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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
Author(s) -
van Dongen W. D.,
van Bommel J. H.,
van Wassenaar P. D.,
Heerma W.,
Haverkamp J.
Publication year - 1994
Publication title -
biological mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1052-9306
DOI - 10.1002/bms.1200231105
Subject(s) - chromatography , fast atom bombardment , chemistry , subtilisin , high performance liquid chromatography , enzyme , protease , peptide sequence , amino acid , sequence (biology) , mass spectrometry , biochemistry , gene
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.