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.