Mass Spectrometry of Protein Assemblies

蛋白质组装体的质谱分析

基本信息

批准号：
7247828
负责人：
Joseph A Loo
金额：
$ 28.67万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-15 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7247828
关键词：
Address Affinity Affinity Chromatography Architecture Binding Binding Sites Biochemical Biochemical Pathway Biological Biology Biomedical Research Cell physiology Cells Charge Complex Detection Devices Disease Dissociation Electrospray Ionization Endopeptidases Fruit Gases Genome Goals Human Genome Ions Life Ligand Binding Ligands Macromolecular Complexes Mass Spectrum Analysis Measures Methods Molecular Molecular Analysis Multi-Drug Resistance Multiprotein Complexes Parkinson Disease Peptide Hydrolases Phase Play Process Protein Chemistry Proteins Proteome Proteomics Protons Reproducibility Research Ribonucleoproteins Role Site Solutions Source Spectrometry Spectrometry, Mass, Electrospray Ionization Staging Structure System Techniques Technology Testing Time aqueous base capsule crosslink functional group improved macromolecular assembly mass spectrometer microchip molecular mass molecular recognition multicatalytic endopeptidase complex nanoengineering new technology particle protein crosslink protein degradation protein function protein protein interaction protein purification small molecule stoichiometry structural biology structural genomics synuclein tandem mass spectrometry tool

项目摘要

DESCRIPTION (provided by applicant): Comprehending the relationship between the body's proteins and how they change is essential to understand disease. With the completion of the human genome, defining the functional role of proteins and protein interactions is the next stage of proteomics and structural biology. Most cellular processes are performed and regulated by proteins acting in macromolecular complexes. New technologies based on mass spectrometry (MS) will be developed to facilitate the characterization of the proteome and its protein assemblies. The application of electrospray ionization mass spectrometry (ESI-MS) for studying noncovalent complexes has utility in biomedical research, and is a powerful means for investigating protein molecular recognition. Measuring molecular mass provides structural information on macromolecular assemblies, as it provides a direct determination of the stoichiometry of the binding partners in the complex. Mass spectrometry and gas phase electrophoretic mobility molecular analysis (GEMMA) will be used to advance the characterization of large macromolecular machines with greater sensitivity than currently practiced. The specific aims of this proposal are: (1) Develop ESI-MS methods with improved sensitivity for measuring large protein complexes (0.5-15 MDa). Collisional cooling and a microfabricated microchip ESI source will increase the sensitivity and reproducibility of protein complex analysis. (2) Develop direct ESI-MS methods to obtain structurally-relevant information on protein complexes and the energetics of the interactions. Develop crosslinking methods, affinity purification, and GEMMA-based techniques for isolating large protein complexes. (3) Apply ESI-MS and ESI-GEMMA to characterize protein complexes, such as the proteasome, the large protease that is responsible for protein degradation, proteins involved in Parkinson's disease (PD), and the ribonucleoprotein vault, a 14 MDa particle implicated in multidrug resistance.

描述（由申请人提供）：理解人体蛋白质之间的关系以及它们如何改变对于理解疾病至关重要。随着人类基因组的完成，定义蛋白质和蛋白质相互作用的功能作用是蛋白质组学和结构生物学的下一个阶段。大多数细胞过程都是由作用在大分子复合物中的蛋白进行和调节的。将开发基于质谱（MS）的新技术，以促进蛋白质组及其蛋白质组件的表征。电喷雾电离质谱法（ESI-MS）在研究非共价复合物中的应用具有生物医学研究的实用性，并且是研究蛋白质分子识别的强大手段。测量分子质量提供了有关大分子组件的结构信息，因为它可以直接确定络合物中结合伙伴的化学计量法。质谱和气相电泳迁移率分子分析（GEMMA）将用于推进比目前实践更高灵敏度的大型大分子机器的表征。该提案的具体目的是：（1）开发具有提高灵敏度的ESI-MS方法，用于测量大蛋白质复合物（0.5-15 MDA）。碰撞冷却和微生物微芯片ESI来源将提高蛋白质复合物分析的敏感性和可重复性。（2）开发直接的ESI-MS方法，以获取有关蛋白质复合物和相互作用能量的结构相关的信息。开发交联方法，亲和力纯化和基于Gemma的技术，用于分离大型蛋白质复合物。（3）应用ESI-MS和ESI-GEMMA来表征蛋白质复合物，例如蛋白酶体，导致蛋白质降解的大蛋白酶，涉及帕金森氏病（PD）的蛋白质以及核糖核蛋白库，涉及多种糖耐药性的14 MDA颗粒蛋白。