Dual Spray Reactor for Biological Mass Spectrometry

描述（由申请人提供）：质谱仪已变得越来越复杂，分析功能强大的仪器，为肽测序和蛋白质组学应用提供了成熟的MS/MS方法。然而，尽管有令人印象深刻的高性能仪器阵列，仍然需要扩展针对目标应用的质谱策略的灵活性以及增强选择性和可调性。该提案的目的是开发一种新的前端方法，用于在引入质谱仪之前操纵离子，从而为串联质谱工作流程增加多功能性。该策略是基于双喷雾反应器，其允许分析物离子与试剂离子或分子的第二群体的交叉，促进在大气压下的气态离子-分子和离子-离子反应。喷雾反应器将促进分析物离子的在线衍生化、基于酸碱化学的电荷状态的操纵以及用于定量和校准目的的内标物的添加。对于自下而上的蛋白质组学应用，双喷雾反应器将通过允许用于操纵电荷状态和化学衍生化的方便手段来扩展激活方法的灵活性。此外，促进选择性反应以添加可裂解的报告标签、连接发色团或方便地添加内标物以提高精度的能力包括对离子化学的前端控制提供实验灵活性的附加维度的一些方式。一系列离子-离子和离子-分子反应将通过双喷雾反应器进行，包括：（i）通过酸/碱化学和添加可电离基团来操纵电荷状态，（ii）基于官能团的选择性反应性，（iii）添加发色团以增强光解离，（iv）连接报告标签以促进通过SRM扫描跟踪离子（数据依赖模式），和（v）注入参考标准以提高运行间精度。自下而上的蛋白质组学应用为扩展现有高性能质谱仪的能力提供了新的范例，并为扩展蛋白质组学应用的标准ESI-MS平台的可调性、灵活性和多功能性提供了巨大的潜力。1 公共卫生相关性（由申请人提供）：串联质谱仪的分析性能和灵敏度的进步使得与人类疾病的起源和进展相关的日益具有挑战性的生物学问题得以解决。拟议的研究将通过利用离子的在线化学操作，扩展高性能质谱仪的灵活性和多功能性，用于大规模和有针对性的蛋白质组学应用。

DESCRIPTION (provided by applicant): Mass spectrometers have become increasingly sophisticated, analytically powerful instruments offering well-established MS/MS methods for peptide sequencing and proteomics applications. However, despite the impressive array of high performance instruments, there remains a need for extending the flexibility of mass spectrometric strategies for targeted applications as well as enhancing both the selectivity and tunability. The objective of this proposal is the development of a new front-end method for manipulation of ions prior to introduction into the mass spectrometer, thus adding versatility to tandem mass spectrometry workflows. The strategy is based on a dual spray reactor that allows intersection of analyte ions with a second population of reagent ions or molecules, promoting gaseous ion-molecule and ion-ion reactions at atmospheric pressure. The spray reactor will facilitate on-line derivatization of analyte ions, manipulation of charge states based on acid-base chemistry, and addition of internal standards for quantitative and calibration purposes. For bottom-up proteomics applications, the dual spray reactor will expand the flexibility of activation methods by allowing a convenient means for manipulation of charge states and chemical derivatization. Morever, the capabilities of promoting selective reactions to add cleavable reporter tags, to attach chromophores, or to conveniently add internal standards to improve precision, comprise some of the ways that front-end control over ion chemistry offers an added dimension of experimental flexibility. An array of ion-ion and ion-molecule reactions will be implemented via the dual spray reactor including: (i) manipulation of charge states via acid/base chemistry and addition of ionizable groups, (ii) selective reactivity based on functional group, (iii) addition of chromophores to enhance photodissociation, (iv) attachment of reporter tags to facilitate tracking ions via SRM scans (data-dependent mode), and (v) infusion of reference standard to improve run-to-run precision. Adaptation for targeted bottom-up proteomics applications offers a new paradigm for extending the capabilities of existing high performance mass spectrometers and affords significant potential for extending the tunability, flexibility, and versatility of the standard ESI-MS platform for proteomics applications. 1 PUBLIC HEALTH RELEVANCE (provided by applicant): Advances in the analytical performance and sensitivity of tandem mass spectrometers have allowed increasingly challenging biological questions related to the origins and progression of human diseases to be addressed. The proposed research will extend the flexibility and versatility of high performance mass spectrometers for broad-scale and targeted proteomics applications by exploiting on-line chemical manipulation of ions.