Mass Spectrometry and Enzyme Processes

质谱分析和酶处理

基本信息

批准号：
7195464
负责人：
JULIE Ann LEARY
金额：
$ 22.44万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
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/7195464
关键词：
Affinity Labels Area Biological Catalysis Cellular biology Chemistry Data Educational process of instructing Electrospray Ionization Ensure Enzyme Kinetics Enzymes Equipment 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 Personal Satisfaction Positioning Attribute Procedures Process Production Protons Reaction Research Screening procedure Site Specificity Spectrometry Spectrometry, Mass, Electrospray Ionization Students Sulfatases System Techniques Time Tyrosine Unspecified or Sulfate Ion Sulfates affinity labeling analog base chromophore concept expression cloning fundamental research indexing inhibitor/antagonist instrument instrumentation method development professor protein purification protein-tyrosine sulfotransferase sulfation sulfotransferase

项目摘要

DESCRIPTION (provided by applicant): 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 proposed for obtaining biological data on phosphoisomerase systems; one of which is highly difficult to classify kinetically and mechanistically.

描述（由申请人提供）：这种竞争更新的长期目标是开发方法，可以使用质谱分析各种酶促过程。本文提出的主要领域包括：1）磷酸己糖 - 异构酶的动力学和催化机理测定。 2）动力学，底物测定和机理测定酪蛋白硫代转移酶。这还将包括一个新的程序，以明确确定天然底物上硫化位点。 3）研究底物和抑制剂与新发现的人内膜酶HSULF-2的相互作用。这些区域中的每一个都使用LTQ离子陷阱或FTICR仪器使用电喷雾电离（ESI）质谱法（MS）来测量各种动力学参数，例如KM，VMAX，KI，KI，KCAT和特异性常数。有人提出，可以使用ESI-MS计算动力学参数，从而消除了传统分光光度计技术要求的色彩团的需求。同样重要的是，可以使用本文概述的基于MS的方法来收集以前棘手或难以测量的动力学信息。分析硫酸化生物分子的问题在质谱中得到了很好的认识。拟议的研究将减轻与电喷雾电离后硫酸盐损失相关的问题，在正离子模式下MS/MS。同样，提出了在磷酸异构酶系统上获得生物学数据的方法。其中之一很难在动力学和机械上进行分类。