| Proteomic and immunoblotting methods were developed to use a tethered metal chelate as a probe to study protein-protein interactions. Tethered proteases have been used extensively to study sigma 70 contacts on E. coli RNA polymerase and other biomolecular interactions associated with transcription. In a typical experiment, a probe is attached to one interacting species and is activated through a series of redox reactions to chemically cleave and/or oxidatively modify its partner. In the case of cleavage, fragments are resolved via SDS-PAGE and identified by immunoblotting methods. Recently, a chemical tagging strategy was developed that allows tandem mass spectrometric identification of the stable carbonyl products resulting from oxidative marking by the probe. A library of single-cysteine mutants of sigma 70 was used to examine the effect of tethering the chelate to different regions of a protein on the oxidation pattern. Also, the binding of sigma 70 on E.coli RNA polymerase was used as a model to apply the method to the study protein-protein interactions. |
