An approach to speciation of selenium incorporated in yeast proteins was developed. ne 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 (M-r 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 (M-r, 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.
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