Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry
Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry
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DOI：
10.1038/nprot.2007.73
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发表时间：
2007-01-01
期刊：
影响因子：
14.8
通讯作者：
Robinson, Carol V.
中科院分区：
文献类型：
--
作者：
Hernandez, Helena;Robinson, Carol V.
越来越多的应用来确定蛋白质复合体的化学计量、相互作用甚至亚单位结构，这表明现在可以提出一些通用的指导方针。在该方案中，我们描述了维持气相亚单位之间相互作用所需的必要步骤。我们从制备适合电喷雾(ES)的溶液开始，然后考虑络合物通过质谱仪的各个阶段的传输，直到它们被检测出来。还描述了后续步骤，包括将这些络合物解离成多个子络合物以生成相互作用网络。贯穿始终，我们强调决定成功的关键实验因素。总体而言，我们开发了一种通用方案，可以使用商业上可用的ES质谱仪执行，而无需进行广泛的修改。
The growing number of applications to determine the stoichiometry, interactions and even subunit architecture of protein complexes from mass spectra suggests that some general guidelines can now be proposed. In this protocol, we describe the necessary steps required to maintain interactions between subunits in the gas phase. We begin with the preparation of suitable solutions for electrospray (ES) and then consider the transmission of complexes through the various stages of the mass spectrometer until their detection. Subsequent steps are also described, including the dissociation of these complexes into multiple subcomplexes for generation of interaction networks. Throughout we highlight the critical experimental factors that determine success. Overall, we develop a generic protocol that can be carried out using commercially available ES mass spectrometers without extensive modification.