Negative Electron Transfer Dissociation for Phospho-Histidine Analysis

用于磷酸组氨酸分析的负电子转移解离

• 批准号: 8862700
• 负责人: JOSHUA J COON
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8862700
• 关键词: Accounting; Acids; Acute; Amino Acids; Anions; Antibodies; Bacteria; Biology; Cataloging; Catalogs; Cations; Cell Cycle; Cell Mobility; Cells; Chronic; Cleaved cell; Complement; Complex; Development; Disease; Dissociation; Drops; Electron Transport; Electrons; Enzymes; Eukaryota; Fasting; Functional disorder; Gases; Histidine; Ions; Knowledge; Mass Spectrum Analysis; Measures; Metabolic; Metabolism; Methodology; Methods; Mitochondria; Mitochondrial Diseases; Modification; Mus; Noise; Obesity; Organelles; Peptides; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Post Translational Modification Analysis; Post-Translational Modification Site; Post-Translational Protein Processing; Prevalence; Process; Proteins; Proteome; Proteomics; Reaction; Reagent; Regulation; Research; Signal Transduction; Site; Staging; System; Technology; Tissues; Vertebral column; Work; analog; base; carboxylation; glycosylation; insight; novel; phosphohistidine; photoactivation; protein aminoacid sequence; public health relevance; research study

 DESCRIPTION (provided by applicant): Post-translational modification (PTM) of proteins is a ubiquitous form of cell signaling that orchestrates numerous processes, including metabolism, cell mobility, cell cycle, and differentiation. As a result, improper regulation of PTMs is widely implicated in aberrant development and disease. Mechanistically, PTMs provide a rapid and largely reversible means to modulate protein activity and transduce signals. Thus, the proteome and its modifications represent a rich and informative experimental plane; research that seeks to understand PTM dynamics has, and continues to, advance our understanding of fundamental biology and disease. With its high sensitivity and capacity to localize modifications to a single amino acid, mass spectrometry (MS) is well-suited to global protein PTM analysis. And MS does not require a priori knowledge of PTM sites; as such, MS has been used to catalog the complexity of various PTMs with great detail and terrific impact. That said, major challenges remain, for example, the field of proteomics almost uniformly relies on peptide cation analysis (i.e., positive electrospray) so that today's paradigm for high-throughput proteomics relies solely on the analysis of gas-phase cations. Our inability to sequence peptide anions has led to an underrepresentation of acidic portions of proteomes. This bias is exacerbated for PTMs that are chemically acidic and/or potentially labile during MS/MS, such as phosphorylation, sulfonation, carboxylation, glycosylation, and succinylation. Histidine phosphorylation (pHis), for example, accounts for ~6% of total phosphorylated proteins in eukaryotes; however, it is currently not possible to globally measure pHis as the modification is rapidly lost when pH drops below 7 (proteomic separations are routinely performed at pH 2.5-3). Here we propose new MS technology that will permit the first global analysis of proteomes in the negative ion mode. To accomplish this we shall develop the negative ion analog of electron transfer dissociation - NETD. In this approach peptide anions are oxidized by NETD reagent cations, promoting electron rearrangements that cleave the C-Ca backbone bond to produce a*- and x-type product ions for sequence interpretation.

 描述（由适用提供）：蛋白质的翻译后修饰（PTM）是一种无处不在的细胞信号形式，它策划了许多过程，包括代谢，细胞迁移率，细胞周期和分化。结果，PTM的调节不当与异常的发育和疾病有关。从机械上讲，PTM提供了一种快速且在很大程度上可逆的手段来调节蛋白质活性和传递信号。这是蛋白质组及其修饰代表了丰富而有益的实验平面。试图了解PTM动力学的研究已经并且继续促进了我们对基本生物学和疾病的理解。质谱法（MS）凭借其对单个氨基酸进行定位的高灵敏度和能力，非常适合全球蛋白质PTM分析。 MS不需要对PTM站点的先验知识；因此，MS已被用来对各种PTM的复杂性进行分类，并具有很大的细节和极大的影响。也就是说，例如，仍然存在主要挑战，例如，蛋白质组学领域几乎均依赖于肽阳离子分析（即阳性电喷雾），因此当今的高通量蛋白质组学的范式完全依赖 关于气相阳离子的分析。我们无法对肽阴离子进行测序，从而导致蛋白质酸性部分的体现不足。对于在MS/MS期间具有化学酸性和/或潜在标记的PTM（例如磷酸化，硫酸化，硫酸化，羧化，糖基化和琥珀酰化）的PTM，这种偏差会加剧。例如，组氨酸磷酸化（PHIS）占真核生物中总磷酸化蛋白的约6％。但是，目前不可能在全球测量pHI，因为当pH下降到7（蛋白质组学分离以通常在pH 2.5-3下进行）时，pH值迅速丢失。在这里，我们提出了新的MS技术，该技术将允许在负离子模式下首次对蛋白质组进行全球分析。为此，我们将发展电子传递解离的负离子类似物-Netd。在这种方法中，肽阴离子被NETD试剂阳离子氧化，促进电子重排清除C-CA骨架键以产生A* - 和X型产物离子以进行序列解释。