BioPlex: an ORFeome-based, Mass Spectrometry-driven, Human Protein Interaction Network
Because a cell's phenotype reflects its underlying proteome, mass-spectrometry-enabled proteomic studies can provide essential biological insights. Whereas surveys of protein expression and select post-translational modifications have achieved near-comprehensive scope, mass-spectrometry-based protein interaction profiles have typically examined small protein families while leaving vast swaths of the interaction landscape unexplored. To achieve more comprehensive examination, we have established a high-throughput pipeline capable of elucidating interactions for several hundred baits per month and are now systematically mapping human protein interactions at unparalleled depth and breadth. The emerging interaction network is providing unique insights into both normal and pathological biological processes by revealing novel functions for familiar proteins and providing tantalizing clues into roles of unknown proteins.
Our high-throughput platform for protein interaction profiling relies upon expressing HA-tagged versions of human proteins within HEK293T cells; following immunoprecipitation, the baits and their interacting partners are identified using Q-Exactive mass spectrometers aided by customized data analysis techniques. To date we have completed analysis of over 3,200 bait proteins and their interacting partners, defining an interaction network that spans 33,000 interactions among over 8,000 proteins. The resulting network reveals clusters matching known subcellular structures such as the proteasome, signalsome, and mediator complexes, while suggesting biological functions for unknown proteins and highlighting shared functional and regulatory clusters across the interactome. Whether viewed individually or in aggregate, these interaction profiles offer unique insights into both known and unknown proteins while also illuminating larger patterns of proteomic regulation.