Department of Chemistry and Biochemistry, Ohio State University, 484 W. 12th Ave., Columbus, Ohio 43210, USA.
Anal Chem. 2013 Sep 3;85(17):8262-7. doi: 10.1021/ac401497c. Epub 2013 Aug 20.
Tandem mass spectrometry is a tool to dissect noncovalent protein complexes into smaller substructures for quaternary structure analysis. The commonly used activation method, collision induced dissociation (CID), often provides limited structural information from the typical dissociation pattern where unfolded monomers are ejected from the protein complex. In contrast, surface-induced dissociation (SID) has been shown to be very effective at dissociating protein complexes with less unfolding than CID. We present here SID of a large noncovalent tetradecamer protein, GroEL (801 kDa). A wide variety of products, including heptamers representative of the native topology, are released from the precursor upon SID, significantly different from the ubiquitous monomer ejection in CID. Enhanced dissociation into heptamers is observed when the charge states of the GroEL precursor are reduced by adding triethylammonium acetate into the spraying buffer. Ion mobility is utilized after SID to separate products overlapping in m/z to simplify the SID spectra. Compact heptamers from the charge-reduced tetradecamer are clearly distinguished from other overlapping species. SID can be very useful for quaternary structure studies of large noncovalent protein complexes, as manifested by the GroEL data where the tetradecamer dissociates into heptamers, reflecting the native topology of the complex.
串联质谱是一种将非共价蛋白质复合物分解为较小亚结构的工具，用于四级结构分析。常用的激活方法，碰撞诱导解离（CID），通常从典型的解离模式中提供有限的结构信息，其中未折叠的单体从蛋白质复合物中弹出。相比之下，表面诱导解离（SID）已被证明在解离蛋白质复合物方面非常有效，其解折叠程度低于 CID。我们在此展示了一种大型非共价十四聚体蛋白质 GroEL（801 kDa）的 SID。在 SID 后，从前体中释放出包括代表天然拓扑结构的七聚体在内的各种产物，与 CID 中普遍存在的单体弹出显著不同。当在喷雾缓冲液中添加三乙铵乙酸盐以降低 GroEL 前体的电荷状态时，观察到增强的七聚体解离。在 SID 之后利用离子淌度将在 m/z 上重叠的产物分离，以简化 SID 谱。来自电荷降低的十四聚体的紧凑七聚体与其他重叠的物质明显区分开来。SID 可非常有用地用于大型非共价蛋白质复合物的四级结构研究，如 GroEL 数据所示，其中十四聚体解离成七聚体，反映了复合物的天然拓扑结构。
