A STRATEGY FOR RAPID, HIGH CONFIDENCE PROTEIN IDENTIFICATION

A procedure is described for rapid, high-confidence identification of proteins using matrix-assisted laser desorption/ionization tandem ion trap mass spectrometry in conjunction with a genome database searching strategy. The procedure involves excision of copper-stained bands or spots from electrophoretic gels, in-gel trypsin digestion of the prote ins, single-stage mass spectrometric analysis of the resultant mixture of tryptic peptides, followed by tandem ion trap mass spectrometric a nalysis of selected individual peptides, and database searching of the relevant genomic database using the program PepFrag. The scheme provi des sensitive, real-time protein identification as well. As facile ide ntification of modifications, A single operator can unambiguously iden tify 5-10 proteins/day from an organism whose genome is known at a lev el of >0.5 pmol of protein loaded on a gel. The utility of the techniq ue was demonstrated by the identification and characterization of a ba nd from a human HTLV-I preparation and 11 different proteins from a ye ast RNA polymerase II C-terminal repeat domain-affinity preparation. T he technology has great potential for postgenome biological science, w here it promises to facilitate the dissection and anatomy of macromole cular assemblages, the definition of disease state markers, and the in vestigation of protein targets in biological processes such as the cel l cycle and signal transduction.