Chapter 5: LC-MS(/MS) of Trypsin-Hydrolysed Proteins

Protein hydrolysis is now a fairly well established process, both industrially and in the laboratory. Among the objectives of enzyme hydrolysis of food proteins are the production of bioactive peptides and modification of food structure and functional properties. The properties of bioactive peptides are determined by their amino acid composition and its permutation in the peptide. It is important to monitor the evolution of peptides during hydrolysis, and preserve those that are desired. An enzyme such as trypsin (EC 3.4.21.4) is popular because of its narrow specificity, which enables peptides of predictable amino acid composition to be produced. Although many protocols exist for use in the determination of the amino acid composition of peptides, mass spectrometry is a versatile tool which can be hyphenated with high-performance liquid chromatography and used for peptide analysis. With a protein such as bovine β-lactoglobulin (β-Lg) with known amino acid sequence, it is possible to monitor the evolution of peptides over time if desired. The choice of the method to stop the enzyme is important: e.g. pH adjustment is instantaneous, but in most cases the enzyme may recover its activity when the pH is adjusted back to the range of native protein structure. Before the commencement of hydrolysis, confirm that the set pH and temperature are stable because enzymes, being proteins, are sensitive to pH and/or temperature, resulting in denaturing processes. Temperature is easily controlled using a thermostatic water bath. The pH is controlled by either manual or automatic addition of an acid such as hydrochloric acid if the pH increases during hydrolysis (e.g. when pepsin is used) or sodium hydroxide if hydrolysis leads to decreases in the pH (e.g. during trypsin hydrolysis of proteins). Long storage of the enzyme may lead to conversion of trypsin to pseudo-trypsin. Notably, even though trypsin is usually treated to remove residual chymotrypsin activity, it deviates from its narrow specificity (cleavage of lysyl and arginyl residues on the C-terminals) to cleave peptide bonds like tyrosyl-seryl. Enzymes may harm the user if not handled carefully and safely. Several enzymes cause asthma if inhaled and it is important to avoid the enzyme dust. It is important to wear protective gloves and goggles when weighing powdered enzymes.