Protein correlation profiling-SILAC to study protein-protein interactions.
Methods Mol Biol
; 1188: 263-70, 2014.
Article
in En
| MEDLINE
| ID: mdl-25059617
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.
Full text:
1
Database:
MEDLINE
Main subject:
Proteins
/
Protein Interaction Mapping
/
Amino Acids
/
Isotope Labeling
Limits:
Humans
Language:
En
Year:
2014
Type:
Article