Sensitive Deuterium Exchange MS for Membrane Proteins

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起重要作用。某些趋化因子gpcr的功能障碍有助于许多疾病的病理，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，阻断趋化因子受体功能的药物的开发是制药工业的一个主要焦点。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。在本项目中，我们将开发一种高灵敏度的膜蛋白结构分析技术——氘交换质谱（DXMS）。虽然DXMS在可溶性蛋白结构分析中非常有用，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尚未应用于膜蛋白。这项工作将为DXMS研究创建一个微流控平台，有效地整合DXMS实验的各个步骤。它将建立最小化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。一旦开发出来，这项技术将用于研究一些重要的趋化因子受体和与之结合的配体。

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.