Development of Multidimensional, Linear and Differential Ion Mobility MS Separations for Middle-Down Proteoforms

项目摘要/摘要 随着蛋白质组学进入第三个十年，初级蛋白质的鉴定和定量 测序已经成为一门成熟的科学。然而，在测绘过程中仍然存在许多分析挑战 蛋白质的翻译后修饰(PTM)，特别是在其天然状态下。例如, 完全破译组蛋白密码需要识别和本地化不同的 使用自上而下策略的PTM。虽然已经取得了一些进展，但传统的LC-MS/MS方法 不能完全分离具有同分异构体PtM位置变体的组蛋白；这导致需要进一步 开发新的、快速和正交的分离方法，可以很容易地与质谱学相结合，用于 “组蛋白密码”的特征。在本项目中，我们的目标是检验这样一种假设：“PTMS 诱导组蛋白的结构变化，使它们能够通过离子的差异进行分离和鉴定 迁移性性质(特别是位置异构体)和碎裂模式“。了解 PTMS对组蛋白结构和功能的影响是表观遗传调控的核心。因此，要启用 在表观遗传学的进一步进展，我们将开发新的多维离子迁移率分离顶部- 向下异构型组蛋白分析。该项目的最终目标是将技术专家聚集在一起 后电离，正交分离和自上而下的质谱学开发了一种综合性的， 多维分析平台，能够使用原生的、例如 与蛋白质降解的组蛋白相反。为实现这一目标，我们将努力实现以下目标： 1)将多级、非线性和线性离子迁移率分离与自上而下的质量相结合 光谱分析(IMSN-MS/MS)；2)开发液相色谱(离线和在线)策略 与IMSN-MS/MS兼容；以及3)评估生物系统中组蛋白PTM的丰度。 LC-MS/MS和LC-IMSN-MS/MS策略。为了支持这些目标，重大技术突破 将在每个综合领域实现：i)非线性IMS(具有3个新的FAIMS几何结构 实施和评估)，ii)线性IMS(具有更高的分辨率、更大的移动范围和更高 实现和评估灵敏度TIMS单元)，以及iii)非遍历自顶向下分段(即，EXD 和UVPD)兼容在线、本地LC和移动工作流。完成这项任务的目标 提案将提供新的创新和有利的分析解决方案和仪器，这将 使整个蛋白质组学社区受益，并为功能自上而下的蛋白质组学打开新的大门。

PROJECT SUMMARY/ABSTRACT With proteomics entering the third decade, the identification and quantification of primary protein sequences has become a mature science. However, many analytical challenges remain during the mapping of post-translational modifications (PTMs) of proteins, especially in their native state. For example, complete deciphering of the "histone code" entails the identification and localization of the different PTMs using top down strategies. While some advances have been made, traditional LC-MS/MS methods cannot fully separate histones with isomeric PTM position variants; this leads to the need to further develop new, fast, and orthogonal separations that can be easily integrated with mass spectrometry for the characterization of the “histone code”. In the present project, we aim to test the hypothesis that “PTMs induce structural changes in histones, allowing their separation and identification by the difference in ion mobility properties (particularly, for positional isomers), and fragmentation patterns”. Understanding the effect of PTMs on histone structure and function is central to the epigenetic regulation. Thus, to enable further advances in epigenetics, we will develop new multidimensional ion mobility separations for top- down isoform histone analyses. The ultimate goal of this project is to bring together technology experts in post-ionization, orthogonal separations and top-down mass spectrometry to develop an integrative, multidimensional analytical platform, capable of characterizing the “histone code” using native, as opposed to proteolytically digested, histones. To accomplish this goal, we will pursue the following aims: 1) To integrate multi-stage, non-linear, and linear ion mobility separations with top-down mass spectrometry (IMSn-MS/MS); 2) To develop liquid chromatography (offline and online) strategies compatible with IMSn-MS/MS.; and 3) To evaluate histone PTM abundances in biological systems using LC-MS/MS and LC-IMSn-MS/MS strategies. To support these aims, major technological breakthroughs in each of the integrated areas will be achieved: i) non-linear IMS (with 3 new FAIMS geometries implemented and evaluated), ii) linear IMS (with a higher resolution, larger mobility range, and higher sensitivity TIMS cell implemented and evaluated), and iii) non-ergodic top-down fragmentation (i.e., ExD and UVPD) compatible with online, native LC and mobility workflows. Completing the aims of this proposal will provide new innovative and enabling analytical solutions and instrumentation, that will benefit the proteomics community at large and open new doors for functional top-down proteomics.