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 Ba cillus subtilisin protease) produced by protein engineering were ident ified using High-performance Liquid Chromatographic/Fast Atom Bombardm ent Mass Spectrometric (HPLC/FAB MS) techniques. The variant and the h ighly homologous wild-type enzyme were treated with CNBr followed by t ryptic digestion. The resulting peptides were analysed using HPLC/frit FAB MS. The peptides with molecular masses beyond the range of the HP LC/MS system under the chosen scanning conditions were collected using HPLC and subsequently analysed 'off-line' using static FAB MS. This p rocedure allowed the complete amino acid sequence determination of the variant protease using the known amino acid sequence of the wild-type enzyme as reference.