Abstract
Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.
Highlights
In cells, most proteins perform their functions through transitory protein-protein interactions or by forming stable protein assemblies
Native protein complexes around a protein of interest are isolated by affinity purification using antibodies against an epitope tag and competitive elution
The complexes are resolved by Blue native polyacrylamide gel electrophoresis (BN-PAGE), and the whole gel lane is excised into 48 sections, and prepared for shotgun LC-MS/MS
Summary
Most proteins perform their functions through transitory protein-protein interactions or by forming stable protein assemblies. The advent of MS-cleavable cross-linking reagents should facilitate mapping of the amino acid residues that are in close proximity in interacting proteins [6,7] Another alternative is to combine affinity purification with prior orthogonal separation techniques [8,9]. Blue native polyacrylamide gel electrophoresis (BN-PAGE) has been widely applied to investigate native protein interactions, typically those involving mitochondrial membrane protein complexes 14 This separation technique was recently used in combination with label-free protein quantification and correlation profiling, on mitochondrial complexes [15,16,17], and for unravelling other protein complexes from whole cells [18,19]. Www.jove.com multiple interactions a protein engages in, resolving them into distinct assemblies based on their correlation profiles, whilst requiring no more work than conventional geLC-MS 20
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
