Localization of anO-glycosylated site in the recombinant barley α-amylase 1 produced in yeast and correction of the amino acid sequence using matrix-assisted laser desorption/ionization mass spectrometry of peptide mixtures

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Abstract
Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) of peptide mixtures was used to characterize recombinant barley α‐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 MALDIMS. Based on the three mass spectrometric peptide maps, an error in the sequence deduced from cDNA, resulting in a mass difference of 28 Da, was located to a sequence stretch of 5 amino acid residues; furthermore, a dihexose substituent was identified on Thr410. Subsequent Edman degradation of two selected peptides isolated from the endoproteinase Lys‐C digest corrected the sequence to be Val instead of Ala in position 284 and confirmed the O‐glycosylation. These results demonstrate that the direct peptide mixture analysis by MALDI‐MS is a rapid and sensitive method for protein characterization and provides valuable information before further characterization.