Histone H2AX Y142 phosphorylation is a low abundance modification

We employ targeted mass spectrometry to compare the levels of H2AX S139 phosphorylation (?H2AX) and Y142 phosphorylation. We use synthetic peptides to facilitate MS optimisation and estimate relative detection efficiencies for the different modifications. Despite phosphopeptide enrichment from large amounts of starting material, we are unable to detect endogenous H2AX Y142 phosphorylation, indicating that it is present in low abundance (<1%). We also calculate the relative levels of H2AX compared to other H2A isoforms and quantify the proportion of H2AX that is phosphorylated on S139 (?H2AX) after ionising radiation.