Comparison of in-gel digestion methods for histone PTM analysis

In order to optimize the histone digestion procedure for low-abundance clinical samples, we compared different in-gel digestion protocols. Histones are usually digested in-gel using “Arg-C-like” strategies, which involve chemical acylation of lysines followed by trypsin digestion (17). Acylated lysines are not recognized by trypsin, which -as a consequence- cuts only at the C-terminus of arginines and generates peptides of suitable length for MS analysis. We compared four Arg-C-like protocols: 1) D3 protocol (deuterated acetic anhydride as acylating agent); 2) PRO protocol (propionic anhydride as acylating agent); 3) PRO-PRO protocol (propionic anhydride as acylating agent and peptide N-terminal derivatization); 4) PRO-PIC protocol (propionic anhydride as acylating agent and phenyl-isocyanate as peptide N-terminal derivatization). We also used an Arg-C in solution digestion as a reference, and tested the performance of the methods with decreasing sample amounts.