Strategy for identifying protein-protein interactions of gel-separated proteins and complexes by mass spectrometry

A strategy for identifying and characterizing protein interactions among gel-separated proteins and complexes has been developed and tested. The method involves the efficient recovery of proteins or complexes from native gels without affecting their conformational integrity. The use of limited proteolysis of protein complexes, isolated from the gel or formed from the interaction of gel-recovered proteins with potential binding partners, has enabled local binding domains to be efficiently identified using a combination of microfiltration and mass spectrometric analysis. The application of mass spectrometry affords high detection sensitivities, enabling the strategy to be applied to low levels of protein and protein mixtures. The approach is demonstrated for both antigen-antibody and peptide-protein complexes for which protein-binding regions are characterized among simple peptide mixtures and proteolytic digests. The strategy can be easily adapted to achieve high sample throughput and automation using gel-excision robotics and provides a means to study protein interactions in complex biological mixtures and extracts.