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。质谱法（MS）是获得这些信息的金标准技术。串联质谱（MS/MS）是其核心组成部分。电子转移解离（ETD）是一种相对较新的质谱/质谱解离方法，由于其与先前难以处理的肽/蛋白质类的相容性而引起了极大的兴奋。五年前，m/z范围、质量精度和质量分辨率极大地限制了ETD的应用。我们最初的RO1方案通过将ETD与orbitrap质量分析仪耦合，成功地消除了这一限制。所得到的系统常规分析多肽和蛋白质，有或没有不稳定的PTMs，具有高保真读数（orbitrap）。结果，它实现了许多我们预期的结果，并创造了许多无法预见的机会。仅在PI的实验室中，后一组包括基于数据的解离方法选择（即决策树），z型离子独特化学成分的发现，使用ETD试剂的内部光谱校准，活化离子ETD以及一些生物学应用。到2008年，我们的技术的商业应用开始覆盖全球的研究人员，到目前为止已经接近300人，这使得我们能够解决许多以前难以解决的问题，例如绘制精氨酸甲基化位点，增加低分子量蛋白质的覆盖范围，提供明确的PTM位点分配，以及筛选糖肽库等。我们详细介绍了两项新目标，这些目标建立在我们最初筹资期的高影响力成果的基础上。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？我们将继续在3.5年前描述的广泛使用的ETD-orbitrap平台上进行仪器、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. PUBLIC HEALTH RELEVANCE: Cutting edge MS based technology, Electron transfer dissociation (ETD), continues to be developed. This new MS/MS dissociation method enables previously intractable peptide/protein classes to be sequenced and identified, have their sites of post-translational modification (PTM) mapped, and assess their abundances. This is central to modern biology and has relevance for research ranging from human disease to evolution.