Next-generation Mass Spectrometry Technologies for Integrated Structural Proteomics

 描述（由申请人提供）：在每种生物体内，蛋白质都在发挥作用，进行影响细胞功能各个方面的活动，从复制到细胞死亡。实现广泛功能和高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结构对于理解正常和疾病状态以及药物开发至关重要。然而，大多数结构仍然是未知的，并且对当前技术是难处理的。目前的方法，这一挑战主要依赖于通过高通量的X射线或NMR技术，分离的蛋白质复合物的原子细节的结构的直接转换。虽然非常成功，但这些方法需要大量的纯样品，通常优化以生成晶体或去除光谱背景。此外，存在于复杂混合物中的瞬态和多分散组装体不能被分析。替代方法，如电子显微镜（EM）和小角X射线散射（SAXS）允许确定足够尺寸的复合物的表面包络，但这些数据的解释是由复杂的组合物的详细知识的帮助，并限于，在一般情况下，均匀的复合物。因此，有必要开发新的方法，定义亚基的化学计量，组成，界面结构，形状，和明确的生物医学重要性的异质大分子复合物的相互作用动力学。这一提议的更新旨在建造新的，创新的结构质谱技术，1）利用受控的蛋白质复合物破坏和天然质谱来构建蛋白质-蛋白质界面的详细模型2）验证将气相蛋白质解折叠与液相蛋白质结构域结构联系起来的最新观察结果，以创建用于蛋白质亚基模型构建的新工具3）增强化学交叉-连接试剂以增加信息含量并量化体外和体内蛋白质相互作用和构象的变化4）构建能够整合多个结构质谱数据来源的新蛋白质建模工具和5）创建能够对兆道尔顿级完整蛋白质复合物进行全面的自上而下测序或增强稳定性的新化学试剂。所有这些技术都将用于发现一系列选定蛋白质复合物的结构，每一种蛋白质复合物都与人类疾病有着关键的联系。

 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.