Bio-Organic Biomedical Mass Spectromy Resource

描述(由申请人提供)：NIH P41RR001614的这一修订版请求支持，以扩大我们新型化学交联剂的开发，以满足BioDrive对集成的、稳健的方法的新需求，这种方法将产生距离限制，用于对更大的蛋白质复合体和机器的体系结构进行建模。新的生物驱动项目名为“RNA聚合酶II转录起始复合体的结构”，主要研究员是斯坦福大学的罗杰·科恩伯格。我们的计划是以我们最近成功的研发演示为基础，证明赖氨酸侧链上具有e-氨基功能的芳香二醛的还原胺化反应实际上非常适合通过使用Velos Orbitrap平台的电子转移解离(ETD)质谱学进行序列和结构表征。实际上完整的序列是从ETD光谱中获得的，这两种多肽都在交联体中。此外，通过选择用于ETD光谱记录的前体电荷态(低于+4的消除态)来实现多肽交联物的气相浓缩，这是可能的，因为还原胺化生成两个新的仲胺，它们在电喷雾电离中质子化。科恩伯格的实验室拥有高度纯化的复合体，分子大小从大约30种蛋白质到100多种蛋白质不等。利用这一系列具有挑战性的相关复合体，我们将能够优化和利用我们新的综合实验战略的主要固有优势(试剂、交联和后续消化的最佳条件、ETD MS以及促进和加速进展的新软件和生物信息学工具)。一旦使用我们新的集成策略优化了健壮的协议，这种能力将被输出到生物医学研究社区。几乎不计其数的这种蛋白质复合体存在，它们在人类和其他细胞中执行一系列过程和功能。例如，我们刚刚报告了艾滋病毒-人类宿主蛋白相互作用组的特征，这提供了近500个复合体的证据(Jaeger等人，2012年自然，481365-370，DOI：10.1038/Nature10719)。

DESCRIPTION (provided by applicant): This revision of NIH P41RR001614 requests support to expand our development of a new class of chemical cross-linking reagents to address new Biodriver needs for integrated, robust methodology that will yield distance constraints for modeling of the architecture of much larger protein complexes and machines. The new Biodriver Project is entitled, "Architecture of RNA polymerase II Transcription Initiation Complexes," the Principal Investigator is Roger Kornberg of Stanford University. Our plan is to build upon our recent successful TR&D demonstration that reductive amination of aryldialdehydes with e-aminofunctions on lysine side chains is virtually ideally suited to sequence and structural characterization by electron transfer dissociation (ETD) mass spectrometry using the Velos Orbitrap platform. Virtually complete sequences are obtained from ETD spectra for BOTH peptides in the cross-linked species. Also gas phase enrichment of peptide cross-links Is achieved by selection of precursor charge state (eliminate states below +4) for ETD spectral recording, this is possible because reductive amination generates two new secondary amines that are protonated in electrospray ionization. Kornberg's laboratory has highly purified complexes that range in molecular size from some 30 proteins to more than 100. Taking advantage of this challenging array of related complexes, we will be able to optimize and exploit the major inherent advantages of our new integrated experimental strategy (reagents, optimal conditions for cross-linking and subsequent digestion, ETD MS and new software and bioinformatics tools to facilitate and accelerate progress). Once a robust protocol is optimized using our new integrated strategy, this capability will be exported to the biomedical research community. Almost untold numbers of such protein complexes exist that carry out a host of processes and functions in human and other cells. For example, we have just reported the characterization of the HIV-human host protein interactome landscape that provides evidence of almost 500 complexes (Jaeger et al., 2012 Nature, 481, 365-370,doi:10.1038/nature10719).