Measurement of degree of hydrolysis and molecular weight distribution of protein hydrolysates by liquid chromatography-mass spectrometry

Enzymatic hydrolysis of milk protein is an effective way to improve protein digestibility, to reduce their allergenicity and to produce peptides with better functionalities. Among the process, the degree of hydrolysis (DH) and molecular weight distribution (MWD) of protein hydrolysates are two important parameters that need to be monitored. In this work, a new method based on liquid chromatography-mass spectrometry (LC-MS) was developed for the first time to accurately detect the DH and the MWD of proteolytic peptides. With LC-MS, the content of free amine groups released during hydrolysis was acquired by direct analysis of free trinitrobenzene sulfonic acid (TNBS) for DH assay, overcoming the disadvantage of TNBS-based spectral method. Based on this method, the DH% values of five protein hydrolysis samples were determined and consistent with file specification values. Compared to the size-exclusion high-performance liquid chromatography (SE-HPLC) method, LC-MS was capable of measuring MWD (similar results with file specification values) while additionally providing precise molecular weight and amino acid sequence information for each proteolytic peptide. This method was characterized by its simplicity, accuracy, and reproducibility, making it a valuable technology for monitoring proteolysis and producing peptides with better functionality.