Next-generation Mass Spectrometry Technologies for Integrated Structural Proteomics

 描述(由申请人提供)：在每个生物体内，蛋白质都在进行影响细胞功能的方方面面的活动，从复制到细胞死亡。实现广泛功能和高度效率的一个关键因素是蛋白质自组装成大分子“机器”的能力。这些组件的3D结构对于了解正常和疾病状态以及药物开发至关重要。然而，大多数结构仍然未知，对当前的技术来说是难以实现的。目前应对这一挑战的方法主要依赖于通过高通量X射线或核磁共振技术将分离的蛋白质复合体直接转化为原子细节结构。虽然这些方法非常成功，但这些方法需要大量纯样品，通常优化为要么产生晶体，要么去除光谱背景。此外，不能分析存在于复杂混合物中的瞬时和多分散组件。电子显微镜(EM)和小角X射线散射(SAXS)等替代方法可以确定足够尺寸的络合物的表面包络，但这些数据的解释有赖于对络合物组成的详细知识，而且一般限于均质络合物。因此，需要开发新的方法来定义具有明显生物医学重要性的多相大分子络合物的亚单位化学计量、组成、界面结构、形状和相互作用动力学。这项提案更新旨在构建新的、创新的结构质谱学技术，该技术1)利用受控的蛋白质复合体破坏和天然质谱学来建立蛋白质-蛋白质界面的详细模型2)验证最近观察到的将气相蛋白质展开与溶液相蛋白质结构域结构相联系的观察结果，以便创建用于蛋白质亚单位模型构建的新工具3)增强化学交联剂以增加信息含量并量化蛋白质相互作用和构象在体外和体内的变化4)建立新的蛋白质建模工具，能够整合多源的结构质谱学数据，以及5)创建新的化学试剂以实现全面的自上而下测序，或增强的稳定性，上百万吨规模的完整蛋白质复合体。所有这些技术都将被用来发现一系列选定的蛋白质复合体的结构，每个复合体都与人类疾病有关键联系。

 DESCRIPTION (provided by applicant): Within each organism proteins are at work carrying out activities which impact every aspect of cellular function, from replication to cell death. A ke factor in achieving both a wide range of functions and high degrees of efficiencies is the ability of proteins to self-assemble into macromolecular `machines'. The 3D structures of these assemblies are crucial for understanding normal and disease states and for drug development. However, most structures remain unknown and are refractory to current technologies. Current approaches to this challenge mainly rely on the direct conversion of isolated protein complexes into structures of atomic-detail via high-throughput X-ray or NMR technologies. While highly successful, these methods require pure samples in large quantities, typically optimized to either generate crystals or remove spectral background. Furthermore, transient and polydisperse assemblies that exist within complex mixtures cannot be analyzed. Alternative methodologies such as electron microscopy (EM) and small angle X-ray scattering (SAXS) allow determination of the surface envelope of complexes of sufficient dimensions but interpretation of these data is aided by detailed knowledge of complex composition, and is limited, in general, to homogeneous complexes. Consequently there is a need to develop new approaches that define subunit stoichiometry, composition, interface structure, shape, and the interaction dynamics of heterogeneous macromolecular complexes of clear biomedical importance. This proposal renewal seeks to construct new, innovative structural mass spectrometry techniques that 1) leverage controlled protein complex disruption and native mass spectrometry to build detailed models of protein-protein interfaces 2) validate recent observations linking gas-phase protein unfolding to solution-phase protein domain structure in order to create a new tool for protein subunit model construction 3) enhance chemical cross-linking reagents to increase information content and quantify changes in protein interactions and conformations in vitro and in vivo 4) build new protein modeling tools capable of integrating multiple sources of structural mass spectrometry data and 5) create new chemical reagents that enable the comprehensive top-down sequencing, or enhanced stability, of megadalton-scale intact protein complexes. All of this technology will be brought to bear to discover the structures of a series of selected protein complexes, each having a critical link to human disease.