Protein digestion optimization for characterization of drug-protein adducts using response surface modeling.

The formation of drug–protein adducts in vivo may have important clinical and toxicological implications. Consequently, there is a great interest in the detection of these adducts and the elucidation of their role in the processes leading to adverse and idiosyncratic drug reactions. Enzymatic digestion is a crucial step in bottom-up proteomics strategies for the analysis of drug–protein adducts. The chosen proteolytic enzyme and digestion conditions have a large influence on the protein coverage of the modified protein and identification of its modification site. In this work, the enzymatic digestion conditions (pH, temperature and time) of trypsin and thermolysin were optimized specifically for the characterization of Human Serum Albumin (HSA) adducts. Using a Design of Experiments (DOE), it was found that of the three optimized parameters mainly pH and temperature showed strong effects on both responses. The optimized digestion conditions were different from those obtained from the suppliers or literature. Their application to HSA adducts resulted in improved protein coverage and signal intensity regarding the peptide containing the modification site, thereby highlighting the importance of a detailed optimization of digestion conditions.