This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Isolation of protein complexes via affinity-tagged proteins provides a powerful tool for studying biological systems, but the technique is often compromised by co-enrichment of non-specifically interacting proteins. We developed a new technique (I-DIRT) that distinguishes contaminants from bona fide interactors in immunopurifications, overcoming this most challenging problem in defining protein complexes. The basic principle of this technique is mixing isotopically light cells that contain an affinity-tagged protein with isotopically heavy cells that do not contain a tagged protein. After isolation of the affinity-tagged protein complex and trypsin digestion of the co-enriching proteins, specifically interacting proteins are identified by mass spectrometry as isotopically light, whilst non-specifically interacting proteins appear as a mixture of isotopically light and heavy. We have published this work in J. Proteome Res. (2005) 4, 1752-6 and have also published applications of this work (e.g., D.J. Lee, S.J.W. Busby, L.F. Westblade, B.T. Chait Immunoisolation and I-DIRT mass spectrometric analysis of the Escherichia coli O157:H7 Sakai RNA polymerase complex. J. Bacteriology 190 (2008) 1284-1289). We continue to investigate the finer details of this technique, with an emphasis on evaluating the effect of specific interactors that have relatively high offrates.
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