title: Resolving protein interactions and complexes by affinity purification followed by label-based quantitative mass spectrometry.
abstract: Label-based quantitative mass spectrometry analysis of affinity purified complexes, with its built-in negative controls and relative ease of use, is an increasingly popular choice for defining protein-protein interactions and multiprotein complexes. This approach, which differentially labels proteins/peptides from two or more populations and combines them prior to analysis, permits direct comparison of a protein pulldown (e.g. affinity purified tagged protein) to that of a control pulldown (e.g. affinity purified tag alone) in a single mass spectrometry (MS) run, thus avoiding the variability inherent in separate runs. The use of quantitative techniques has been driven in large part by significant improvements in the resolution and sensitivity of high-end mass spectrometers. Importantly, the availability of commercial reagents and open source identification/quantification software has made these powerful techniques accessible to nonspecialists. Benefits and drawbacks of the most popular labeling-based approaches are discussed here, and key steps/strategies for the use of labeling in quantitative immunoprecipitation experiments detailed.
authors.affiliation: Department of Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON, Canada. ltrinkle@uottawa.ca
authors.forename: Laura
authors.initials: L
authors.lastname: Trinkle-Mulcahy
mesh_terms: D000345:Affinity Labels; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*
chemical_list: D000345:Affinity Labels; D011506:Proteins