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

✍ Scribed by Christian Pasquali; Francis Vilbois; Marie-Laure Curchod; Rob Hooft van Huijsduijnen; Fabrizio Arigoni

• Publisher: John Wiley and Sons
• Year: 2000
• Tongue: English
• Weight: 766 KB
• Volume: 21
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/1522-2683(20001001)21:16<3357::aid-elps3357>3.0.co;2-l

✦ Synopsis

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 comprehensive identification of substrates for poorly characterized human protein tyrosine phosphatases (PTPs). We took advantage of so-called "substrate trapping" mutants, a procedure originally described by Flint et al. (Proc. Natl. Acad. Sci. USA 1997, 94, 1680-1685) to identify binding partners of cloned PTPs. This procedure was adapted to a proteome-wide approach to probe for candidate substrates in cellular extracts that were separated by two-dimensional (2-D) gel electrophoresis and blotted onto membranes. Protein-protein interactions were revealed by far-Western immunoblotting and positive binding proteins were subsequently identified from silver-stained gels using tandem mass spectrometry. With this method we were able to identify possible substrates for PTPs without using any radio-labeled cDNA or protein probes and showed that they corresponded to tyrosine phosphorylated proteins. We believe that this method could be generally applied to identify possible protein-protein interactions.