Online, Bottom-up Characterization of Histone H4 4–17\nIsomers

The “Histone\nCode” is comprised of specific types\nand positions of post-translational modifications (PTMs) which produce\nbiological signals for gene regulation and have potential as biomarkers\nfor medical diagnostics. Previous work has shown that electron-based\nfragmentation improves the sequence coverage and confidence of labile\nPTM position assignment. Here, we evaluated two derivatization methods\n(e.g., irreversible - propionylation and reversible–citraconylation)\nfor bottom-up analysis of histone H4 4–17 proteoforms using\nonline liquid chromatography (LC), trapped ion mobility spectrometry\n(TIMS), and electron-based dissociation (ExD) in tandem with mass\nspectrometry. Two platforms were utilized: a custom-built LC-TIMS-q-ExD-ToF\nMS/MS based on a Bruker Impact and a commercial μLC-EAD-ToF\nMS/MS SCIEX instrument. Complementary LC-TIMS preseparation of H4\n4–17 0–4ac positional isomer standards showed that they\ncan be resolved in their endogenous form, while positional isomers\ncannot be fully resolved in their propionylated form; online LC-ExD-MS/MS\nprovided high sequence coverage (>90%) for all H4 4–17 (0–4ac)\nproteoforms in both instrumental platforms. When applied to model\ncancer cells treated with a histone deacetylase inhibitor (HeLa +\nHDACi), both derivatization methods and platforms detected and confirmed\nH4 4–17 (0–4ac) proteolytic peptides based on their\nfragmentation pattern. Moreover, a larger number of HeLa + HDACi H4\n4–17 proteoforms were observed combining LC-TIMS and LC-q-ExD-ToF\nMS/MS due to the positional isomer preseparation in the LC-TIMS domain\nof citraconylated H4 4–17 (0–4ac) peptides.