title: Protein correlation profiling-SILAC to study protein-protein interactions.
abstract: An interactome describes the global organization of protein interactions within a cell and is typically generated using affinity purification-mass spectrometry (AP-MS), yeast two-hybrid screening, or protein-fragment complementation assays (Gavin et al. Nature 440: 631-636, 2006; Krogan et al. Nature 440: 637-643, 2006; Uetz et al. Nature 403: 623-627, 2000; Tarassov et al. Science 320: 1465-1470, 2008). These techniques have been widely used to depict the interactome as we know it today but current models of interactomes do not contain stoichiometric or temporal information. In this chapter we describe size-exclusion chromatography (SEC) combined with protein correlation profiling-stable isotope labeling by amino acids in cell culture (PCP-SILAC) to generate dynamic chromatographs for thousands of proteins (Kristensen et al. Nat Methods 9: 907-909, 2012). Using the precise co-elution of two proteins as evidence that they interact, it is possible to identify similar numbers of protein interactions without overexpression or creating fusion proteins as other high-throughput techniques require. In addition, triplex SILAC allows us to quantify protein stoichiometry and temporal changes to the interactome following perturbation. Finally, SEC-PCP-SILAC is very time efficient since it generates two orders of magnitude fewer samples for LC-MS analysis and avoids the tedious tagging and purification steps, making it possible for everyone with a single mass spectrometer to study the interactome.
authors: Department of Biochemistry & Molecular Biology and Centre for High-Throughput Biology, University of British Columbia, 2125 East Mall, Vancouver, BC, Canada, V6T 1Z4.
authors: Anders R
authors: AR
authors: Kristensen
authors: Leonard J
authors: LJ
authors: Foster
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D053000:Analytic Sample Preparation Methods; D002850:Chromatography, Gel; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D014357:Trypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D011506:Proteins; D014357:Trypsin