Digestion of Enolase and Carbonic Anhydrase as Model Proteins for Therapeutic Proteins in Blood Plasma with Immobilized Thermolysin and Quantification of Some of the Peptides by LC/LC-MS/MS

There is a need for fast method development in the early drug discovery phase of therapeutic proteins. Thermolysin has not been used for quantification of proteins in blood plasma earlier. It is a thermostable protease which permits the use of high temperatures for fast hydrolysis of proteins. Model proteins were digested with immobilized thermolysin on agarose gel. Protein-specific peptides were selected for quantitation and quantified based on stable isotope dilution. Protein digests of blood plasma were cleaned and separated using an automated LC/LC–MS/MS system. Essential digestion parameters that influence thermolysin hydrolytic activity were optimized for high peptide yield. The validated methods were selective, linear, precise and accurate with a limit of detection of 2 nM for both proteins. The proposed strategy for method development could be valuable for quantification of proteins in blood plasma samples. The study underscores and discusses important features of the enzymatic digestion and chromatographic separation.