Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification ofyeast proteins separated by two-dimensional gel electrophoresis

Current analytical techniques in protein identification by mass spectrometr y are based on the generation of peptide mass maps or sequence tags that ar e idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in rese arch on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assi sted laser desorptior/ionization time-of-flight (MALDI-TOF) mass mapping an d liquid chromatography/quadrupole time-of-flight electrospray ionization t andem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in ident ification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots were selected for analysis in order t o collect information for future studies, to cover the whole pl range from 3 to 10, and to evaluate information from spots of different intensities. M ass mapping as a rapid, high-throughput method was in most cases sensitive enough for identification. LC/MS/MS was found to be more sensitive and to p rovide more accurate data, and was very useful when analyzing small amounts of sample. Even one sequence tag acquired by this method could be enough f or unambiguous identification, and, in the present case, successfully ident ified a point mutation. Copyright (C) 2001 John Wiley & Sons, Ltd.