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 of yeast proteins separated by two-dimensional gel electrophoresis.
Current analytical techniques in protein identification by mass spectrometry are based on the generation of peptide mass maps or sequence tags that are idiotypic for the protein sequence. This work reports on the development of the use of mass spectrometric methods for protein identification in research on metabolic pathways of a genetically modified strain of the baker's yeast Saccharomyces cerevisiae. This study describes the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag analysis in identification of yeast proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots were selected for analysis in order to collect information for future studies, to cover the whole pI range from 3 to 10, and to evaluate information from spots of different intensities. Mass 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 provide more accurate data, and was very useful when analyzing small amounts of sample. Even one sequence tag acquired by this method could be enough for unambiguous identification, and, in the present case, successfully identified a point mutation.
Poutanen
M
M
Institute of Biotechnology, P.O. Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland. marjo.poutanen@helsinki.fi
Salusjärvi
L
L
Ruohonen
L
L
Penttilä
M
M
Kalkkinen
N
N
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry*; D006868:Hydrolysis; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D016324:Sequence Tagged Sites; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
D005656:Fungal Proteins; D014357:Trypsin