Mass Spectrometry and Enzyme Processes

质谱分析和酶处理

• 批准号: 7535238
• 负责人: JULIE Ann LEARY
• 金额: $ 22.84万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7535238
• 关键词: Affinity Labels; Area; Biological; Catalysis; Cellular biology; Chemistry; Data; Educational process of instructing; Ensure; Enzyme Kinetics; Enzymes; Equipment; Fourier transform ion cyclotron resonance; Genome; Glean; Goals; Grant; Heparin; Human; Individual; Inorganic Sulfates; Investigation; Ions; Isomerase; Isomerism; Ketoses; Ketosis; Kinetics; Knowledge; Mannose-6-Phosphate Isomerase; Mass Spectrum Analysis; Measurement; Measures; Mentors; Methods; Molecular; Nature; Positioning Attribute; Procedures; Process; Production; Protons; Reaction; Research; Screening procedure; Site; Specificity; Spectrometry, Mass, Electrospray Ionization; Students; Sulfatases; System; Techniques; Time; Tyrosine; Unspecified or Sulfate Ion Sulfates; affinity labeling; analog; base; biological systems; chromophore; expression cloning; fundamental research; indexing; inhibitor/antagonist; instrument; instrumentation; method development; professor; protein purification; protein-tyrosine sulfotransferase; sulfation; sulfotransferase

The long term goal of this competing renewal is the development of methods that allow for the analysis of various enzymatic processes using mass spectrometry. The major areas proposed herein include: 1) Kinetics and catalytic mechanism determination for phosphohexose-isomerases. 2) Kinetics, substrate determination, and mechanism determination of tyrosylprotein sulfotransferases. This will also include a new procedure for unambiguous determination of sites of sulfation on natural substrates. 3) Investigation of substrate and inhibitor interactions with a newly discovered human endosulfatase, Hsulf-2 Each of these areas employs electrospray ionization (ESI)mass spectrometry (MS)using either an LTQ ion trap or an FTICR instrument to measure various kinetic parameters such as Km, Vmax, Ki, kcat and specificity constants. It is proposed that kinetic parameters can be calculated using ESI-MS thus obviating the need for chromophores as required by traditional spectrophotometric techniques. Equally important is the fact that kinetic information, either previously intractable or difficult to measure, can be gleaned using the MS based methods outlined herein. The problem with analyzing sulfated biomolecules is well recognized in mass spectrometry. The proposed research will alleviate those problems associated with loss of sulfate upon electrospray ionization and MS/MS in the positive ion mode. Similarly, methods are proposedfor obtaining biological data on phosphoisomerase systems; one of which is highly difficult to classify kinetically and mechanistically. . .

这种竞争性更新的长期目标是开发允许分析以下因素的方法： 使用质谱的各种酶促过程。本报告建议的主要领域包括： 1)磷酸己糖异构酶的动力学和催化机理研究。 2)酪蛋白磺基转移酶的动力学、底物测定和机理测定。这 还将包括一个新的程序，明确确定网站的硫酸化天然基板。 3)底物和抑制剂与新发现的人endosulfatase Hsulf-2相互作用的研究 这些领域中的每一个都采用电喷雾电离（ESI）质谱（MS），其使用LTQ 离子阱或FTICR仪器来测量各种动力学参数，例如Km、Vmax、Ki、kcat和 特异性常数提出了用电喷雾质谱法计算动力学参数，从而避免了 传统分光光度技术所需的发色团。同样重要的是 事实上，动力学信息，无论是以前棘手或难以测量，可以收集使用 本文概述的基于MS的方法。分析硫酸化生物分子的问题在以下文献中被充分认识到： 质谱分析法来拟议的研究将缓解与硫酸盐损失相关的问题 电喷雾离子化和MS/MS在正离子模式。类似地，方法被提议用于 获得磷酸异构酶系统的生物学数据;其中一种非常难以动力学分类 和机械性。. .