Sensitive Deuterium Exchange MS for Membrane Proteins

用于膜蛋白的灵敏氘交换质谱

• 批准号: 7862290
• 负责人: Tracy M Handel
• 金额: $ 33.82万
• 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/7862290
• 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）是一类膜蛋白，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。某些趋化因子 GPCR 的功能障碍会导致许多疾病的病理，包括哮喘、类风湿性关节炎、多发性硬化症、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行业的主要焦点。 GPCR-趋化因子复合物的结构信息非常重要，但由于产生足够数量的蛋白质进行研究的挑战以及现有生物物理方法的固有局限性，这些信息很难获得。在这个项目中，我们将开发一种使用氘交换质谱 (DXMS) 进行膜蛋白结构分析的高灵敏度技术。虽然 DXMS 对于可溶性蛋白质结构分析非常有用，但由于与灵敏度和保持膜蛋白功能所需的脂质环境相关的问题，DXMS 尚未广泛应用于膜蛋白。这项工作将为 DXMS 研究创建一个微流体平台，该平台将有效地集成 DXMS 实验中的各个步骤。它将建立尽量减少脂质和洗涤剂成分对下游质谱分析影响的方法。这将导致灵敏度提高 100-1000 倍，并将 DXMS 方法扩展到任何涉及完整膜蛋白的系统。一旦开发完成，该技术将用于研究许多重要的趋化因子受体和与其结合的配体。 公共健康相关性：这项工作将提供获取少量膜蛋白的重要结构信息的方法。开发治疗哮喘、类风湿关节炎、多发性硬化症、心脏病和癌症等多种疾病的药物需要这些信息。因此，它将对改善人类健康产生重大影响。