Histone H2AX Y142 phosphorylation is a low abundance modification

•We quantify histone H2AX S139 and Y142 phosphorylation using mass spectrometry.•Endogenous H2AX Y142ph is below the limit of detection.•Using enrichment and synthetic standards we calculate Y142ph has <1% abundance.•Low level H2AX Y142ph implies S139ph will rarely be present on the same H2AX.•We calculate H2AX levels in three human cell lines as between 1 and 6% of total H2A.