New Ion Mobility and Crosslinking Technologies for Analysis of Protein Complexes

描述（由申请人提供）：为了发挥其生物学功能，单个蛋白质通常以一种短暂的方式结合，形成复合物。从分子医学到物理化学，了解复合物的作用方式是一个影响深远的科学目标。虽然通过x射线衍射分析有时可以获得高度详细的结构信息，但这需要有足够数量的均质材料和确定合适的结晶参数。对于大型复合物和膜蛋白来说，这两个条件往往难以满足，因此这些结构在数据库中的数量相对较少。电子显微镜（EM）和小角度X射线散射（SAXS）等替代方法可以确定足够尺寸的配合物的表面包络，但对这些数据的解释需要对复杂成分的详细了解，并且通常仅限于均相配合物。因此，有必要开发新的方法来定义具有生物医学重要性的异质大分子复合物的亚基化学计量、组成、形状和动力学，特别是那些在完整的生物膜和细胞器中。该方案结合了新的交联剂策略和离子迁移率（IM）耦合质谱（MS）作为多蛋白复合物，特别是膜复合物的高通量结构探针。基于小分子化学的新交联剂策略的发展符合质谱和IM的要求，将克服许多现有的蛋白质复合物分析的限制。这是朝着开发一套新的基于高通量质谱的技术迈出的第一步，该技术将能够发现许多以前未知的多蛋白复合物结构，并将提供用于解释结构的肽接近信息，并为蛋白质折叠计算提供限制。重要的是，它也将为开发蛋白质复合物中相互作用动力学的方法提供基础。

DESCRIPTION (provided by applicant): To perform their biological function, individual proteins associate, often in a transient manner, to form complexes. Understanding the way complexes function is a far-reaching scientific goal for disciplines ranging from molecular medicine to physical chemistry. While high- detail structural information can sometimes be obtained by X-ray diffraction analysis, this requires the availability of a sufficient quantity of homogenous material and definition of suitable crystallization parameters. Both conditions are often difficult to meet for large complexes and for membrane proteins and thus the number of these structures deposited in databases remains relatively low. 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, shape, and the dynamics of heterogeneous macromolecular complexes of biomedical importance particularly those in intact biological membranes and organelles. This proposal combines new crosslinker strategies and ion mobility (IM) coupled to mass spectrometry (MS) jointly as high-throughput structural probes for multi-protein complexes, particularly membrane complexes. The development of new crosslinker strategies based on small molecule chemistries consistent with the requirements of MS and IM will overcome many of the existing constraints for analysis of protein complexes. This is a first step towards developing a suite of new high-throughput mass spectrometry-based technologies that will enable the discovery of many previously-unknown multi-protein complex structures and will provide peptide proximity information of use for interpreting structures and providing constraints for protein folding calculations. Importantly, it will also provide a basis for developing methods for following interaction dynamics in protein complexes.