High Throughput Affinity Purification and Mass Spectrometry to Determine Protein Complex Interactions

Abstract

Affinity purification, or immunoprecipitation, followed by mass spectrometry (AP/MS or IP/MS, respectively) is a proper and powerful method to analyze protein complexes. Technological capabilities of mass spectrometry instrumentation have dramatically improved over the last decade, and now allow routine detection of high- and low-abundance components in composite protein mixtures, expanding our ability to use co-immunoprecipitation to study cellular regulatory protein interaction networks. In this review, we summarize several key efforts and accomplishments in applying mass spectrometry towards mapping of a proteome-wide interactome. Emerging high-throughput studies begin to etch a ‘systems biology’ view of protein-protein interactions, where modularity and interconnectivity of cellular protein machinery are unequivocally revealed. Yet, major challenges still remain in the application of high-throughput (HT) affinity purification for studies in higher organisms, particularly in human cells, and in addressing persistent issues associated with analysis, interpretation, and dissemination of massive IP/MS data. We describe some of the conceptual and practical challenges of the HT-IP/MS approach, and discuss feasible solutions in context of the protein complexes involved in transcriptional regulation.