Experimental design-guided development of a stereospecific capillary electrophoresis assay for methionine sulfoxide reductase enzymes using a diastereomeric pentapeptide substrate

• The first assay for measuring the stereospecificity of methionine sulfoxide reductase enzymes using a peptide substrate has been developed.
• Diastereomer resolution was achieved by cooperative effects of sulfated β-cyclodextrin and the achiral crown ether 15-crown-5 as buffer additives.
• The assay was applied to the characterization of recombinant human methionine sulfoxide reductase enzymes.
• The separation of peptide sulfoxide diastereomers by capillary electrophoresis is reported for the first time.

A capillary electrophoresis method has been developed and validated to evaluate the stereospecific activity of recombinant human methionine sulfoxide reductase enzymes employing the C-terminally dinitrophenyl-labeled N-acetylated pentapeptide ac-KIFM(O)K-Dnp as substrate (M(O) = methionine sulfoxide). The separation of the ac-KIFM(O)K-Dnp diastereomers and the reduced peptide ac-KIFMK-Dnp was optimized using experimental design with regard to the buffer pH, buffer concentration, sulfated β-cyclodextrin and 15-crown-5 concentration as well as capillary temperature and separation voltage. A fractional factorial response IV design was employed for the identification of the significant factors and a five-level circumscribed central composite design for the final method optimization. Resolution of the peptide diastereomers as well as analyte migration time served as responses in both designs. The resulting optimized conditions included 50 mM Tris buffer, pH 7.85, containing 5 mM 15-crown-5 and 14.3 mg/mL sulfated β-cyclodextrin, at an applied voltage of 25 kV and a capillary temperature of 21.5 °C. The assay was subsequently applied to the determination of the stereospecificity of recombinant human methionine sulfoxide reductases A and B2. The Michaelis–Menten kinetic data were determined. The pentapeptide proved to be a good substrate for both enzymes. Furthermore, the first separation of methionine sulfoxide peptide diastereomers is reported.