Quantifying Modified Histones by Mass Spectrometry

Histone samples were TCA precipitated, acetone washed, and prepared for mass spectrometry analysis as previously described (Kuo and Andrews, 2013 (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 to 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 2 and the peptides used are listed in Supplementary file 1; the transitions for the xenopus peptides have been previously reported (Kuo et al., 2014 (link)).