Proteomics Defines Protein Interaction Network of Signaling Pathways

Protein interactions play fundamental roles in signaling transduction. Analysis of protein–protein interaction (PPI) has contributed numerous insights to the understanding of the regulation of signal pathways. Different approaches have been used to discover PPI and characterize protein complexes. In addition to conventional PPI methods, such as yeast two-hybrid (YTH), affinity purification coupled with mass spectrometry (AP-MS) is emerging as an important and popular tool to unravel protein complex and elucidate protein function through the interaction partners. With the AP-MS method, protein complexes are prepared first by affinity purification directly from cell lysates, followed by characterization of their components by mass spectrometry. In contrast to most PPI methods, AP-MS reflects PPI under near physiological conditions in the relevant organism and cell type. AP-MS is also able to probe dynamic PPI dependent on protein posttranslational modifications, which is common for signal transduction. AP-MS mapping protein interaction network of various signal pathways has dramatically increased in recent years. Here, I’ll present the strategies toward obtaining an interactome map of signal pathway and the methodology, detailed protocols, and perspectives of AP-MS.