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

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Abstract

Ser139-phosphorylated histone H2AX (γH2AX) is a useful biomarker of DNA double strand breaks. γH2AX has been conventionally detected by immunology-based methods using anti-γH2AX 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 γH2AX (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 γH2AX (ATQA(pS)QEY) per vehicle-treated HeLa S3 cell was 9.4 × 104 and increased to 6.2 × 105 molecules/cell after exposure to the DNA-damaging agent camptothecin (10 μM) for 1 h. The estimated total amount of H2AX (ATQA(pS)QEY + ATQASQEY) was 3.3–3.6 × 106 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.

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Acknowledgments

This study was supported by KAKENHI (23221006) from the Japan Society for the Promotion of Science and Grants-in-Aid for Scientific Research on Innovative Areas.

Conflict of interest

The authors declare no conflict of interest.

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Authors and Affiliations

  1. Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, 520-0811, Japan

    Shun Matsuda & Tomonari Matsuda

  2. Radiation Biology Center, Kyoto University, Yoshidakonoecho, Sakyo-ku, Kyoto, 606-8501, Japan

    Tsuyoshi Ikura

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  1. Shun Matsuda
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  2. Tsuyoshi Ikura
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  3. Tomonari Matsuda
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Correspondence to Tomonari Matsuda.

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Matsuda, S., Ikura, T. & Matsuda, T. Absolute quantification of γH2AX using liquid chromatography–triple quadrupole tandem mass spectrometry. Anal Bioanal Chem 407, 5521–5527 (2015). https://doi.org/10.1007/s00216-015-8725-z

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  • DOI: https://doi.org/10.1007/s00216-015-8725-z

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