Optimization of Enzymolytic Conditions and Evaluation of Enzymolytic Properties in Determination of Bovine Casein by UPLC in Hyphenation with MS/MS Tandem

Improving recovery of MS determination of protein is one of the factors to raise the accuracy of the method, hence a study on optimization and establishment of optimum conditions for enzymolysis is of utmost importance. Seven methods for enzymolysis were designed and studied comparatively. Soy-bean milk was used as negetive matrix in the testing. The direct enzymolysis was found to be the best one among the 7 enzymolytic methods proposed, and its procedure was given below:1 g of soybean milk powder is dissolved in 10 mL of 50 mmol·L^-1 Tris-HCl buffer solution to give the soy-bean milk extract. 200 μL of 200 nmol·L^-1 β-casein solution are added to 300 μL of the soy-bean milk extract together with 10 μg of trypsin (MS-grade). After mixing, the solution was enzymolyzed at 37℃ for 16 h. At the end of enzymolysis, 10 μL of 10% (φ) formic acid solution were added to stop the reaction. Volume of the solution was made up to 1.0 mL. The solution is contrifuged at high rate for 10 min. The supernatant is used for UPLC-MS/MS analysis to take down the peak-area of the characteristic peptide segment of VLPVPQK. Efficiency of enzymolysis found for this method was 106% with RSD (n=5) of 5.1%. Optimal ratio of mass of trypsin added (w_trypsin) to mass of β-casein present was found to be 1 to 20. Reduction and alkylation were not applied in the optimization of the method proposed.