Figure 2

The isolation of protein complexes and the identification of components. (a) Approaches for the isolation of protein complexes. Prior to the MS-based identification of individual polypeptides, physically associated protein complexes can be isolated from crude extracts using either: (i) co-purification (AP) of stably associated protein interactors of a tagged bait protein that is expressed in a cell; (ii) antibody-based pull-down (co-IP) of complexes containing a protein target of interest; or (iii) biochemical co-fractionation of protein complexes using native chromatographic separation. (b) Liquid chromatography (LC)-MS-based identification is then performed to characterize the co-purifying protein complex components. (i) Proteins are initially cleaved by a protease (normally trypsin) to generate peptides, which are subjected to reverse-phase LC separation followed by electrospray ionization prior to MS analysis. (ii) In the first mass analyzer (MS1) charged peptides with the highest intensity are sequentially selected (one by one) for collision-induced fragmentation. The second mass analyzer (MS2) records the mass of peptide fragments (with signal peaks expressed as mass to charge ratios (m/z)). (iii) MS1 and MS2 data for each peptide are then used together to search a cognate protein sequence database to produce a list of confidently identified peptides and proteins.