Mass Spectrometry of Protein Assemblies

蛋白质组装体的质谱分析

• 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
• 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

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）：理解身体蛋白质之间的关系以及它们如何变化对于理解疾病至关重要。随着人类基因组的完成，定义蛋白质的功能作用和蛋白质相互作用是蛋白质组学和结构生物学的下一阶段。大多数细胞过程是由在大分子复合物中起作用的蛋白质执行和调节的。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装体的表征。应用电喷雾电离质谱 (ESI-MS) 研究非共价复合物在生物医学研究中具有实用性，并且是研究蛋白质分子识别的有力手段。测量分子量可以提供大分子组装体的结构信息，因为它可以直接确定复合物中结合配偶体的化学计量。 质谱和气相电泳迁移率分子分析（GEMMA）将用于推进大型高分子机器的表征，其灵敏度比目前的实践更高。该提案的具体目标是： (1) 开发具有更高灵敏度的 ESI-MS 方法，用于测量大蛋白质复合物 (0.5-15 MDa)。碰撞冷却和微型芯片 ESI 源将提高蛋白质复合物分析的灵敏度和重现性。 (2) 开发直接 ESI-MS 方法以获得蛋白质复合物的结构相关信息和相互作用的能量学。开发交联方法、亲和纯化和基于 GEMMA 的技术来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（负责蛋白质降解的大型蛋白酶）、与帕金森病 (PD) 相关的蛋白质，以及核糖核蛋白穹窿（涉及的 14 MDa 颗粒）在多重耐药性中。