Abstract
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.
Original language | English |
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Journal | Biological Mass Spectrometry |
Volume | 23 |
Issue number | 9 |
Pages (from-to) | 547-54 |
Number of pages | 7 |
ISSN | 1052-9306 |
DOIs | |
Publication status | Published - 1. Sept 1994 |
Keywords
- Amino Acid Sequence
- Animals
- Base Sequence
- Chromatography, High Pressure Liquid
- Cricetinae
- Cyanogen Bromide
- DNA, Plant
- Glucose
- Hordeum
- Lasers
- Mass Spectrometry
- Molecular Sequence Data
- Peptides
- Recombinant Proteins
- Saccharomyces cerevisiae
- Spectrophotometry, Ultraviolet
- alpha-Amylase