Dataset of proteomics using broad specificity proteases

Abstract

Trypsin is a gold standard protease in bottom-up proteomics, but its limited proteome coverage is due to its inability to access many protein sequences. In this study, we propose a fast and robust protocol using subtilisin, protease K, and thermolysin, which are proteases with broad specificity, to overcome this limitation. We demonstrate that broad-specificity proteases allow relative quantitation of proteomes after only minutes of digestion. We used DDA and DIA to evaluate workflows based on SP3 and STRAP for each of the three enzymes, and we measured 20 concatenated fractions from a first-dimension high-pH reversed-phase separation of Jurkat cells digested with each enzyme by DDA for deep profiling.