Protein structure and function by hydrogen exchange mass spectrometry analysis

描述（由申请人提供）：蛋白质化学家面临的主要挑战是解释蛋白质如何实现其有限的结构和生物物理特性， 产生了无数的功能。与任何其他实验室方法不同，氢交换（HX）行为编码详细的定量信息，在氨基酸分辨率，生物物理因素，产生蛋白质功能-结构，结构变化，相互作用，动力学和能量。该NIH资助的先前版本通过使用各种NMR方法在开发HX领域中发挥了重要作用，但常规NMR分析仅限于大量和高浓度的小的高度可溶性蛋白质。发展中的氢交换质谱技术（HX MS）有望将这种已被证明的能力扩展到更大和更复杂的蛋白质系统，使生物学工作，同时仅需要亚微摩尔浓度的皮摩尔蛋白质。本申请旨在完善HX MS技术，并将其应用于重要蛋白质体系的研究。它们是：1）载脂蛋白E3和E4，当无脂质和脂质结合时，对于心血管和阿尔茨海默病是重要的; 2）获得性TTP疾病中自身免疫抗体表位的定义，更一般地与抗体治疗和蛋白质相互作用相关; 3）Hsp 104蛋白解聚酶，与蛋白质聚集和淀粉样蛋白加工的控制相关。HX MS的全面发展及其对困难但重要的蛋白质系统的演示将提供一种独特的强大方法，可广泛应用于蛋白质结构-功能问题的研究。

DESCRIPTION (provided by applicant): The major challenge for the protein chemist is to explain how proteins implement their limited repertoire of structural and biophysical properties to produce their myriad functions. Unlike any other laboratory method, hydrogen exchange (HX) behavior encodes detailed quantitative information, at amino acid resolution, on the biophysical factors that produce protein function -- structure, structure change, interactions, dynamics, and energetics. Previous versions of this NIH grant have been instrumental in developing the HX field by using a variety of NMR methods, but routine NMR analysis is limited to small highly soluble proteins in substantial quantity and high concentration. A developing hydrogen exchange - mass spectrometry technology (HX MS) promises to extend this proven capability to the larger and more complex protein systems that make biology work while requiring only picomoles of protein at sub-micromolar concentrations. This application proposes to perfect the HX MS technology and apply it in studies of important protein systems. They are: 1) apolipoproteins E3 and E4, when lipid- free and lipid-bound, important for cardiovascular and Alzheimer's diseases; 2) the definition of autoimmune antibody epitopes in acquired TTP disease, relevant to antibody therapeutics and to protein interactions more generally; 3) the Hsp104 protein disaggregase, relevant to the control of protein aggregation and amyloid processing. The full development of HX MS and its demonstration with difficult but centrally important protein systems will provide a uniquely powerful methodology that is widely applicable to the study of protein structure - function problems.