Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry.
Two-dimensional gel electrophoresis, bioinformatics, and mass spectrometry are key analysis tools in proteome analysis. The further characterization of post-translational modifications in gel-separated proteins relies fully on data obtained by mass spectrometric analysis. In this study, stress-induced changes in protein expression in Saccharomyces serevisiae were investigated. A total of eleven spots on a silver-stained two-dimensional (2-D) gel were identified by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping to represent C and/or N-terminal processed forms of enolase 2. The processing sites were determined by MALDI peptide mass mapping using a variety of proteolytic enzymes, by optimizing the sample preparation procedure and by specific labeling of all C-termini derived from in-gel digestion using a buffer containing 16O:18O (1:1). Out of eleven processed forms of enolase 2, six were fully characterized and the approximate processing sites identified for the remaining five.
Larsen
M R
MR
Department of Biochemistry Molecular Biology, University of Southern Denmark, Odense University.
Larsen
P M
PM
Fey
S J
SJ
Roepstorff
P
P
D000595:Amino Acid Sequence; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D010450:Endopeptidases; D007553:Isotope Labeling; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods; D010751:Phosphopyruvate Hydratase / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin
D010450:Endopeptidases; D014357:Trypsin; D010751:Phosphopyruvate Hydratase