Mapping and identification of protein-protein interactions by two-dimensional far-Western immunoblotting

Studies of protein-protein interactions have proved to be a useful approach to link proteins of unknown function to known cellular processes. In this study we have combined several existing methods to attempt the comprehensiv e identification of substrates for poorly characterized human protein tyros ine phosphatases (PTPs). We took advantage of so-called "substrate trapping " mutants, a procedure originally described by Flint et al. (Proc. Natl. Ac ad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PT Ps. This procedure was adapted to a proteome-wide approach to probe for can didate substrates in cellular extracts that were separated by two-dimension al (2-D) gel electrophoresis and blotted onto membranes. Protein-protein in teractions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tande m mass spectrometry. With this method we were able to identify possible sub strates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We beli eve that this method could be generally applied to identify possible protei n-protein interactions.