LOCALIZATION OF AN O-GLYCOSYLATED SITE IN THE RECOMBINANT BARLEY ALPHA-AMYLASE-1 PRODUCED IN YEAST AND CORRECTION OF THE AMINO-ACID-SEQUENCE USING MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY OF PEPTIDE MIXTURES

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-M S) of peptide mixtures was used to characterize recombinant barley alp ha-amylase 1, produced in yeast. Three peptide mixtures were generated by cleavage with CNBr, digestion with endoproteinase Lys-C and Asp-N, respectively, and analyzed directly by MALDI-MS. Based on the three m ass spectrometric peptide maps, an error in the sequence deduced from cDNA, resulting in a mass difference of 28 Da, was located to a sequen ce stretch of 5 amino acid residues; furthermore, a dihexose substitue nt was identified on Thr(410). Subsequent Edman degradation of two sel ected peptides isolated from the endoproteinase Lys-C digest corrected the sequence to be Val instead of Ala in position 284 and confirmed t he O-glycosylation. These results demonstrate that the direct peptide mixture analysis by MALDI-MS is a rapid and sensitive method for prote in characterization and provides valuable information before further c haracterization.