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.

Jens S. Andersen, M Søgaard, B Svensson, P Roepstorff

Research output: Contribution to journalJournal articleResearchpeer-review

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 languageEnglish
JournalBiological Mass Spectrometry
Volume23
Issue number9
Pages (from-to)547-54
Number of pages7
ISSN1052-9306
DOIs
Publication statusPublished - 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

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