DIRECT ANALYSIS AND IDENTIFICATION OF PROTEINS IN MIXTURES BY LC MS/MS AND DATABASE SEARCHING AT THE LOW-FEMTOMOLE LEVEL/

A method to directly identify proteins contained in mixtures by microc olumn reversed-phase liquid chromatography electrospray ionization tan dem mass spectrometry (LC/MS/MS) is studied. In this method, the mixtu re of, proteins is digested with a proteolytic enzyme to produce a lar ge collection of peptides. ?Pie complex peptide mixture is then separa ted on-line with a tandem mass spectrometer, acquiring large numbers o f tandem mass spectra, The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from sta ndard protein mixtures show that proteins present in simple mixtures c an be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within th e mixture can be identified at low femtomole levels. Based on these st udies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interactio n with a protein-protein fusion product, and specific interaction with a macromolecular complex, The approach described in this article prov ides a rapid method for the direct identification of proteins in mixtu res.