Absolute quantification of histone PTM marks by MRM-based LC-MS/MS

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Histone N-terminal tails are crucial for chromatin regulation via post-translational modifications (PTMs).
• Profiling histone PTMs offers insights into epigenetic events, cancer, and other diseases.
• Previous work enhanced histone peptide chromatography for LC/MS analysis.

Purpose of the Study:

• To develop and validate a multiple reaction monitoring (MRM)-based LC-MS/MS method for quantifying targeted histone peptides.
• To measure the absolute concentrations of tryptic peptides from H3 histone proteins in cancer cell lines.
• To investigate the function of histone lysine methyltransferase NSD2 in multiple myeloma cells.

Main Methods:

• Developed a novel multiple reaction monitoring (MRM)-based LC-MS/MS assay targeting 42 histone peptides.
• Utilized stable isotopic labeled peptides as internal standards for absolute quantification.
• Validated the method's accuracy and reproducibility using recombinant proteins and cellular samples.

Main Results:

• Achieved a linear dynamic range of 3 orders of magnitude (1 nM to 1 μM) for all targeted peptides.
• Demonstrated excellent intrabatch and interbatch reproducibility (<15% CV).
• Successfully quantified individual and combinatorial histone marks in parental and NSD2 knockout KMS11 multiple myeloma cells.

Conclusions:

• The developed MRM LC-MS/MS method provides accurate and reproducible quantification of histone PTMs.
• This method enables the study of epigenetic alterations in cancer, such as NSD2 function in multiple myeloma.
• The findings contribute to understanding the role of histone modifications in disease pathogenesis.