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

项目概要/摘要 随着蛋白质组学进入第三个十年， 序列已经成为一门成熟的科学。然而，在制图过程中仍然存在许多分析挑战 蛋白质的翻译后修饰（PTM），尤其是在其天然状态下。比如说， “组蛋白密码”的完全破译需要鉴定和定位不同的 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 不能完全分离具有异构PTM位置变体的组蛋白;这导致需要进一步分离具有异构PTM位置变体的组蛋白。 开发新的，快速的，正交的分离，可以很容易地与质谱集成， “组蛋白密码”的特征。在本项目中，我们的目标是测试的假设，“PTM 诱导组蛋白的结构变化，允许它们通过离子的差异进行分离和鉴定。 流动性（特别是位置异构体）和裂解模式”。了解 PTM对组蛋白结构和功能的影响是表观遗传调控的核心。因此，为了使 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，用于顶级 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，以开发一个综合的， 多维分析平台，能够使用天然的，如 与蛋白水解消化的组蛋白相反。为了实现这一目标，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集成 2）开发液相色谱（离线和在线）策略 与IMSn-MS/MS兼容。和3）使用以下方法评估生物系统中的组蛋白PTM丰度： LC-MS/MS和LC-IMSn-MS/MS策略。为了支持这些目标，重大的技术突破 i）非线性IMS（具有3种新的FAIMS几何形状 ii）线性IMS（具有更高的分辨率、更大的迁移率范围和更高的分辨率）， 实现和评估的灵敏度TIMS单元），和iii）非遍历自顶向下分段（即，EXD 和UVPD），与在线、本地LC和移动工作流兼容。为了实现这一目标， 该提案将提供新的创新和有利的分析解决方案和仪器， 使蛋白质组学社区受益，并为功能性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.