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Mol Cell Proteomics. 2014 Jun;13(6):1510-22. doi: 10.1074/mcp.M113.033902. Epub 2014 Apr 10.

Controlling for gene expression changes in transcription factor protein networks.

Author information

1
From the ‡Stowers Institute for Medical Research, Kansas City, Missouri 64110;
2
From the ‡Stowers Institute for Medical Research, Kansas City, Missouri 64110; §Departments of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas 66160 mpw@stowers.org.

Abstract

The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein-protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions.

PMID:
24722732
PMCID:
PMC4047470
DOI:
10.1074/mcp.M113.033902
[Indexed for MEDLINE]
Free PMC Article
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