Abstract
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).
Highlights
Proteins rarely work as monomers to carry out all the biological processes needed for cells to function
To improve the efficiency of chromatography-MS based global analyses of protein complexes and circumvent the under-representation of membrane complexes and complexes tightly bound to cell substructures, we have developed a methodology that combines in vivo protein crosslinking prior to cell lysis with subsequent size-exclusion chromatography (SEC) fractionation and MS analysis (Fig. 1A)
To determine a suitable amount of formaldehyde for use in U2OS cells, we first titrated the concentration applied to intact, adherent U2OS cells, aiming for a concentration of formaldehyde that resulted in isolation of tubulin as predominantly multimers i.e. larger than dimers, while simultaneously recovering GAPDH predominantly in complexes not larger than tetramers (Fig. 1B)
Summary
Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*□S. The methods available for the proteome-wide analysis of protein interactions have developed swiftly over the last ten years This field is dominated by affinity-enrichment based approaches, using either tagged constructs, or antibodies specific for endogenous proteins. Each of these affinity enrichments will be performed in only one type of buffer system, which is unlikely to be compatible with the maintenance of all protein-protein interactions Another dimension to the analytical problem is that many proteins are expressed as different sized isoforms and/or in different post-translationally modified forms, resulting in formation of multiple, related, but functionally distinct complexes, with different combinations of interaction partners [6]. Using affinity-enrichment/pull-down methods alone makes it difficult to resolve such mixtures of different forms of related protein complexes, complicating a detailed understanding of biological response mechanisms
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
