title: Mass spectrometric analysis of peptides from an immobilized lipase: focus on oxidative modifications.
abstract: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to study the primary structure of immobilized Candida antarctica lipase B (Novozym(R)435) without detaching the enzyme from the carrier. The immobilized enzyme packed in a miniature column was subjected to proteolysis and the peptides released were injected into the mass spectrometer for analysis. The set-up was utilized to determine amino acid oxidation after treatment of the biocatalyst with hydrogen peroxide. In total, sequence coverage of more than 90% was obtained, containing almost all of the amino acids sensitive to oxidation. Oxidation of methionine, tryptophan and cystine residues was observed. The flow system also allowed evaluation of the enzyme activity prior to peptide analysis. The developed method is general and should be applicable to other immobilized enzyme systems and to different treatments.
authors.affiliation: Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. ulrika.tornvall@biotek.lu.se
authors.forename: Ulrika
authors.initials: U
authors.lastname: Törnvall
authors.forename: Camilla Melin
authors.initials: CM
authors.lastname: Fürst
authors.forename: Rajni
authors.initials: R
authors.lastname: Hatti-Kaul
authors.forename: Martin
authors.initials: M
authors.lastname: Hedström
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins; D008049:Lipase / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D010455:Peptides; D008049:Lipase; C431349:lipase B, Candida antarctica