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.
|Journal||Biological Mass Spectrometry|
|Publication status||Published - 1994|
Andersen, J. S., Søgaard, M., Svensson, B., & Roepstorff, P. (1994). 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. Biological Mass Spectrometry, 23, 547-554. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abstract&list_uids=7948047&query_hl=21&itool=pubmed_docsum