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.
Andersen
J S
JS
Department of Molecular Biology, Odense University, Denmark.
Søgaard
M
M
Svensson
B
B
Roepstorff
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003488:Cyanogen Bromide; D018744:DNA, Plant / Q000032:analysis; D005947:Glucose / Q000032:analysis; D001467:Hordeum / Q000201:enzymology*; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D000516:alpha-Amylases / Q000096:biosynthesis / Q000737:chemistry*
D018744:DNA, Plant; D010455:Peptides; D011994:Recombinant Proteins; D000516:alpha-Amylases; D005947:Glucose; D003488:Cyanogen Bromide