Arg-C Ultra Simplifies Histone Preparation for LC-MS/MS

Arginine-specific cleavage is the primary method used to prepare lysine-rich histone proteins in bottom-up proteomics. As the Arg-C enzyme has demonstrated suboptimal specificity, cleavage at the carboxyl side of arginine residues is typically achieved through the chemical derivatization of lysines followed by trypsin digestion. Recent improvements in proteolytic enzymes are reflected in the introduction of Arg-C Ultra, a recombinant proteinase with a substantially improved digestion specificity. Here, using mammalian histone extract, we demonstrate that Arg-C Ultra facilitates histone preparation for LC-MS/MS. We show the performance of Arg-C Ultra in terms of digestion specificity, number of modified forms identified, and yield of quantitative information compared with Arg-C and trypsin digestion combined with chemical derivatization with trimethylacetic anhydride. Importantly, we show that chemical derivatization at the peptide level, i.e., after Arg-C Ultra digestion, is still necessary to improve the quantification of short histone peptidoforms as well as positional isomers.