Identification of water-soluble selenium-containing proteins in selenized yeast by size-exclusion-reversed-phase HPLC/ICPMS followed by MALDI-TOF and electrospray Q-TOF mass spectrometry.
An approach to speciation of selenium incorporated in yeast proteins was developed. The tryptic digest of a water-soluble protein fraction isolated by size-exclusion chromatography was analyzed by reversed-phase HPLC/ICPMS. The selenopeptides selected owing to the detector's elemental specificity were then analyzed by MALDI-TOFMS in order to select target ions for collision-induced dissociation MS. The latter, carried out with an electrospray Q-TOF spectrometer, enabled the sequencing of the selenopeptides detected by HPLC/ICPMS. The approach allowed for the first time the identification of a family of Se-containing proteins resulting from the replacement by selenomethionine of 2-9 methionine residues in a salt-stress-induced protein SIP18 (Mr 8874). The presence of these proteins was confirmed by MALDI-TOFMS of the original (nondigested) protein fraction. Another selenium protein identified was a heat-shock protein HSP12 (Mr 11693) in which the only methionine residue was replaced by selenomethionine. These two Se-containing proteins accounted for more than 95% of selenium in the water-soluble protein fraction.
Encinar
Jorge Ruiz
JR
CNRS UMR 5034, Hélioparc, 2 Avenue Pr. Angot, F-64053 Pau, France.
Ouerdane
Laurent
L
Buchmann
William
W
Tortajada
Jeanine
J
Lobinski
Ryszard
R
Szpunar
Joanna
J
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006360:Heat-Shock Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D012643:Selenium / Q000032:analysis* / Q000737:chemistry; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*
D006360:Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D012643:Selenium