Histone post-translational modifications (hPTMs) belong to decisive epigenetic marks affecting, among others, cell cycle and protein interactions. hPTMs have been studied by both antibody- and mass spectrometry-based methods for years, however, those analyses are still challenging, mainly due to multiple histone variants and their modification diversity arising from high content of reactive amine groups in the amino acid sequence. Here, we introduce trimethylacetic anhydride (TMA) as a new reagent for efficient histone alkylation, a necessary approach prior to hPTMs analysis using bottom-up proteomics. TMA reagent is applied for derivatization of unmodified amine groups at lysine residues as well as for subsequent alkylation of newly generated amine groups at peptide N-termini after trypsin digestion. Derivatization reaction is facilitated by microwave irradiation, which also reduces incubation time to minute intervals. We demonstrate that histone alkylation with TMA represents a reliable technique with high yield of fully derivatized peptides, hence an effective alternative to conventionally used methods. More than 98 % and 99 % labelling efficiency of TMA was achieved for histone H4 and H3, respectively, ensuring an accurate quantification of peptide forms. Trimethylacetylation substantially improves chromatographic separation of peptide forms which is a prerequisite for direct quantification based on signal extracted from MS1 data. For this purpose, software tools widely adopted within proteomics community can be used without a need of additional computational advances. Based on a thorough side-by-side comparison of TMA performance with well-adopted propionylation we comment on the impact of histone derivatization on the hPTMs monitoring in biological samples.

Histones extracted from chronic lymphocytic leukemia cell line MEC-1 were alkylated by trimetylacetic anhydride and digested by trypsin. Alkylation reaction was enhanced by irradiation in microwave oven. Peptides were desalted and underwent LC-MS/MS analysis with Orbitrap Fusion Lumos Tribrid mass spectrometer. Histone forms precursors, that co-elute on RP-C18 column, were selected for tandem mass spectrometry PRM analysis. Signals of histone form specific fragments were used for determination of peptide form quantity in co-eluting peak.

The chronic lymphocytic leukemia cell line MEC-1 treated with Entinostat (enti) and without treatment (ctrl). Histone proteins acidic extraction. Sequential alkylation of unmodified NH2- groups at histone proteins, followed by digestion and another sequential alkylation of newly generated NH2- groups. Alkylation reaction enhanced by irradiation in microwave oven.