Year: 2024
Type: article
Abstract: The "Histone Code" is comprised of specific types and positions of post-translational modifications (PTMs) which produce biological signals for gene regulation and have potential as biomarkers for medical diagnostics. Previous work has shown that electron-based fragmentation improves the sequence coverage and confidence of labile PTM position assignment. Here, we evaluated two derivatization methods (e.g., irreversible - propionylation and reversible-citraconylation) for bottom-up analysis of histone H4 4-17 proteoforms using online liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), and electron-based dissociation (ExD) in tandem with mass spectrometry. Two platforms were utilized: a custom-built LC-TIMS-q-ExD-ToF MS/MS based on a Bruker Impact and a commercial μLC-EAD-ToF MS/MS SCIEX instrument. Complementary LC-TIMS preseparation of H4 4-17 0-4ac positional isomer standards showed that they can be resolved in their endogenous form, while positional isomers cannot be fully resolved in their propionylated form; online LC-ExD-MS/MS provided high sequence coverage (>90%) for all H4 4-17 (0-4ac) proteoforms in both instrumental platforms. When applied to model cancer cells treated with a histone deacetylase inhibitor (HeLa + HDACi), both derivatization methods and platforms detected and confirmed H4 4-17 (0-4ac) proteolytic peptides based on their fragmentation pattern. Moreover, a larger number of HeLa + HDACi H4 4-17 proteoforms were observed combining LC-TIMS and LC-q-ExD-ToF MS/MS due to the positional isomer preseparation in the LC-TIMS domain of citraconylated H4 4-17 (0-4ac) peptides.
Source: Analytical Chemistry
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: National Science Foundation, Florida International University, National Institute of General Medical Sciences, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, Division of Chemistry, Division of Human Resource Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Core Research for Evolutional Science and Technology
Awards: G1312641614, G1492610919, G1564983540, G2476700337, G301209757, G3137425078, G4507699080, CREST Phase II: Center for Aquatic Chemistry and Environment (CAChE), CREST: Center for Aquatic Chemistry and the Environment, G5292893421, G5613623313, G5838312420, G7511355324, Collaborative Research: MFB: Deciphering the Logic of PTM Crosstalk via Novel Chemical Technology: Histones and Beyond, Development of biological structural tools for the study of protein-DNA complexes, Quantitative mass spectrometry for comprehending epigenetic mechanisms in a new underlying neurological developmental disorder
OpenAlex ID: W4403543349