Using mass spectrometry to quantify changes in histone post-translational modifications as a result of DNA double-strand breaks

The structure of DNA is under constant threat from both extracellular and intracellular sources, causing mutations that could result in the reduced viability of the cell. In the nucleus of eukaryotes, DNA wraps around histone proteins 1.67 times. Two copies of Histone H2A, H2B, H3 and H4 congregate to form an octomer: along with the DNA strand together make the nucleosome. It has been shown that via histone post-translational modifications (HPTM), the nucleosome will signal to recruit biological machinery to process DNA such as during transcription or repair. One of the most characterised HPTM is phosphorylation of serine 139 of H2AX (?H2AX), which is indicative of a DNA strand break — single or double. Subsequent to the phosphorylation, the cell’s DNA damage response (DDR) will proceed to recruit proteins that repair the damage. Recent studies have implicated a myriad of HPTMs in the DDR. However, many of the putative DDR HPTM were described with various immunoassays such as western blot and immunofluorescence. Immunoassays are notorious for reproducibility issues and experimental design biases. Comparatively, mass spectrometry is renowned for its precision and its ability to accommodate high throughput analysis of many datasets in an unbiased manner. We utilise MS to quantify ?H2AX changes after DNA damage and show that H2AX tyrosine 142 phosphorylation is not as abundant as previously shown. We also developed a method for simultaneously quantifying many HPTMs changes that occur as a result of the DDR at both proximal to the site of damage and a genomic scale. The developed method of quantifying DDR HPTM accommodates future experiments such as determining whether DDR HPTMs are old or new (with SILAC) or be adapted to analyse HPTMs as a result of transcription, translation or development. A translational aspect would be to quantify the epigenetic marks of cancer patients before and post-treatment.