Journal
PROTEOMICS
Volume 12, Issue 10, Pages 1623-1638Publisher
WILEY
DOI: 10.1002/pmic.201100438
Keywords
Affinity purification; Animal proteomics; Interactome; Isotopes; Quantitative Proteomics; SILAC
Funding
- Canadian Cancer Research/Terry Fox Foundation [20148]
- NSERC [372370]
- CIHR
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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 proteinprotein 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.
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