I-DIRT: A Novel Method to Distinguish Specific Protein Interactions from Contaminants in Biological Complexes
There have been significant advances in protein research, specifically in the increased ease in isolating protein complexes from biological systems of interest using an affinity-tagged member, and then identifying the constituents of those same complexes using mass spectrometry. The main problem is the co-enrichment of proteins that associate non-specifically with the affinity-tagged complex of interest.
Our scientists have developed a method to overcome this challenge using metabolic labeling, termed Isotopic Differentiation of Interactions as Random or Targeted or I-DIRT for short. First, cells that contain the affinity-tagged protein are grown in regular media, while cells that do not contain the tagged protein are grown in a heavy isotopic medium so that the resulting proteins are labeled at a specific amino acid with a heavier isotope that can be distinguished easily as a visible mass shift during mass spectrometry. The two cell cultures are combined in equivalent amounts, lysed, and a standard immunoisolation procedure is used to obtain the affinity-tagged protein of interest along with its partners. The resulting complex is then digested and analyzed by mass spectrometry to identify those proteins that have a large ratio (60% or more) of light isotopes, which are the proteins that specifically interact with the tagged member.
This method enables researchers to isolate protein complexes under non-stringent conditions, thus preserving the integrity of the complex, while allowing for the discrimination between specific and non-specific interactors within the complex. This method is straightforward, efficient, and adaptable to most biological systems that commonly use immunoisolation methods.
Area of Application
Proteomic research, specifically the analysis of isolated protein complexes from biological systems utilizing mass spectrometry for individual protein identification.
Stage of Development
Proof of concept – the method has been used to successfully identify specific interactions within a yeast DNA polymerase complex and a bacterial RNA polymerase complex.
Brian Chait & Dr. Michael Rout
Patent Information and References
- S. Patents 7,968,299 (issued June 28, 2011) and 8,227,198 (issued July 24, 2012)
- Tackett, et al. 2005. Prot. Res., vol. 4:1752-1756.
- Lee, et a 2008. J. Bacteriol., vol. 190:1284-1289.