Absolute quantification of γH2AX using liquid chromatography-triple quadrupole tandem mass spectrometry

Ser139-phosphorylated histone H2AX (gamma H2AX) is a useful biomarker of DNA double strand breaks. gamma H2AX has been conventionally detected by immunology-based methods using anti-gamma H2AX antibody, but quantitative analysis is difficult to perform with such methods. Here, we describe an absolute quantification method using liquid chromatography-triple quadrupole tandem mass spectrometry that is applicable to peptides derived from gamma H2AX (ATQA(pS)QEY) and unphosphorylated H2AX (ATQASQEY). Our method was successfully applied to histones extracted from human cervix adenocarcinoma HeLa S3 cells. The estimated number of molecules of gamma H2AX (ATQA(pS)QEY) per vehicle-treated HeLa S3 cell was 9.4 x 10(4) and increased to 6.2 x 10(5) molecules/cell after exposure to the DNA-damaging agent camptothecin (10 mu M) for 1 h. The estimated total amount of H2AX (ATQA(pS)QEY + ATQASQEY) was 3.3-3.6 x 10(6) molecules/cell. Due to its broad adaptability and throughput performance, we believe that our method is a powerful tool for mechanistic studies of the DNA-damage response as well as for genotoxicity testing, cancer drug screening, clinical studies, and other fields.