Format

Send to

Choose Destination
See comment in PubMed Commons below
Anal Chem. 2011 Jul 15;83(14):5511-8. doi: 10.1021/ac200950k. Epub 2011 Jun 15.

Stable isotope labeling with amino acids in cell culture based mass spectrometry approach to detect transient protein interactions using substrate trapping.

Author information

1
Department of Radiation Oncology, University of Maryland, Baltimore, Maryland, United States. stthomas@som.umaryland.edu

Abstract

The analysis of protein interactors in protein complexes can yield important insight into protein function and signal transduction. Thus, a reliable approach to distinguish true interactors from nonspecific interacting proteins is of utmost importance for accurate data interpretation. Although stringent purification methods are critical, challenges still remain in the selection of criteria that will permit the objective differentiation of true members of the protein complex from nonspecific background proteins. To address these challenges, we have developed a quantitative proteomic strategy combining stable isotope labeling with amino acids in cell culture (SILAC), affinity substrate trapping, and gel electrophoresis followed by liquid chromatography-tandem mass spectrometry (geLC-MS/MS) protein quantitation. ATP hydrolysis-deficient vacuolar protein sorting-associated protein 4B (Vps4B) was used as the "bait" protein which served as a substrate trap since its lack of ATP hydrolysis enzymatic activity allows the stabilization of its transiently associated interacting proteins. A significant advantage of our approach is the use of our new in-house-developed software program for SILAC-based mass spectrometry quantitation, which further facilitates the differentiation between the bait protein, endogenous bait-interacting proteins, and nonspecific binding proteins based on their protein ratios. The strategy presented herein is applicable to the analysis of other protein complexes whose compositions are dependent upon the ATP hydrolysis activity of the bait protein used in affinity purification studies.

PMID:
21619060
PMCID:
PMC3136636
DOI:
10.1021/ac200950k
[Indexed for MEDLINE]
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for American Chemical Society Icon for PubMed Central
    Loading ...
    Support Center