Combination of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Substrate Trapping for the Detection of Transient Protein Interactions.

Antibody-based affinity purification is a recognized method for use in studying protein-protein interactions. There are four different classes of proteins that are typically identified with such affinity purification workflows: bait protein, proteins that specifically interact with the bait protein, proteins nonspecifically associated with the antibody, and proteins that cross-react with the antibody. Mass spectrometry can be used to differentiate these classes of proteins in affinity-purified mixtures. Here we describe the use of stable isotope labeling by amino acids in cell culture, substrate trapping, and mass spectrometry to enable the objective identification of the components of affinity-purified protein complexes.