Global Membrane Protein Interactome Analysis using In vivo Crosslinking and MS-based Protein Correlation Profiling

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes, in addition to soluble complexes, allowing identification and analysis of complexes not previously accessible in native extracts. Using this approach, we demonstrate how these data may be applied to the analysis of membrane protein complexes and to discriminate between interactions of different protein isoforms. The entire proteome-wide dataset of in vivo crosslinked protein complexes from U2OS cells is available via a searchable online database (www.peptracker.com/epd).