Sequential Ion/Ion Reactions for Large Peptide and Whole Protein Characterization

描述（由申请人提供）：对蛋白质进行测序和鉴定、绘制其翻译后修饰（PTM）位点并评估其丰度的能力是现代生物学的核心。质谱（MS）是获得这些信息的金标准技术。串联质谱（MS/MS）是主要组成部分。电子转移解离（ETD），一个相对较新的MS/MS解离方法，产生了显着的兴奋与以前棘手的肽/蛋白质类的兼容性。五年前，m/z范围，质量精度和质量分辨率大大限制了ETD的应用。我们最初的RO 1提案成功地消除了ETD耦合到轨道阱质量分析器的限制。由此产生的系统常规分析肽和蛋白质，有和没有不稳定的PTM，具有高保真度读出（orbitrap）。结果，它实现了我们预期的许多结果，并创造了许多意想不到的机会。仅在PI的实验室中，后一组包括解离方法的数据依赖性选择（即，决策树），发现z型离子的独特化学组成，使用ETD试剂的内部光谱校准，活化离子ETD和几种生物应用。到2008年，我们的技术的商业实施开始覆盖全球的研究人员-迄今为止已有近300人-能够解决许多以前难以解决的问题，例如绘制Arg甲基化位点，增加低分子量蛋白质的覆盖范围，提供明确的PTM位点分配，以及筛选糖肽文库等。我们详细介绍了两个新的目标，建立在我们最初的融资期的高影响力的结果。目标1，我们如何扩大ETD在生物医学研究中的应用？目的2，气相纯化在定量蛋白质组学中的作用是什么？我们继续在我们3.5年前描述的广泛使用的ETD轨道阱平台上构建仪器，方法，信息和应用项目的平衡。

DESCRIPTION (provided by applicant): The ability to sequence and identify proteins, map their sites of post-translational modification (PTM), and assess their abundances is central to modern biology. Mass spectrometry (MS) is the gold standard technology by which this information is obtained. Serving as the centerpiece, tandem MS (MS/MS) is a principal component. Electron transfer dissociation (ETD), a relatively new MS/MS dissociation method, has generated significant excitement for its compatibility with previously intractable peptide/protein classes. Five years ago m/z range, mass accuracy, and mass resolution considerably restricted the application of ETD. Our initial RO1 proposal successfully eliminated this limitation by coupling ETD to the orbitrap mass analyzer. The resulting system routinely analyzes peptides and proteins, with and without labile PTMs, with a high-fidelity readout (orbitrap). As a result, it realized many of our anticipated outcomes and created numerous unforeseen opportunities. Just in the PI's laboratory, the latter set includes data-dependent selection of dissociation method (i.e., Decision Tree), discovery of the unique chemical compositions of z-type ions, internal spectral calibration using ETD reagents, activated-ion ETD, and several biological applications. By 2008, the commercial implementation of our technology began to reach researchers across the globe-nearly 300 to date-enabling access to numerous previously intractable problems such as mapping Arg methylation sites, increasing coverage of low molecular weight proteins, providing unambiguous PTM site assignment, and screening glycopeptide libraries, among many others. We detail two new aims that build upon the high impact results of our initial funding period. Aim 1, how do we broaden the utility of ETD for biomedical research? Aim 2, what is the role of gas- phase purification in quantitative proteomics? We continue with a balance of instrumentation, method, informatic, and applied projects constructed upon the widely used ETD-orbitrap platform we described 3.5 years ago.