An overview of a typical membrane protein purification and preparation for Mass spectrometry
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An overview of a typical membrane protein purification and preparation, as well as analysis by mass spectrometry of the intact complex.(a) Purified membranes containing an overexpressed membrane protein fused to GFP and a His-tag; the protein is solubilized in a detergent of interest. Solubilized membrane protein is purified using immobilized metal affinity chromatography (IMAC) and concentrated before preparation for native MS. (b) Detergent can be screened or optimized starting with a purified membrane protein; follow the procedure outlined here to exchange into a different detergent. Furthermore, membrane proteins can be prepared from solubilized membranes in different detergents, followed by purification using IMAC. (c) Purified membrane proteins are buffer-exchanged into an MS-compatible buffer containing two times the CMC of the detergent of interest. Typically, buffer-exchange centrifugal devices, such as a Bio-Spin device, are used. Alternatively, buffer exchange can also be achieved using a small analytical gel filtration column. (d) Theoretical mass spectrum for a tetrameric membrane protein complex (200 kDa) with a protein monomeric mass of 50 kDa. Under MS conditions, the oligomeric or tetrameric complex, centered around 8,500 m/z, undergoes collision induced disassociation. Activation results in the ejection of a highly charged monomer, centered around 3,500 m/z, and a monomer-stripped oligomer or oligomeric complex minus the ejected monomer, spanning the region 10,000–25,000 m/z.
Reference: Mass spectrometry of intact membrane protein complexes. Nat Protoc. 2013 Apr;8(4):639-51. doi: 10.1038/nprot.2013.024. Epub 2013 Mar 7.
