Sensitive and Precise Characterization of Combinatorial Histone Modifications by Selective Derivatization Coupled with RPLC-EThcD-MS/MS

Decipheringthe combinatorial histone codeshas been a long-standing interest in the epigenetics field, which requires the reliable and robust characterization of the post-translational modifications (PTMs) coexisting on histones. To this end, weak cation exchange hydrophilic interaction liquid chromatography is commonly used in middle-down liquid chromatography–mass spectrometryapproaches for online separation of variously modified histonepeptides. Here we provide a novel strategy that combines the selective histonepeptide derivatizationusing N-hydroxysuccinimidepropionate ester with reversed-phase liquid chromatography (RPLC) for the robust, sensitive, and reliable characterization of combinatorial histonePTMs. Derivatizationamplifies the subtle physical differences between similarly modified histonepeptides, thereby allowing baseline separation of these peptides by standard RPLC. Also, the sensitivity of MS is enhanced greatly by derivatizationdue to the increased peptide hydrophobicity and co...