Rapid identification of specific mutations in the sequence of an enzyme variant produced by protein engineering using high-performance liquid chromatographic/fast atom bombardment mass spectrometric techniques.
Unknown, specific mutations in the sequence of an enzyme variant (a Bacillus subtilisin protease) produced by protein engineering were identified using High-performance Liquid Chromatographic/Fast Atom Bombardment Mass Spectrometric (HPLC/FAB MS) techniques. The variant and the highly homologous wild-type enzyme were treated with CNBr followed by tryptic digestion. The resulting peptides were analysed using HPLC/frit FAB MS. The peptides with molecular masses beyond the range of the HPLC/MS system under the chosen scanning conditions were collected using HPLC and subsequently analysed 'off-line' using static FAB MS. This procedure allowed the complete amino acid sequence determination of the variant protease using the known amino acid sequence of the wild-type enzyme as reference.
van Dongen
W D
WD
Bijvoet Center for Biomolecular Research, Department of Mass Spectrometry, Utrecht University, The Netherlands.
van Bommel
J H
JH
van Wassenaar
P D
PD
Heerma
W
W
Haverkamp
J
J
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D005818:Genetic Engineering; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013381:Subtilisins / Q000032:analysis / Q000235:genetics*; D014357:Trypsin
D010446:Peptide Fragments; D011994:Recombinant Proteins; D013381:Subtilisins; D014357:Trypsin; D003488:Cyanogen Bromide