛的应用。正如溶液中的生物缀合化学已被分子生物学研究界广泛用于广泛的测量策略中一样，实现气相生物缀合的能力将使许多新颖的结构表征方法成为可能。气相方法的有趣特征之一是它是快速的（即，几十毫秒的时间 规模），它完全避免了样品操作问题。这项工作将涉及新的选择性反应的开发，其中许多将受到丰富的生物缀合文献的启发，肽离子应用的开发，以及整个蛋白质/蛋白质复合物离子应用的开发，如具体目标所反映的那样：具体目标1：发现/开发反应性氨基酸侧链的官能团特异性离子/离子反应，包括但不限于赖氨酸（和N-末端），具有酸性侧链的残基（即，谷氨酸和天冬氨酸）和C-末端、精氨酸和半胱氨酸，以及常见的翻译后修饰，如磷酸和硫酸。具体目标2：开发在肽离子的MSn中使用选择性离子/离子反应的应用，以支持基于肽的（即，自下而上）方法进行蛋白质鉴定/表征。具体目标3：开发在完整蛋白质和蛋白质复合物离子的MSn中使用选择性离子/离子反应的应用，以支持基于蛋白质的（即，自上而下）方法进行蛋白质鉴定/表征。 基于选择性气相反应的新型化学和方法学的发展将与生物医学研究的许多领域相关，因为串联质谱法已经在健康相关科学的各个领域发挥作用。本提案中的工作必须集中于蛋白质分析。然而，官能团特异性气相反应也可以导致在其他领域的新应用，如代谢组学，糖组学，脂质组学等。因此，这项工作的潜在生物医学影响是非常广泛的。

DESCRIPTION (provided by applicant): The overall thrust of this research initiative is to develop new and improved means for the structural characterization of polypeptide ions, including those formed from peptides derived from enzymatic or chemical digestion, whole proteins, and protein complexes. The near-term objective is to develop a new direction in tandem mass spectrometry that will enable a wide range of novel strategies to deal with protein 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 development of a very wide range of 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 time scale) 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: Discover/develop functional group specific ion/ion reactions for reactive amino acid side chains, including, but not limited to, lysine (and N-termini), residues with acidic side-chains (i.e., glutamic and aspartic acids) and C-termini, arginine, and cysteine, as well as common post-translational modifications, such as phosphate and sulfate. Specific Aim 2: Develop applications for the use of selective ion/ion reactions in the MSn of peptide ions in support of peptide-based (i.e., bottom-up) approaches in protein identification/characterization. Specific Aim 3: Develop applications for the use of selective ion/ion reactions in the MSn of intact protein and protein complex ions in support of protein-based (i.e., top-down) approaches in protein identification/characterization. The development of novel chemistries and methodologies based on selective gas-phase reactions will be relevant to many areas of bio-medical research by virtue of the role that tandem mass spectrometry already plays across the spectrum of health related science. The work in this proposal must necessarily be focused, and will be directed to protein analysis. However, functional group-specific gas-phase reactions can also lead to new applications in other areas, such as metabolomics, glycomics, lipidomics, etc. The potential bio- medical impact for this work, therefore, is unusually broad.