Abstract
Trypsin is by far the most commonly used protease in proteomics. Even though the amount of protease used in each experiment is very small, digestion of large amounts of protein prior to enrichment can be rather costly. The price of commercial trypsin is highly dependent on the quality of the enzyme, which is determined by its purity, activity, and chemical modifications. In this study we evaluated several strategies for improving the quality of crude trypsin by reductive methylation and affinity purification. We present a protocol applicable to most proteomics laboratories for obtaining a highly stable and pure trypsin preparation using reductive methylation and purification by benzamidine-sepharose. The entire workflow can be performed within a day and yields ~4 mg per batch but is completely scalable. The methylated product was benchmarked against sequencing grade trypsin from Promega and they were found to be comparable for one hour digestions at elevated temperatures, where residual chymotryptic activity was found to be negligible.