Mass spectrometric quantification of histone H2A phosphorylation induced by DNA damage

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The posttranslational modifications of histone proteins in chromatin play a significant role in the control of gene expression. One specific modification, the phosphorylation of serine 129 in histone H2A of the yeast Saccharomyces cerivisiae (139 in mammals), has gained prominence with a putative direct link to DNA damage and repair. Using antibody-based methods, researchers have correlated this phosphorylation event with formation of frank double-stranded breaks, though a more complex scenario involving clustered DNA lesions as arise with ionizing radiation and other oxidants has not been ruled out. More recently, a neighboring serine (122) and threonine (126) in yeast H2A have also been shown to be phosphorylated differentially depending on the nature of the DNA damaging agent, which suggests that a pattern among these three sites might discern the underlying lesions or fine-tune the cellular response to the agents. Unfortunately, the use of antibodies, while extremely sensitive, is often hindered by cross-reactivity and a phenomenon known as epitope occlusion in which some nearby amino acid modifications could interfere with proper detection. To more directly monitor the status of these and other modification sites within H2A, we have developed LC-MS/MS to rigorously characterize and quantify the different DNA damage-induced H2A phosphorylation events, using an approach that involves isotope dilution mass spectrometric analysis of tryptic fragments of H2A isolated from cells treated with a variety of DNA damaging agents. This sensitive and specific approach should provide novel insights into the relationship between H2A phosphorylation and the cellular response to DNA damage.