PROTEIN IDENTIFICATION BY MASS SPECTRAL ANALYSIS OF PEPTIDES WITH SUBSEQUENT DATABASE SEARCHING HAS BECOME AN IMPORTANT TECHNIQUE IN A NUMBER OF LABORATORIES. THE SUCCESS OF THIS METHODOLOGY IS DUE TO RECENT IMPROVEMENTS IN MASS SPECTROMETRY, IMPROVEMENTS IN SAMPLE HANDLING, IMPROVEMENTS IN SEARCHING ALGORITHMS, AND THE DRAMATIC GROWTH IN THE SEQUENCE DATABASES. MASS SPECTROMETRY WILL BE A VITAL PART OF THE RESOURCE. IT WILL BE USED TO IDENTIFY PROTEINS FROM 2D GELS FOLLOWING PROTEOLYTIC DIGESTION VIA ESTABLISHED PROTOCOLS, SOME DEVELOPED BY DR. AEBERSOLD. MASS SPECTROMETRY WILL UTILIZE AN ELECTROSPRAY IONIZATION-ION TRAP MASS SPECTROMETER WITH THE PEPTIDE MIXTURES INTRODUCED BY CAPILLARY LC OR CAPILLARY ELECTROPHORESIS. AUTOMATED, ON-LINE SEPARATION WITH TANDEM MASS SPECTROMETRY WILL PRODUCE FRAGMENTATION SPECTRA FOR EACH PEPTIDE. EACH SPECTRUM IS USED TO SEARCH A SEQUENCE DATABASE AUTOMATICALLY WITH A PROGRAM DEVELOPED BY DR. YATES LABORATORY, AUTOSEQUEST. IN ADDITION TO THE PROTEIN PREPARATION BY 2D GEL ELECTROPHORESESIS, PROTEINS MAY ALSO BE PREPARED BY SOME DEGREE OF ENRICHMENT, E.G., IMMUNOPRECIPITATION OR AFFINITY CHOMATOGRAPHY. THE MIXTURE ANALYSIS CAPABILITIES OF DR. YATES DATABASE SEARCHING APPROACH MAKE THIS LATTER APPROACH POSSIBLE WITHOUT HIGH RESOLUTION PROTEIN SEPARATION.
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