Journal article
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-MS) of peptide mixtures was used to characterize recombinant barley alpha-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 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.
Language: | English |
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Year: | 1994 |
Pages: | 547-54 |
ISSN: | 10969888 , 10765174 , 10529306 and 23763876 |
Types: | Journal article |
DOI: | 10.1002/bms.1200230904 |