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

中下蛋白质组多维、线性和微分离子淌度 MS 分离的开发

• 批准号: 10019582
• 负责人: Francisco Fernandez-Lima
• 金额: $ 36.34万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10019582
• 关键词: Amino Acid Sequence; Area; Atmospheric Pressure; Biological; Cell physiology; Cells; Communities; Custom; Development; Devices; Dissociation; Electrons; Epigenetic Process; Epithelial Cells; Gases; Geometry; Germ-Free; Goals; Histone Code; Histones; Isomerism; Knowledge; Language; Liquid Chromatography; Liquid substance; Mass Spectrum Analysis; Methodology; Methods; Mus; Pattern; Phase; Positioning Attribute; Post-Translational Protein Processing; Property; Protein Isoforms; Proteins; Proteomics; Resolution; Science; Speed; Structure; Technology; Testing; Transcription Process; Variant; Work; analytical tool; base; biological systems; epigenetic regulation; gut bacteria; innovation; instrumentation; ion mobility; ionization; liquid chromatography mass spectrometry; next generation; pressure; success; tandem mass spectrometry; Amino Acid Sequence; Area; Atmospheric Pressure; Biological; Cell physiology; Cells; Communities; Custom; Development; Devices; Dissociation; Electrons; Epigenetic Process; Epithelial Cells; Gases; Geometry; Germ-Free; Goals; Histone Code; Histones; Isomerism; Knowledge; Language; Liquid Chromatography; Liquid substance; Mass Spectrum Analysis; Methodology; Methods; Mus; Pattern; Phase; Positioning Attribute; Post-Translational Protein Processing; Property; Protein Isoforms; Proteins; Proteomics; Resolution; Science; Speed; Structure; Technology; Testing; Transcription Process; Variant; Work; analytical tool; base; biological systems; epigenetic regulation; gut bacteria; innovation; instrumentation; ion mobility; ionization; liquid chromatography mass spectrometry; next generation; pressure; success; tandem mass spectrometry

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.

项目摘要/摘要 随着蛋白质组学进入第三个十年，原代蛋白的识别和定量 序列已成为一门成熟的科学。但是，在映射期间仍然存在许多分析挑战 蛋白质的翻译后修饰（PTM），尤其是在其本地状态。例如， 完全解密“组蛋白代码”实体的识别和本地化不同 PTM使用自上而下的策略。尽管已经取得了一些进步，但传统的LC-MS/MS方法 无法完全分离具有异构体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，可以轻松地与质谱法集成 “组蛋白代码”的表征。在本项目中，我们旨在检验“ PTMS”的假设 诱导组蛋白的结构变化，从而通过离子的差异使它们的分离和鉴定 移动性特性（尤其是用于位置异构体）和破碎模式”。了解 PTM对组蛋白结构和功能的影响是表观遗传调节的核心。那，启用 表观遗传学的进一步进步，我们将开发新的多维离子迁移率分离 下降同工蛋白分析。该项目的最终目标是将技术专家汇集在一起 离子化后，正交分离和自上而下的质谱法，以形成综合性， 多维分析平台，能够使用本机表征“组蛋白代码” 与蛋白水解消化的相反，希腊。为了实现这一目标，我们将追求以下目标： 1）与自上而下的质量整合多阶段，非线性和线性离子迁移率 光谱法（IMSN-MS/MS）; 2）制定液相色谱法（离线和在线）策略 与IMSN-MS/MS兼容。 3）使用生物系统中的组蛋白PTM抽象使用 LC-MS/MS和LC-IMSN-MS/MS策略。为了支持这些目标，主要的技术突破 将在每个集成领域中实现：i）非线性IMS（有3个新的Faims几何技术 实施和评估），ii）线性IMS（分辨率更高，移动性范围较大，较高 敏感性TIMS细胞实现和评估），以及iii）非共进自上而下的碎片（即EXD 和UVPD）与在线，本机LC和Mobility Workfrows兼容。完成目标 提案将提供新的创新和启用分析解决方案和仪器，这将提供 在大型开放式新门上受益于功能性自上而下的蛋白质组学。