Identification of two-dimensional gel electrophoresis resolved yeast proteins by matrix-assisted laser desorption ionization mass spectrometry.
Protein extract from yeast cells growing exponentially in saline medium was separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with the separation in the first dimension on a wide range immobilized pH (3-10) gradient. From one preparative 2-D gel a number of previously identified proteins were used as test material for our initial matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) efforts on large scale rapid protein spot identification. Sample preparation via in-gel trypsin digestion was slightly modified to be compatible to MS analysis, and via this modified procedure MS generated peptide mass profiles could, in most cases with good precision, identify the protein in question. Preferential ionization was tested on a yeast aldehyde dehydrogenase (ALD7), and it was shown that the ionization of some peptides was clearly suppressed by the presence of others. Roughly 50% of the observed peptide masses was found by the search routines in the database, and the mass measurement accuracy of the peptides was within 0.5 Da. Silver-stained gels could be used with good results for the generation of peptides to be analyzed by MALDI-MS. For one of the 2-D resolved proteins, glycerol 3-phosphatase (GPP1), the post-source decay (PSD) spectrum proved crucial in identification.
Larsson
T
T
Göteborg University, Sweden.
Norbeck
J
J
Karlsson
H
H
Karlsson
K A
KA
Blomberg
A
A
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000032:analysis*; D005782:Gels; D007202:Indicators and Reagents / Q000737:chemistry; D007477:Ions; D008969:Molecular Sequence Data; D012394:Rosaniline Dyes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005656:Fungal Proteins; D005782:Gels; D007202:Indicators and Reagents; D007477:Ions; D012394:Rosaniline Dyes; C048139:Coomassie blue