Sensitive Deuterium Exchange MS for Membrane Proteins

用于膜蛋白的灵敏氘交换质谱

• 批准号: 8325676
• 负责人: Tracy M Handel
• 金额: $ 31.36万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325676
• 关键词: Agonist; Amides; Arthritis; Asthma; CCR1 gene; California; Cells; Cities; Complex; Complication; Crystal Formation; Custom; Detergents; Deuterium; Development; Device or Instrument Development; Devices; Digestion; Disease; Disseminated Malignant Neoplasm; Drug Industry; Elements; Endocrine; Environment; Excision; Family; G-Protein-Coupled Receptors; G-substrate; Goals; HIV; Health; Heart Diseases; Human; Hydrogen; Immune; Immune system; Insecta; Integral Membrane Protein; Knowledge; Label; Laboratories; Ligand Binding; Ligands; Lipids; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Membrane Proteins; Methods; Micelles; Microfluidic Microchips; Microfluidics; Molecular; Multiple Sclerosis; Neuraxis; Nuclear Magnetic Resonance; Pathology; Pepsin A; Peptides; Physiological; Post-Translational Protein Processing; Process; Proteins; Proteolysis; Proteomics; Regulation; Research; Resolution; Resources; Rheumatoid Arthritis; Role; Sampling; Scaffolding Protein; Site; Solid; Solutions; Spectrometry; Stimulus; Structural Protein; Structure; System; Systems Analysis; Technical Expertise; Technology; Testing; Universities; Wood material; Work; X-Ray Crystallography; base; cell motility; chemokine; chemokine receptor; drug development; expectation; improved; insight; interest; ion source; liquid chromatography mass spectrometry; member; new technology; protein folding; protein function; protein protein interaction; prototype; public health relevance; receptor; receptor function; receptor structure function; research and development; research study; three dimensional structure; transmission process

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are a class of membrane proteins that have an essential role in development and function of the endocrine, immune and central nervous systems. The malfunction of certain chemokine GPCRs contribute to the pathology of many diseases including asthma, rheumatioid arthritis, multiple sclerosis, heart disease and metastatic cancer. Consequently the development of drugs to block chemokine receptor function is a major focus in the pharmaceutical industry. Structural information on the GPCR-chemokine complexes is of great importance, but such information is difficult to obtain because of the challenges of producing sufficient quantities of protein to study and the inherent limitations of existing bio-physical methods. In this project we will develop a highly sensitive technology for membrane protein structural analysis using deuterium exchange mass spectrometry (DXMS). While very useful for soluble protein structural analysis, DXMS has not been applied much to membrane proteins because of issues related to sensitivity and the lipid environment needed to preserve membrane protein function. This work will create a microfluidic platform for DXMS studies that will efficiently integrate the various steps in a DXMS experiment. It will establish methods for minimizing the impact of lipid and detergent components on the downstream mass spectral analysis. This will result in a 100-1000 fold increase in sensitivity and extend DXMS methods to any system involving integral membrane proteins. Once developed, the technology will be used to study a number important chemokine receptors and ligands that bind to them. PUBLIC HEALTH RELEVANCE: This work will provide the means to obtain vital structural information on small amounts membrane proteins. Such information is needed for the development of drugs to treat many diseases such as asthma, rheumatoid arthritis, multiple sclerosis, heart disease, and cancer. Consequently, it will have a major effect on improving human health.

描述（由申请人提供）：G蛋白偶联受体（GPCR）是一类在内分泌、免疫和中枢神经系统的发育和功能中具有重要作用的膜蛋白。某些趋化因子GPCR的功能障碍有助于许多疾病的病理学，包括哮喘、类风湿性关节炎、多发性硬化、心脏病和转移性癌症。因此，开发阻断趋化因子受体功能的药物是制药工业的主要焦点。关于GPCR-趋化因子复合物的结构信息是非常重要的，但是由于生产足够量的蛋白质以进行研究的挑战和现有生物物理方法的固有限制，这样的信息难以获得。在这个项目中，我们将开发一种高灵敏度的技术，用于膜蛋白结构分析，使用氘交换质谱（DXMS）。虽然对于可溶性蛋白质结构分析非常有用，但由于与敏感性和保护膜蛋白功能所需的脂质环境相关的问题，DXMS尚未被大量应用于膜蛋白。这项工作将为DXMS研究创建一个微流体平台，该平台将有效地整合DXMS实验中的各个步骤。它将建立最大限度地减少脂质和洗涤剂组分对下游质谱分析的影响的方法。这将导致灵敏度增加100-1000倍，并将DXMS方法扩展到涉及整合膜蛋白的任何系统。一旦开发出来，该技术将用于研究一些重要的趋化因子受体和与它们结合的配体。 公共卫生相关性：这项工作将提供获得少量膜蛋白的重要结构信息的方法。这些信息是开发治疗许多疾病的药物所必需的，如哮喘、类风湿性关节炎、多发性硬化症、心脏病和癌症。因此，它将对改善人类健康产生重大影响。