Quantifying Histone Post-Translational Modifications

Acid-extracted histone samples were TCA-precipitated, acetone-washed, and prepared for mass spectrometry analysis as previously described (Kuo et al., 2014 (link)). A Waters (Milford, MA) Acquity H-class UPLC system coupled to a Thermo (Waltham, MA) TSQ Quantum Access triple-quadrupole (QqQ) mass spectrometer was used to quantify modified histones. Selected reaction monitoring was used to monitor the elution of the acetylated and propionylated tryptic peptides. Transitions were created to study acetylation of pombe H3 wild type and mutants as well as the H4 tails. The detailed transitions for peptides of H3 that vary in sequence from Xenopus are reported in Supplementary file 3, the transitions for the Xenopus peptides and pombe H3G34R have been previously reported (Kuo et al., 2014 (link); Yadav et al., 2017 (link)).