Department of Chemistry and Biochemistry and Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, Ohio 43210, United States.
J Am Chem Soc. 2021 May 26;143(20):7698-7706. doi: 10.1021/jacs.1c00855. Epub 2021 May 13.
Characterizing protein-protein interactions, stoichiometries, and subunit connectivity is key to understanding how subunits assemble into biologically relevant, multisubunit protein complexes. Native mass spectrometry (nMS) has emerged as a powerful tool to study protein complexes due to its low sample consumption and tolerance for heterogeneity. In nMS, positive mode ionization is routinely used and charge reduction, through the addition of solution additives, is often used, as the resulting lower charge states are often considered more native-like. When fragmented by surface-induced dissociation (SID), charge reduced complexes often give increased structural information over their "normal-charged" counterparts. A disadvantage of solution phase charge reduction is that increased adduction, and hence peak broadening, is often observed. Previous studies have shown that protein complexes ionized using negative mode generally form lower charge states relative to positive mode. Here we demonstrate that the lower charged protein complex anions activated by surface collisions fragment in a manner consistent with their solved structures, hence providing substructural information. Negative mode ionization in ammonium acetate offers the advantage of charge reduction without the peak broadening associated with solution phase charge reduction additives and provides direct structural information when coupled with SID. SID of 20S human proteasome (a 28-mer comprised of four stacked heptamer rings in an αββα formation), for example, provides information on both substructure (e.g., splitting into a 7α ring and the corresponding ββα 21-mer, and into α dimers and trimers to provide connectivity around the 7 α ring) and proteoform information on monomers.
鉴定蛋白质-蛋白质相互作用、化学计量和亚基连接性对于理解亚基如何组装成具有生物学意义的多亚基蛋白质复合物至关重要。由于其低样品消耗和对异质性的容忍度，天然质谱（nMS）已成为研究蛋白质复合物的有力工具。在 nMS 中，通常使用正离子模式进行电离，并且通过添加溶液添加剂来进行电荷还原，因为所得的较低电荷状态通常被认为更具有天然样性。当通过表面诱导解离（SID）进行碎片化时，电荷还原的复合物通常比其“正常带电”对应物提供更多的结构信息。溶液相电荷还原的一个缺点是，通常会观察到增加的加合，从而导致峰展宽。先前的研究表明，使用负离子模式电离的蛋白质复合物通常相对于正离子模式形成较低的电荷状态。在这里，我们证明了通过表面碰撞激活的较低电荷的蛋白质复合物阴离子以与其已解决结构一致的方式进行碎片化，从而提供亚结构信息。在乙酸铵中进行负离子化具有电荷还原的优势，而没有与溶液相电荷还原添加剂相关的峰展宽，并在与 SID 结合时提供直接的结构信息。例如，20S 人蛋白酶体（由四个堆叠的七聚体环以αββα形式组成的 28 -mer）的 SID 提供了关于亚结构（例如，分裂成 7α环和相应的ββα 21-mer，以及分裂成α二聚体和三聚体以提供围绕 7α环的连接性）和单体的蛋白质形式信息。
