Mapping Protein–Protein Interactions by Quantitative Proteomics - LC-MS/MS in Proteomics: Methods and Applications - page 267

Fig. 16.1 Generic approach for the analysis of protein interactions using quantitative MS. ( A ) Cells are SILAC labeled
using Arg0/Lys0 (light) and Arg10/Lys8 (heavy), respectively. Light and heavy labeled cells are differentially manipulated,
as required by the experiments set up, mixed at equal proportions, and the protein complexes of interest are purified
by a suitable affinity purification procedure. Purified complexes can by digested in-solution and directly analyzed by
LC-MS/MS. Alternatively, the eluted protein complexes can first be separated by SDS-PAGE. The whole lane is then
cut in slices, proteins are digested using trypsin, and resulting peptide mixtures are analyzed by LC-MS/MS. ( B ) The
experimental design follows the one outlined in panel ( A ). However, affinity purifications are carried out in parallel using,
e.g., either unmodified or PTM-modified version of a specific peptide as bait. This approach is also useful for analysis
of weak interactions with fast exchange rates. Performing the purification step in separate tubes allows to preserve the
ratios of such dynamic binding partners. However, the standard deviations of the ratios of unspecific binders are generally
higher in these experiments. ( C ) Exemplified results. Each of the identified proteins is represented as a diamond and
plotted based on the observed SILAC ratio. Specific interaction partners show ratios significantly different from 1 (gray
diamonds) and can be easily distinguished from the large pool of unspecific binders (blackdiamonds) as those maintain
ratios similar to the original mixing ratio.