Anal Bioanal Chem DOI 10.1007/s00216-015-8725-z

RESEARCH PAPER

Absolute quantification of γH2AX using liquid chromatography–triple quadrupole tandem mass spectrometry Shun Matsuda 1 & Tsuyoshi Ikura 2 & Tomonari Matsuda 1

Received: 18 March 2015 / Revised: 16 April 2015 / Accepted: 17 April 2015 # Springer-Verlag Berlin Heidelberg 2015

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.

Keywords γH2AX . Multiple reaction monitoring/selected reaction monitoring (MRM/SRM) . Absolute quantification . DNA damage

* Tomonari Matsuda [email protected] 1

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

2

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

Introduction Histone H2AX is phosphorylated at Ser139 in response to DNA double strand breaks and replication fork collapse [1, 2]. Ser139-phosphorylated H2AX (γH2AX) rapidly accumulates at the damage site (within 1 min after damage occurs), and these γH2AX aggregates can be visualized as discrete nuclear foci by immunostaining using anti-γH2AX antibody [3]. γH2AX recruits proteins involved in DNA repair and other DNA-damage responses to the site of damage, where it serves as a binding target of these proteins (reviewed in [4]). The findings that one γH2AX focus corresponds to one double strand break [3, 5] and that a broad spectrum of mutagens can induce γH2AX (reviewed in [6]) ensures that γH2AX is a sensitive indicator of DNA damage. In regulatory science, γH2AX is expected to be a useful biomarker of genotoxicity of chemicals [7–12] and is also used for detection of genotoxic environmental pollutants such as polyaromatic hydrocarbons and cigarette smoke [13–15]. In pharmacology, γH2AX is a good indicator of efficacy of cancer drugs which damage DNA and also may be a potential target of cancer drugs [16, 17]. Also in clinical study, γH2AX is useful for detection of DNA double strand breaks induced by radiotherapy and chemotherapy as a marker of therapy efficacy [18, 19]. γH2AX is also measured to determine the efficiency of DNA repair and to predict cancer sensitivity and resistivity to drugs [17]. On the other hand, since DNA damages are one of the major factors of carcinogenesis, γH2AX is also useful for detection of precancerous regions [20, 21]. γH2AX is conventionally detected by immunology-based methods such as western blotting, immunostaining, enzymelinked immunosorbent assay, and flow cytometry. However, these methods have several limitations. First, all rely on the anti-γH2AX antibody whose quality can vary between sources. Second, the staining protocol is burdensome, making high-throughput analysis difficult. Third, these methods are

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not quantitative compared with other methods such as liquid chromatography–tandem mass spectrometry (LC/MS/MS). Using LC/MS/MS, several excellent quantitative methods of proteins have been developed such as the stable isotope labeling in culture (SILAC) [22], the isotope coded affinity tag (iCAT) [23], and the isobaric tags for relative and absolute quantitation (iTRAQ) [24]. These Bglobal^ quantification methods allow relative quantification of protein amounts, and iTRAQ is capable of absolute quantification. The limitations of these methods arise from the semi-random acquiring of MS/MS spectra for peptides in which low-abundance peptides yield less precise measurements. These methods also have a limitation particularly in high-throughput sample analysis. Recently, there have been increasing reports concerning Btargeted^ quantification of proteins using multiple reaction monitoring (MRM). In this case, a specific protease (generally trypsin)-digested product (peptide) of a target protein is quantified. Standard peptides of appropriate length, including their isotope-labeled counterparts, can be synthesized, thus making absolute quantification of proteins of interest possible [25–27]. Absolute quantification permits stoichiometric analysis of proteins and thus can provide mechanistic insights in related fields such as molecular biology and systems biology. Here, we developed a sensitive and reliable method for absolute quantification of γH2AX using LC/MS/MS with MRM. Furthermore, we successfully applied this method to human cell samples.

Materials and methods Materials β-Glycerophosphate was purchased from Merck Millipore, MA, USA. Tablets of phosphate-buffered salts were purchased from Takara, Shiga, Japan. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Life Technologies, CA, USA. Camptothecin (CPT) and newborn calf serum (FCS) were purchased from Sigma-Aldrich, MO, USA. Synthetic peptides representing the sequences of two trypsin cleavage products of H2AX were ordered: ATQASQEY and ATQA(pS)QEY (Eurofins Genomics, Tokyo, Japan). Internal standards of these peptides containing 13C- and 15N-labeled amino-terminal alanine, [13C3, 15N]ATQASQEY and [13C3, 15 N]ATQA(pS)QEY, were also ordered. The other chemicals were purchased from Wako, Osaka, Japan. A stock solution of CPT was dissolved in dimethyl sulfoxide (DMSO). Cell culture Human cervix adenocarcinoma HeLa S3 cells were cultured in DMEM supplemented with 10 % FCS at 37 °C in a humidified 5 % CO2 incubator.

Histone extraction Acid extraction of histones from cells followed a previous report [28]. HeLa S3 cells (6×106 cells) were treated with vehicle (0.1 % (v/v) DMSO) or CPT for 1 h. The chemical solution was directly added to the medium. The cells were washed with phosphate-buffered saline (PBS), collected in 1 ml PBS, and pelleted by centrifugation at 1000 rpm for 2 min at 4 °C. The cell pellet was resuspended in 1 ml hypotonic buffer (10 mM Tris-HCl pH 8.0, 1 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride, and 10 mM β-glycerophosphate) and rotated for 30 min at 4 °C. The cell nuclei were pelleted by centrifugation at 10,000×g for 10 min at 4 °C, resuspended in 400 μl 0.4 N H2SO4, and rotated for 30 min at 4 °C. After centrifugation at 16,000×g for 10 min at 4 °C, 132 μl 100 % (w/v) trichloroacetic acid was added to the supernatant followed by incubation for 30 min on ice to precipitate histones. The histones were pelleted by centrifugation at 16,000×g for 10 min at 4 °C, washed with 500 μl acetone twice, and air-dried for 20 min at room temperature. In-solution tryptic digestion In-solution tryptic digestion followed a previous report [29]. The histone pellet was dissolved in 150 μl 50 mM ammonium bicarbonate and digested at a sequencing-grade trypsin/sample ratio of 1:50 overnight at 37 °C. After addition of sequencinggrade trypsin at a sequencing-grade trypsin/sample ratio of 1:100, the sample was incubated for a further 3 h at 37 °C. The reaction was stopped by adding 3 μl 20 % trifluoroacetic acid. After addition of 3 μl of two isotope-labeled peptides (approximately 6.3 pmol each) dissolved in 5 % acetonitrile, the sample was concentrated and desalted on reversed-phase C18 StageTips [30]. Peptides were eluted with 40 μl elution buffer (80 % acetonitrile and 0.5 % trifluoroacetic acid), concentrated by a centrifugal concentrator CC-105 (Tomy, Tokyo, Japan) at room temperature for 10 min, and adjusted to approximately 20 μl with 5 % acetonitrile. LC/MS/MS Mass spectrometric analysis was performed using a Xevo TQS (Waters, Manchester, UK) with an ACQUITY UPLC system (Waters). One microliter of each sample was separated on an ACQUITY UPLC BEH C18 1.7 μm 2.1×50 mm column (Waters) at a flow rate of 0.5 ml/min, and subsequently eluted as follows (solvent A, 0.1 % formic acid; solvent B, acetonitrile): 0–0.5 min, isocratic with 0 % B; 0.5–7 min, linear gradient to 30 % B; 7–9 min, linear gradient to 80 % B; 9–10 min, isocratic with 80 % B; 10–12 min, isocratic with 0 % B. MRM was performed in positive ion mode using nitrogen as the nebulizing gas. Experimental conditions were set as follows: ion source temperature, 150 °C; desolvation temperature,

Absolute quantification of γH2AX Table 1 Set of MRM transitions Peptide sequence

MS1 (m/z)

MS2 (m/z)

Collision energy (eV)

Used for quantificationa

ATQASQEY

449.2

451.2

10 10 10 10 10 10 10 13 15

○

[13C3, 15N]ATQASQEY

526.2 311.1 182.1 526.2 311.1 182.1 440.1 431.5 182.2 442.1 433.5 182.2

10 13 15

○

(internal standard) ATQA(pS)QEY

489.2

[13C3, 15N]ATQA(pS)QEY

491.2

(internal standard) a

○

Circles indicate transitions used for quantification

650 °C; desolvation gas flow rate, 1000 L/h; capillary voltage, 0.5 kV; cone voltage, 35 V; cone gas flow rate, 150 L/h; collision gas, argon; collision gas flow rate, 0.15 ml/min. The conditions of MRM transitions, including collision energy, are shown in Table 1. The amount of each peptide was quantified by calculating the peak area ratio of the target peptide and its isotope-labeled internal standard. The calibration curve was obtained by using an authentic standard peptide spiked with its isotope-labeled internal standard.

Results and discussion Method development Because it is often difficult to directly detect protein levels by LC/MS/MS due to the large molecular weight of polypeptides and possible post-translational modifications (phosphorylation, acetylation, methylation, etc.), proteins are generally digested with proteases such as trypsin to a workable size before LC/MS/MS analysis. We selected trypsin cleavage products unique to the carboxy-terminus of H2AX including Ser139 (ATQASQEY, Ser139 is underlined) for quantification. We synthesized four peptides, the unphosphorylated Table 2

○

form (ATQASQEY), the Ser139-phosphorylated form (ATQA(pS)QEY) representing γH2AX, and their respective isotope-labeled peptides, as internal standards. To optimize MRM transitions for these peptides, their MS/MS spectra were manually obtained by LC/MS/MS analysis. Three MRM transitions with optimized collision energy were selected for each peptide (Table 1). We obtained calibration curves from each transition per peptide and the sum of three transitions per peptide, and compared their precision and accuracy (data not shown). As a result, we concluded that the transition m/z 449.2>526.2 for ATQASQEY and the transition m/z 489.2>440.1 for ATQA(pS)QEY were suitable for quantification (Table 1). Calibration curves were linear over the entire range of 1.5–3345 fmol/injection for ATQASQEY and 1.4– 1280 fmol/injection for ATQA(pS)QEY. The correlation coefficients were 0.9992 for ATQASQEY and 0.9975 for ATQA(pS)QEY. Next, precision and accuracy were calculated at three levels for each peptide. The obtained precision and accuracy based on eight replicate experiments ranged from 1.8 to 5.4 %CV and from −0.3 to 1.9 %Bias for ATQASQEY, and from 3.4 to 7.6 %CV and from −3.7 to 9.6 %Bias for ATQA(pS)QEY (Table 2). These data indicate that our method has acceptable accuracy and precision. Since a previous report noted that peptide decay, which would be caused by

Accuracy and precision data (n=8)

Peptide sequence

Expected amount/injection (fmol)

Calculated amount/injection (fmol)a

%CV

%Bias

ATQASQEY

41 372 1115 47 427 1280

41±2 374±7 1136±40 52±4 462±16 1233±43

5.4 1.8 3.5 7.6 3.4 3.5

−0.3 0.6 1.9 9.6 8.2 −3.7

ATQA(pS)QEY

a

Mean±SD

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Fig. 1 Evaluation of peptide decay. a Experimental outline for evaluation of decay of the peptides during tryptic digestion. Internal standards (IS) of each peptide were added as indicated. b

Quantification result of the peptides in the standard sample and the tryptic digestion sample shown in Fig. 1a. Mean±SD, n=3

degradation and precipitation of peptides, may affect the quantitative accuracy [31], we tested the stability of the peptides during tryptic digestion (Fig. 1a). As shown in Fig. 1b, the decay effect was negligible in both peptides in our condition.

thus γH2AX formation. Histones were extracted from the cells and subjected to tryptic digestion, after which we applied our method to try to detect ATQASQEY and ATQA(pS)QEY. Note that any cell-derived proteases, which could affect peptide degradation, in histone samples should be inactivated since any proteins in the samples were denatured by sulfuric acid and trichloroacetic acid. As shown in Fig. 2, obvious peaks representing ATQASQEY and ATQA(pS)QEY were observed at the same retention times as their respective internal standards (2.91 min for ATQASQEY and 3.16 min for

Application to histones extracted from cells We tested whether our method can be applied to a biological sample. HeLa S3 cells were treated for 1 h with the topoisomerase I inhibitor CPT to induce DNA double strand breaks and Fig. 2 MS/MS chromatograms of the analytes from the standard peptides (1.1 pmol for ATQA SQEY and 1.3 pmol for ATQA(pS)QEY) and from histones extracted from HeLa S3 cells treated with 10 μM CPT for 1h

Absolute quantification of γH2AX

Fig. 3 MS/MS chromatograms of the analytes and their internal standards (IS), from histones extracted from HeLa S3 cells treated with DMSO (vehicle) or 1 μM CPT for 1 h. Peaks representing analytes or IS are indicated by arrows. The value on the left side of each peak represents its area

ATQA(pS)QEY). Furthermore, the ratio of peak areas among the three transitions of ATQASQEY in the histone extract was well correlated with that in its standard. The same holds for

ATQA(pS)QEY. Next, we compared CPT-treated cells with vehicle (DMSO)-treated cells. As shown in Fig. 3, CPT treatment significantly increased ATQA(pS)QEY relative to

Fig. 4 Dose response of CPT. a After HeLa S3 cells (6×106 cells) were treated with 0, 0.5, 1, 5, and 10 μM CPT for 1 h, absolute quantification of two H2AX-derived peptides was performed using our method. After calculation of the total amount of each peptide in the sample, the peptide

amount in a cell was estimated according to the equation described in the text. Mean±SD, n=3. b Ratio of γH2AX (ATQA(pS)QEY) amount to total H2AX (ATQASQEY and ATQA(pS)QEY) amount based on Fig. 4a. Mean±SD, n=3

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vehicle treatment as expected, clearly indicating induction of DNA damage and subsequent γH2AX formation. On the other hand, ATQASQEY was slightly decreased by CPT treatment. Given these data, we conclude that our method is able to specifically detect these peptides from cell samples.

Quantitative analysis of cell samples Next, we attempted to quantify the absolute amounts of ATQASQEY and ATQA(pS)QEY in histones extracted from HeLa S3 cells (6×106) which had been exposed to various concentrations of CPT for 1 h. The total amount of each peptide in the samples was calculated, and the number of molecules of each peptide in a HeLa S3 cell was estimated according to the following equation: . total peptide amountðmolÞ molecules cell≅  6:02 6  106 ðcell numberÞ  1023 ðAvogadro constantÞ In the Bno damage^ (vehicle) condition, the amount of ATQASQEY (3.3×106 molecules/cell) was about 30 times more than the amount of ATQA(pS)QEY (9.4×104 molecules/cell) (Fig. 4a). In cells exposed to CPT, the amount of ATQA(pS)QEY increased in a CPT dose-dependent manner but reached a plateau at doses higher than 1 μM CPT despite there being considerable ATQASQEY remaining. The amount of ATQA(pS)QEY at the highest CPT concentration (10 μM) was 6.2×105 molecules/cell. These data indicate that the apparent velocity of H2AX phosphorylation, which is catalyzed by ataxia–telangiectasia mutated (ATM) [32], ataxia– telangiectasia and Rad3-related (ATR) [2], and DNAdependent protein kinase [33], has an obvious limit. In contrast to ATQA(pS)QEY, the amount of ATQASQEY was found to decrease slightly in a CPT dose-dependent manner up to 5 μM CPT. Furthermore, the sum of the amounts of ATQASQEY and ATQA(pS)QEY changed very little across the dosing range, suggesting that the reduction in the amount of ATQASQEY is roughly accounted for by the increase in the amount of ATQA(pS)QEY. Given this observation, we hypothesized that the total amount of H2AX equals the sum of the amounts of ATQASQEY and ATQA(pS)QEY, and calculated the ratio of γH2AX to total H2AX (Fig. 4b). As a result, we estimate the total number of molecules of H2AX (ATQASQEY +ATQA(pS)QEY) in a HeLa S3 cell to be 3.3–3.6×106. Whereas 2.8 % of H2AX was in the γH2AX form in the Bno damage^ (vehicle) condition, the ratio of γH2AX to total H2AX increased to 17.2 % at 10 μM CPT. An earlier report roughly estimated the number of molecules of total H2AX and γH2AX in a HeLa cell to be 1.4×106 and 4.5×105, respectively, 30 min after exposure to 25 Gy ionizing radiation, based on imaging of a stained gel prepared by

two-dimensional electrophoresis [1]. The results of our highly quantitative estimate are consistent with the previous estimation. Our method has several limitations, especially concerning other modification(s) in the carboxy-terminus of H2AX. For example, Tyr142 phosphorylation of H2AX and its role in DNA damage responses were reported [34, 35]. Thus, there could be at least two additional types of the carboxy-terminus of H2AX other than Ser139-phosphorylated H2AX, including Tyr142-phosphorylated H2AX (ATQASQE(pY)) and dip h o s p h o r y l a t e d a t b o t h S e r 1 3 9 a n d Ty r 1 4 2 (ATQA(pS)QEY(pY)). We could not detect these types of H2AX because of their low sensitivity in LC/MS/MS analysis (data not shown). These modification patterns may be minor relative to total H2AX at least in our condition, since the sum of the amounts of ATQASQEY and ATQA(pS)QEY was almost constant across the dosing range (Fig. 4b).

Conclusion We developed an LC/MS/MS-based method for absolute quantification of γH2AX, which is a useful biomarker of DNA damage. This method could accurately and precisely quantitate Ser139-unphosphorylated H2AX and γH2AX at ranges of 1.5–3345 and 1.4–1280 fmol/injection, respectively. We successfully applied this method to human cell samples. Due to its broad adaptability and throughput performance, we believe that our method is a powerful tool for studying DNA damage-response mechanisms, genotoxicity testing, cancer drug screening, clinical studies, and other fields. 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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