Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-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 accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).
From the ‡Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom;
Mark
M
Larance
From the ‡Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom;
Kathryn J
KJ
Kirkwood
§Biological Chemistry and Drug Discovery Division, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Michele
M
Tinti
From the ‡Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom;
Alejandro
A
Brenes Murillo
§Biological Chemistry and Drug Discovery Division, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Michael A J
MA
Ferguson
From the ‡Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom; a.i.lamond@dundee.ac.uk.
Angus I
AI
Lamond
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D003432:Cross-Linking Reagents; D008565:Membrane Proteins