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Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry
Analytical Biochemistry 413 (2011) 164–170
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Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry Jun-jie Zhang a, Lijian Zhang a, Keyuan Zhou a, Xiaoxia Ye a, Chunan Liu a, Liangtao Zhang a, Jingxuan Kang b, Chun Cai a,⇑ a b
Guangdong Medical College, Zhanjiang, Guangdong 524023, People’s Republic of China Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA
a r t i c l e
i n f o
Article history: Received 23 December 2010 Received in revised form 24 January 2011 Accepted 25 January 2011 Available online 9 March 2011 Keywords: DNA methylation Mass spectrum Hydrophilic interaction chromatography (HILIC) Ultra high-pressure liquid chromatography (UHPLC)
a b s t r a c t We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol–chloroform, hydrolyzed using 88% formic acid at 140 °C, spiked with cytosine-2,4-13C15N2 as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC–MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1– 50 and 1–100 ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70–4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC–MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation. Ó 2011 Elsevier Inc. All rights reserved.
DNA methylation [1], carried out by the addition of a methyl group to position 5 of cytosine by DNA methyltransferase, not only is emerging as an important player in the regulation of genes but also is crucial for maintaining genome stability [2]. Changes in the methylation status of genomic DNA can play a central role in processes related to several diseases [3,4], especially tumorigenesis. With the increasing interest in DNA methylation, there currently is a lot of information on the detection of DNA methylation, such as Southern blot analyses [5,6], combined bisulfite restriction analysis (COBRA)1 [7], methylight [8], and capillary zone electrophoresis [9], but each method has its own deficiency [10,11]. Compared with the above methods, accurate genomic methylation can be determined by chromatographic techniques. The technique separates the different nucleosides or nucleobases obtained following ⇑ Corresponding author. Fax: +1 86 759 2284104. E-mail address: [email protected] (C. Cai). Abbreviations used: COBRA, combined bisulfite restriction analysis; GC, gas chromatography; MS, mass spectrometry; RPLC, reversed phase liquid chromatography; MS/MS, tandem mass spectrometry; HILIC, hydrophilic interaction chromatography; UHPLC, ultra high-pressure liquid chromatography; Cyt, cytosine; 5mCyt, 5methylcytosine; IS, internal standard; Ura, uracil; LC, liquid chromatography; Cyt13C15N2, cytosine-2,4-13C15N2; ESI, electrospray ionization; MRM, multiple reaction monitoring; UV, ultraviolet; CAD, collision-assisted dissociation; RSD, relative standard deviation; LOD, limit of detection; LOQ, limit of quantification. 1
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.01.029
enzymatic or chemical hydrolysis of DNA. Recently, some assays have been developed in the hope of analyzing nucleobases using gas chromatography (GC) and detection with mass spectrometry (MS) [12,13]. Unfortunately, the speed of the method used is also a consideration. Moreover, the method requires conversion of nucleobases to volatile derivatives for separation, so the application of the method is limited. In comparison with GC–MS, high-performance liquid chromatography (HPLC)–MS provides significant advantages in accordance with speed and ease of assay. Recently, several methods have been published with the aim of estimating global DNA methylation through reversed phase liquid chromatography tandem mass spectrometry (RPLC–MS/MS) [14–16]. However, due to nucleosides as target compounds in these methods, they cannot overcome the inconsistencies in the activity and stability of enzyme that lead to incompletion of DNA hydrolysis, so the results using nucleobases to reflect DNA methylation are often so good that the use of nucleosides is unnecessary. Although RPLC is overall the most applied separation technique, it is not adaptable to retain or separate highly polar, ionic, and hydrophilic compounds such as nucleobases. For that reason, nucleobases may elute in void volume or coelute by RPLC, making it difficult to assess them [17]. But the ability of hydrophilic interaction chromatography (HILIC) is particularly useful for these compounds [18–21].
Analysis of DNA methylation by HILIC–LC–MS / J.-j. Zhang et al. / Anal. Biochem. 413 (2011) 164–170
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In this article, we describe the development of an ultra highpressure liquid chromatography (UHPLC)–MS/MS method that hydrolyzes DNA sample by formic acid and separates using a HILIC column for determining the change in the relative amount of cytosine (Cyt) and 5-methylcytosine (5mCyt) in global genomic DNA. Compared with the existing methods, the advantages of this method are as follows: (i) it avoids the derivatization of nucleobases and incompletion of enzymatic hydrolysis; (ii) it avoids the use of ion pair reagents; and (iii) the use of isotopically labeled internal standards (ISs) increases sensitivity and precision.
DNA from samples was isolated by digestion with proteinase K at 45 °C for 24 h, followed by phenol–chloroform and precipitation by the addition of 1/10 volume of 3 M sodium acetate (pH 5.4) and 2.5-fold volume of absolute ethanol. Extracted DNA samples were dried under nitrogen and reconstituted with 0.5 ml of water. DNA purity was confirmed by comparing the ratio of ultraviolet (UV) measurements at 260 and 280 nm with the ratio of pure DNA standards, which should be between 1.7 and 1.9. The concentration of isolated DNA was determined by measuring UV absorbance at 260 nm.
Materials and methods
Sample preparation
Reagents
DNA solution containing 1.0 lg of DNA in a glass vial was dried by nitrogen. The residue was mixed with 0.2 ml of 88% aqueous formic acid and then hydrolyzed at 140 °C for 90 min. To the solution, Cyt13C15N2 was added and then evaporated under nitrogen. The residue was dissolved in methanol and centrifuged at 15,000g for 5 min, and the supernatant was extracted for analysis by HILIC–UHPLC–MS/MS as described above.
Cyt, 5mCyt, uracil (Ura), and ammonium formate (HPLC grade, purity >99%) were purchased from Sigma. The IS was cytosine2,4-13C15N2 (Cyt13C15N2, purity >99%, Toronto Research Chemical). Methanol and acetonitrile (HPLC grade) were purchased from Merck. All other reagents were of analytical grade. For liquid chromatography (LC), distilled water was further purified through passing a Milli-Q Plus apparatus (Millipore).
Percentage of methylation
Preparation of stock solution and calibration standard
The percentage of methylation was calculated using the following expression:
Stock solutions of the individual standards were prepared by dissolving each analyte in methanol at 40 lg/ml with the exception of 5mCyt at a concentration of 2 lg/ml. For assessing the linearity of the method, the stock standard solutions were further diluted in methanol to prepare a series of working standard solutions of the analytes containing Cyt and 5mCyt in the range of 0.1, 0.075, 0.05, 0.04, 0.025, and 0.001 lg/ml (Cyt) and 0.025, 0.01, 0.0075, 0.005, 0.004, 0.0025, and 0.001 lg/ml (5mCyt). Cyt13C15N2 as IS was dissolved in methanol to a final concentration of 0.04 lg/ml.
Methylation% ¼ Q 5mCyt =ðQ 5mCyt þ Q Cyt Þ 100%; where Q5mCyt is the molar quantity of 5mCyt and QCyt is the molar quantity of Cyt determined in the DNA sample. Statistical analysis For statistical analysis, we used SPSS software. Descriptive statistics were used to describe data distribution. Results are expressed as means ± standard deviations.
LC–MS
Results and discussion
LC, consisting of a binary pump, an autosampler, and a thermostatted column compartment, was performed with a BEH HILIC column (1.7 lm, 2.1 100 mm, Waters), preceded by an inline filter, and interfaced with a 6430A triple–quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source and operated in positive mode using nitrogen as nebulizer gas. A sample volume of 5 ll was injected into the HILIC column. Acetonitrile (solvent A) and water with 2.5 mM ammonium formate (solvent B) were used as mobile phase. An isocratic elution program was performed, with solvent A and solvent B being mixed (93:7 proportion) at a flow rate of 0.2 ml/min. The parameters of ESI–MS were as follows: nebulizer gas pressure, 35 psi; curtain gas flow, 10 l/min; capillary voltage, 4000 V; turboprobe temperature, 320 °C. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 112/95 for Cyt, m/z 126/109 for 5mCyt, m/z 115/97 for Cyt13C15N2, and m/z 113/96 for Ura. Alternatively, qualitative chromatograms were acquired in MRM mode by monitoring two transition pairs of m/z 112/69, 126/83, 115/70, and 113/70 for Cyt, 5mCyt, Cyt13C15N2, and Ura, respectively.
Mass spectrometric characterization of Cyt and 5mCyt
Biological samples and DNA extraction Tumor tissue and adjacent normal tissue in six patients with colorectal cancer were obtained from the affiliated hospital of Guangdong Medical College (Zhanjiang, China). The samples were stored at –70 °C until analysis.
Acquisition parameters were determined by means of direct infusion into the mass spectrometer of a 1-lg/ml solution of the analytes and a 40-ng/ml solution of Cyt13C15N2 at a flow rate of 0.3 ml/min, with the ESI source operating in both positive and negative modes. For evaluating the performance of the signal in each ionization mode, full scan mass spectra was recorded in the m/z range 100–200. The results indicated higher responses in positive mode than in negative mode for analytes. Consequently, ESI in positive mode was selected for subsequent experiments. The most abundant ions of each compound were selected by continuous infusion in product ion scan mode to quantitation. Fig. 1A reports a full scan mass spectrum of Cyt. As shown in this figure panel, the most intense ion at m/z 112 corresponded to the protonated molecular ion [M + H]+. The collision-assisted dissociation (CAD) spectrum of the [M + H]+ of Cyt is presented in Fig. 1B. From this figure panel, it is noted that the product ions m/z 95 and 69 were generated from the precursor ion m/z 112. This indicates that the most abundant product ion at m/z 95 was a good candidate to monitor the quantitative transition of m/z 112/95 during the CAD reaction. Moreover, the multiple second transitions of m/z 112/69 can be used as a confirmatory ion. Fig. 1C reveals that 5mCyt exhibited the precursor ion at m/z 126 in the full scan mode spectra, coinciding with the compound’s protonated molecular ion [M + H]+. The ion at m/z 109 as the most abundant daughter ion was obtained under collision energy of
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Fig.1. Full scan and product ion scan of Cyt (A and B), 5mCyt (C and D), and Cyt13C15N2 (E and F).
20 eV. Therefore, the transition m/z 126/109 was chosen for quantification of 5mCyt in the MS/MS experiment, whereas the transition m/z 126/83 was used as qualitative ions. Fig. 1E displays that Cyt13C15N2 showed the precursor ion at m/z 115 in the full scan mode spectra, conforming to the compound’s protonated molecular ion [M + H]+. A common fragmentation pattern in Fig. 1F, leading to product ions with m/z 97 and 70, was observed for Cyt13C15N2. The product ion at m/z 97 was the quantification ion, and the product ion at m/z 70 was the confirmatory ion.
Liquid chromatographic characterization of Cyt and 5mCyt To set up the HILIC–UHPLC method, acetonitrile and water with 2.5 mM ammonium formate as modifier were used to try to separate Cyt and 5mCyt. The concentration of modifier was varied from 80:20 to 93:7, as displayed in Fig. 2. From this figure, following the increase in scales of acetonitrile/water with 2.5 mM ammonium formate, we found that the two nucleobases were easily resolved. Under these conditions, the back-pressure is very low.
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Fig.2. Chromatograms of Cyt and 5mCyt standard solutions obtained by using acetonitrile/2.5 mM ammonium formate at 0.2 ml/min. The mobile phase composition used for separation Cyt and 5mCyt is also shown: (A) 80:20 mixture of acetonitrile/2.5 mM ammonium formate; (B) 85:15 mixture of actonitrile/2.5 mM ammonium formate; (C) 90:10 mixture of acetonitrile/2.5 mM ammonium formate; (D) 93:7 mixture of acetonitrile/2.5 mM ammonium formate.
Interestingly, this may provide a way to obtain high efficiency of sub-2-lm particle packed columns on a conventional HPLC system as well. To optimize the UHPLC condition, the following factors must be considered: the LC elution program, the mobile phase composition, and flow rates [22,23]. Fig. 3 provides details of the test results. On the basis of the chromatographic results, 2.5 mM ammonium formate and acetonitrile (7:93 proportion) by isocratic elution at 0.2 ml/min was chosen as mobile phase. The HILIC– UHPLC conditions reduced the total chromatographic analysis time reported in other studies from 10 to 60 min [18,24,25]. Sample preparation In this method, the absence of RNA from samples used for analysis is crucial. So, we checked the Ura in the sample to estimate the
RNA residue in the sample. Fig. 4 reports the MRM chromatogram of 1 pg Ura standard (Fig. 4A) and sample (Fig. 4B). From the results of chromatograms, the retention time of Ura is 1.80 min and used samples are free of Ura, showing that there is no RNA residue or only trace amounts. Calibration curve Calibration was performed in MRM mode under the previously described optimized conditions. For the sake of quantifying the relative amounts of nucleobases, the HILIC–UHPLC–MS/MS method used an IS. The assay was found to have good linearity in the tested range for all analytes. The coefficient of determination was typically higher than 0.99. The experimental results for the calibration curve are demonstrated in Fig. 5. Good performance was found for
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Fig.3. Optimization of chromatographic parameters for sensitivity of 5mCyt and Cyt by hydrophilic interaction liquid chromatography. (A) The ammonium formate dependency of sensitivity was tested with Cyt and 5mCyt, and an eluent consisting of acetonitrile and water was adjusted to different ammonium formate concentrations. (B) To investigate the impact of formic acid, eluents containing 2.5 mM ammonium formate and the indicated concentrations of formic acid was applied. (C) A solution consisting of 2.5 mM ammonium formate and acetonitrile was used to record the flow rate dependency of sensitivity. (D) The proportion of acetonitrile was varied in eluents containing 2.5 mM ammonium formate.
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Precision was expressed as relative standard deviation (RSD) of five replicate determinations of the same standard solutions. Results described precision as being counted at the range of 0.70– 4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. Accuracy was in the range of 85–105%. For manifesting the precision of this method, we performed different experiments to determine the interday and intraday precision. The results were obtained by analyzing standard solution. The intraday precision had RSD values of 1.86% and 4.67% for Cyt and 5mCyt, respectively. The interday precision, expressed as RSD, were 3.72% and 4.68% for Cyt and 5mCyt, respectively. From the results, both the interassay variability and the intraassay variability were better than, or approximately the same as, those of other published methods [22,26,27]. The intraday and interday precision of the samples was tested. Intraday precision ranged from 3.73% to 4.12%, whereas interday precision ranged from 5.58% to 6.06%.
Fig.4. MRM chromatogram of 1 pg of Ura standard (A) and real sample (B). The retention time of Ura is 1.80 min.
Fig.5. Calibration curve for Cyt (A) and 5mCyt (B).
DNA methylation (%)
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6 Tumor tissue
Fig.6. Box plot of DNA methylation in adjacent normal tissue and tumor tissue in patients with colorectal cancer. N = 6.
For the recovery experiment, known amounts of Cyt and 5mCyt (1, 10, and 50 ng/ml) were spiked into 200 ll of DNA hydrolyzate (n = 3). After redissolution by methanol, UHPLC–MS/MS was carried out in triplicate for each sample. The recoveries of the known spiked amounts of Cyt ranged from 87.38% to 105.14%, whereas the recoveries of 5mCyt ranged from 86.52% to 94.52%. Limits of detection and quantification Based on a signal/noise ratio higher than 3, limit of detection (LOD) and limit of quantification (LOQ) were calculated. Attributed to excellent sensitivity by the use of IS, this method was applied to determine nucleobases in biological samples reaching an LOD of 0.05 pg for Cyt and 5mCyt, and the LOQ value for Cyt and 5mCyt was 0.5 pg. Considering the LOQ of 5mCyt, an LOQ of 0.21% for global methylation was estimated. Such a low methylation level is not expected in both physiological and pathological conditions (low expected methylation of 2%), so the assay could also be detected by a lower amount of DNA. However, the amount of DNA used in this assay (i.e., 1.0 lg) is lower than that required in a previous GC–MS assay (2.5 lg). Application to real samples According to the previously described and validated method, the laboratory study of both adjacent normal and tumor tissues in six patients with colorectal cancer was carried out to elucidate the utility and performance of this method. Characteristic results from the analysis of clinical samples are shown in Fig. 6. The degree of DNA methylation in adjacent normal or tumor tissue of patients with colorectal cancer was 5.20 ± 0.22% or 3.62 ± 0.19%, respectively, consistent with other authors’ reported data [28,29]. Conclusion A rapid, sensitive, and specific HILIC–UHPLC–MS/MS method for determination of Cyt and 5mCyt was applied to measure the alteration of DNA methylation. The method was fully validated in terms of selectivity, linearity, sensitivity, precision, accuracy, and recovery. Due to nucleobases as target compounds on UHPLC, this technique has the advantage of not only simplifying the derivative process but also being less expensive and reflecting accurate global methylation on account of no enzyme. The analytical procedure showed the determination of target compounds in the low picograms per microgram of DNA. The results obtained from real samples could be considered as the best way to measure differences in the degree of methylation among DNA samples. Considering the sensitivity, accuracy, and rapidity of our HILIC–UHPLC–MS/MS assay, this assay is suitable for application in extensive and routine analysis.
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[26] M.B. Vandegehuchte, F. Lemiere, C.R. Janssen, Quantitative DNA-methylation in Daphnia magna and effects of multigeneration Zn exposure, Comp. Biochem. Physiol. C 150 (2009) 343–348. [27] H. Ma, W. Zhang, J. Hu, Z. Yu, Y. Chen, Q. Luo, X. Shi, Y. Zhang, R. Song, Z. Zhou, G. Shen, J. Fu, Analysis of global DNA methylation levels in human blood using high-performance liquid chromatography/tandem electrospray ionization mass spectrometry, Eur. J. Mass Spectrom. (Chichester) 15 (2009) 555–561.
[28] F.J. Hernandez-Blazquez, M. Habib, J.M. Dumollard, C. Barthelemy, M. Benchaib, A. de Capoa, A. Niveleau, Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis, Gut 47 (2000) 689–693. [29] F. Lyko, D. Stach, A. Brenner, S. Stilgenbauer, H. Dohner, M. Wirtz, M. Wiessler, O.J. Schmitz, Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients, Electrophoresis 25 (2004) 1530–1535.
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Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry Jun-jie Zhang a, Lijian Zhang a, Keyuan Zhou a, Xiaoxia Ye a, Chunan Liu a, Liangtao Zhang a, Jingxuan Kang b, Chun Cai a,⇑ a b
Guangdong Medical College, Zhanjiang, Guangdong 524023, People’s Republic of China Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA
a r t i c l e
i n f o
Article history: Received 23 December 2010 Received in revised form 24 January 2011 Accepted 25 January 2011 Available online 9 March 2011 Keywords: DNA methylation Mass spectrum Hydrophilic interaction chromatography (HILIC) Ultra high-pressure liquid chromatography (UHPLC)
a b s t r a c t We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol–chloroform, hydrolyzed using 88% formic acid at 140 °C, spiked with cytosine-2,4-13C15N2 as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC–MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1– 50 and 1–100 ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70–4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC–MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation. Ó 2011 Elsevier Inc. All rights reserved.
DNA methylation [1], carried out by the addition of a methyl group to position 5 of cytosine by DNA methyltransferase, not only is emerging as an important player in the regulation of genes but also is crucial for maintaining genome stability [2]. Changes in the methylation status of genomic DNA can play a central role in processes related to several diseases [3,4], especially tumorigenesis. With the increasing interest in DNA methylation, there currently is a lot of information on the detection of DNA methylation, such as Southern blot analyses [5,6], combined bisulfite restriction analysis (COBRA)1 [7], methylight [8], and capillary zone electrophoresis [9], but each method has its own deficiency [10,11]. Compared with the above methods, accurate genomic methylation can be determined by chromatographic techniques. The technique separates the different nucleosides or nucleobases obtained following ⇑ Corresponding author. Fax: +1 86 759 2284104. E-mail address: [email protected] (C. Cai). Abbreviations used: COBRA, combined bisulfite restriction analysis; GC, gas chromatography; MS, mass spectrometry; RPLC, reversed phase liquid chromatography; MS/MS, tandem mass spectrometry; HILIC, hydrophilic interaction chromatography; UHPLC, ultra high-pressure liquid chromatography; Cyt, cytosine; 5mCyt, 5methylcytosine; IS, internal standard; Ura, uracil; LC, liquid chromatography; Cyt13C15N2, cytosine-2,4-13C15N2; ESI, electrospray ionization; MRM, multiple reaction monitoring; UV, ultraviolet; CAD, collision-assisted dissociation; RSD, relative standard deviation; LOD, limit of detection; LOQ, limit of quantification. 1
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.01.029
enzymatic or chemical hydrolysis of DNA. Recently, some assays have been developed in the hope of analyzing nucleobases using gas chromatography (GC) and detection with mass spectrometry (MS) [12,13]. Unfortunately, the speed of the method used is also a consideration. Moreover, the method requires conversion of nucleobases to volatile derivatives for separation, so the application of the method is limited. In comparison with GC–MS, high-performance liquid chromatography (HPLC)–MS provides significant advantages in accordance with speed and ease of assay. Recently, several methods have been published with the aim of estimating global DNA methylation through reversed phase liquid chromatography tandem mass spectrometry (RPLC–MS/MS) [14–16]. However, due to nucleosides as target compounds in these methods, they cannot overcome the inconsistencies in the activity and stability of enzyme that lead to incompletion of DNA hydrolysis, so the results using nucleobases to reflect DNA methylation are often so good that the use of nucleosides is unnecessary. Although RPLC is overall the most applied separation technique, it is not adaptable to retain or separate highly polar, ionic, and hydrophilic compounds such as nucleobases. For that reason, nucleobases may elute in void volume or coelute by RPLC, making it difficult to assess them [17]. But the ability of hydrophilic interaction chromatography (HILIC) is particularly useful for these compounds [18–21].
Analysis of DNA methylation by HILIC–LC–MS / J.-j. Zhang et al. / Anal. Biochem. 413 (2011) 164–170
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In this article, we describe the development of an ultra highpressure liquid chromatography (UHPLC)–MS/MS method that hydrolyzes DNA sample by formic acid and separates using a HILIC column for determining the change in the relative amount of cytosine (Cyt) and 5-methylcytosine (5mCyt) in global genomic DNA. Compared with the existing methods, the advantages of this method are as follows: (i) it avoids the derivatization of nucleobases and incompletion of enzymatic hydrolysis; (ii) it avoids the use of ion pair reagents; and (iii) the use of isotopically labeled internal standards (ISs) increases sensitivity and precision.
DNA from samples was isolated by digestion with proteinase K at 45 °C for 24 h, followed by phenol–chloroform and precipitation by the addition of 1/10 volume of 3 M sodium acetate (pH 5.4) and 2.5-fold volume of absolute ethanol. Extracted DNA samples were dried under nitrogen and reconstituted with 0.5 ml of water. DNA purity was confirmed by comparing the ratio of ultraviolet (UV) measurements at 260 and 280 nm with the ratio of pure DNA standards, which should be between 1.7 and 1.9. The concentration of isolated DNA was determined by measuring UV absorbance at 260 nm.
Materials and methods
Sample preparation
Reagents
DNA solution containing 1.0 lg of DNA in a glass vial was dried by nitrogen. The residue was mixed with 0.2 ml of 88% aqueous formic acid and then hydrolyzed at 140 °C for 90 min. To the solution, Cyt13C15N2 was added and then evaporated under nitrogen. The residue was dissolved in methanol and centrifuged at 15,000g for 5 min, and the supernatant was extracted for analysis by HILIC–UHPLC–MS/MS as described above.
Cyt, 5mCyt, uracil (Ura), and ammonium formate (HPLC grade, purity >99%) were purchased from Sigma. The IS was cytosine2,4-13C15N2 (Cyt13C15N2, purity >99%, Toronto Research Chemical). Methanol and acetonitrile (HPLC grade) were purchased from Merck. All other reagents were of analytical grade. For liquid chromatography (LC), distilled water was further purified through passing a Milli-Q Plus apparatus (Millipore).
Percentage of methylation
Preparation of stock solution and calibration standard
The percentage of methylation was calculated using the following expression:
Stock solutions of the individual standards were prepared by dissolving each analyte in methanol at 40 lg/ml with the exception of 5mCyt at a concentration of 2 lg/ml. For assessing the linearity of the method, the stock standard solutions were further diluted in methanol to prepare a series of working standard solutions of the analytes containing Cyt and 5mCyt in the range of 0.1, 0.075, 0.05, 0.04, 0.025, and 0.001 lg/ml (Cyt) and 0.025, 0.01, 0.0075, 0.005, 0.004, 0.0025, and 0.001 lg/ml (5mCyt). Cyt13C15N2 as IS was dissolved in methanol to a final concentration of 0.04 lg/ml.
Methylation% ¼ Q 5mCyt =ðQ 5mCyt þ Q Cyt Þ 100%; where Q5mCyt is the molar quantity of 5mCyt and QCyt is the molar quantity of Cyt determined in the DNA sample. Statistical analysis For statistical analysis, we used SPSS software. Descriptive statistics were used to describe data distribution. Results are expressed as means ± standard deviations.
LC–MS
Results and discussion
LC, consisting of a binary pump, an autosampler, and a thermostatted column compartment, was performed with a BEH HILIC column (1.7 lm, 2.1 100 mm, Waters), preceded by an inline filter, and interfaced with a 6430A triple–quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source and operated in positive mode using nitrogen as nebulizer gas. A sample volume of 5 ll was injected into the HILIC column. Acetonitrile (solvent A) and water with 2.5 mM ammonium formate (solvent B) were used as mobile phase. An isocratic elution program was performed, with solvent A and solvent B being mixed (93:7 proportion) at a flow rate of 0.2 ml/min. The parameters of ESI–MS were as follows: nebulizer gas pressure, 35 psi; curtain gas flow, 10 l/min; capillary voltage, 4000 V; turboprobe temperature, 320 °C. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring a transition pair of m/z 112/95 for Cyt, m/z 126/109 for 5mCyt, m/z 115/97 for Cyt13C15N2, and m/z 113/96 for Ura. Alternatively, qualitative chromatograms were acquired in MRM mode by monitoring two transition pairs of m/z 112/69, 126/83, 115/70, and 113/70 for Cyt, 5mCyt, Cyt13C15N2, and Ura, respectively.
Mass spectrometric characterization of Cyt and 5mCyt
Biological samples and DNA extraction Tumor tissue and adjacent normal tissue in six patients with colorectal cancer were obtained from the affiliated hospital of Guangdong Medical College (Zhanjiang, China). The samples were stored at –70 °C until analysis.
Acquisition parameters were determined by means of direct infusion into the mass spectrometer of a 1-lg/ml solution of the analytes and a 40-ng/ml solution of Cyt13C15N2 at a flow rate of 0.3 ml/min, with the ESI source operating in both positive and negative modes. For evaluating the performance of the signal in each ionization mode, full scan mass spectra was recorded in the m/z range 100–200. The results indicated higher responses in positive mode than in negative mode for analytes. Consequently, ESI in positive mode was selected for subsequent experiments. The most abundant ions of each compound were selected by continuous infusion in product ion scan mode to quantitation. Fig. 1A reports a full scan mass spectrum of Cyt. As shown in this figure panel, the most intense ion at m/z 112 corresponded to the protonated molecular ion [M + H]+. The collision-assisted dissociation (CAD) spectrum of the [M + H]+ of Cyt is presented in Fig. 1B. From this figure panel, it is noted that the product ions m/z 95 and 69 were generated from the precursor ion m/z 112. This indicates that the most abundant product ion at m/z 95 was a good candidate to monitor the quantitative transition of m/z 112/95 during the CAD reaction. Moreover, the multiple second transitions of m/z 112/69 can be used as a confirmatory ion. Fig. 1C reveals that 5mCyt exhibited the precursor ion at m/z 126 in the full scan mode spectra, coinciding with the compound’s protonated molecular ion [M + H]+. The ion at m/z 109 as the most abundant daughter ion was obtained under collision energy of
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Fig.1. Full scan and product ion scan of Cyt (A and B), 5mCyt (C and D), and Cyt13C15N2 (E and F).
20 eV. Therefore, the transition m/z 126/109 was chosen for quantification of 5mCyt in the MS/MS experiment, whereas the transition m/z 126/83 was used as qualitative ions. Fig. 1E displays that Cyt13C15N2 showed the precursor ion at m/z 115 in the full scan mode spectra, conforming to the compound’s protonated molecular ion [M + H]+. A common fragmentation pattern in Fig. 1F, leading to product ions with m/z 97 and 70, was observed for Cyt13C15N2. The product ion at m/z 97 was the quantification ion, and the product ion at m/z 70 was the confirmatory ion.
Liquid chromatographic characterization of Cyt and 5mCyt To set up the HILIC–UHPLC method, acetonitrile and water with 2.5 mM ammonium formate as modifier were used to try to separate Cyt and 5mCyt. The concentration of modifier was varied from 80:20 to 93:7, as displayed in Fig. 2. From this figure, following the increase in scales of acetonitrile/water with 2.5 mM ammonium formate, we found that the two nucleobases were easily resolved. Under these conditions, the back-pressure is very low.
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x10 2 +ESI MRM Frag=114.0V [email protected] (112.0 -> 95.0) standard s… 1 1
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Fig.2. Chromatograms of Cyt and 5mCyt standard solutions obtained by using acetonitrile/2.5 mM ammonium formate at 0.2 ml/min. The mobile phase composition used for separation Cyt and 5mCyt is also shown: (A) 80:20 mixture of acetonitrile/2.5 mM ammonium formate; (B) 85:15 mixture of actonitrile/2.5 mM ammonium formate; (C) 90:10 mixture of acetonitrile/2.5 mM ammonium formate; (D) 93:7 mixture of acetonitrile/2.5 mM ammonium formate.
Interestingly, this may provide a way to obtain high efficiency of sub-2-lm particle packed columns on a conventional HPLC system as well. To optimize the UHPLC condition, the following factors must be considered: the LC elution program, the mobile phase composition, and flow rates [22,23]. Fig. 3 provides details of the test results. On the basis of the chromatographic results, 2.5 mM ammonium formate and acetonitrile (7:93 proportion) by isocratic elution at 0.2 ml/min was chosen as mobile phase. The HILIC– UHPLC conditions reduced the total chromatographic analysis time reported in other studies from 10 to 60 min [18,24,25]. Sample preparation In this method, the absence of RNA from samples used for analysis is crucial. So, we checked the Ura in the sample to estimate the
RNA residue in the sample. Fig. 4 reports the MRM chromatogram of 1 pg Ura standard (Fig. 4A) and sample (Fig. 4B). From the results of chromatograms, the retention time of Ura is 1.80 min and used samples are free of Ura, showing that there is no RNA residue or only trace amounts. Calibration curve Calibration was performed in MRM mode under the previously described optimized conditions. For the sake of quantifying the relative amounts of nucleobases, the HILIC–UHPLC–MS/MS method used an IS. The assay was found to have good linearity in the tested range for all analytes. The coefficient of determination was typically higher than 0.99. The experimental results for the calibration curve are demonstrated in Fig. 5. Good performance was found for
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Fig.3. Optimization of chromatographic parameters for sensitivity of 5mCyt and Cyt by hydrophilic interaction liquid chromatography. (A) The ammonium formate dependency of sensitivity was tested with Cyt and 5mCyt, and an eluent consisting of acetonitrile and water was adjusted to different ammonium formate concentrations. (B) To investigate the impact of formic acid, eluents containing 2.5 mM ammonium formate and the indicated concentrations of formic acid was applied. (C) A solution consisting of 2.5 mM ammonium formate and acetonitrile was used to record the flow rate dependency of sensitivity. (D) The proportion of acetonitrile was varied in eluents containing 2.5 mM ammonium formate.
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Precision was expressed as relative standard deviation (RSD) of five replicate determinations of the same standard solutions. Results described precision as being counted at the range of 0.70– 4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. Accuracy was in the range of 85–105%. For manifesting the precision of this method, we performed different experiments to determine the interday and intraday precision. The results were obtained by analyzing standard solution. The intraday precision had RSD values of 1.86% and 4.67% for Cyt and 5mCyt, respectively. The interday precision, expressed as RSD, were 3.72% and 4.68% for Cyt and 5mCyt, respectively. From the results, both the interassay variability and the intraassay variability were better than, or approximately the same as, those of other published methods [22,26,27]. The intraday and interday precision of the samples was tested. Intraday precision ranged from 3.73% to 4.12%, whereas interday precision ranged from 5.58% to 6.06%.
Fig.4. MRM chromatogram of 1 pg of Ura standard (A) and real sample (B). The retention time of Ura is 1.80 min.
Fig.5. Calibration curve for Cyt (A) and 5mCyt (B).
DNA methylation (%)
Analysis of DNA methylation by HILIC–LC–MS / J.-j. Zhang et al. / Anal. Biochem. 413 (2011) 164–170
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4.00 3.00 2.00 1.00 0.00 N=
6 Normal tissue
6 Tumor tissue
Fig.6. Box plot of DNA methylation in adjacent normal tissue and tumor tissue in patients with colorectal cancer. N = 6.
For the recovery experiment, known amounts of Cyt and 5mCyt (1, 10, and 50 ng/ml) were spiked into 200 ll of DNA hydrolyzate (n = 3). After redissolution by methanol, UHPLC–MS/MS was carried out in triplicate for each sample. The recoveries of the known spiked amounts of Cyt ranged from 87.38% to 105.14%, whereas the recoveries of 5mCyt ranged from 86.52% to 94.52%. Limits of detection and quantification Based on a signal/noise ratio higher than 3, limit of detection (LOD) and limit of quantification (LOQ) were calculated. Attributed to excellent sensitivity by the use of IS, this method was applied to determine nucleobases in biological samples reaching an LOD of 0.05 pg for Cyt and 5mCyt, and the LOQ value for Cyt and 5mCyt was 0.5 pg. Considering the LOQ of 5mCyt, an LOQ of 0.21% for global methylation was estimated. Such a low methylation level is not expected in both physiological and pathological conditions (low expected methylation of 2%), so the assay could also be detected by a lower amount of DNA. However, the amount of DNA used in this assay (i.e., 1.0 lg) is lower than that required in a previous GC–MS assay (2.5 lg). Application to real samples According to the previously described and validated method, the laboratory study of both adjacent normal and tumor tissues in six patients with colorectal cancer was carried out to elucidate the utility and performance of this method. Characteristic results from the analysis of clinical samples are shown in Fig. 6. The degree of DNA methylation in adjacent normal or tumor tissue of patients with colorectal cancer was 5.20 ± 0.22% or 3.62 ± 0.19%, respectively, consistent with other authors’ reported data [28,29]. Conclusion A rapid, sensitive, and specific HILIC–UHPLC–MS/MS method for determination of Cyt and 5mCyt was applied to measure the alteration of DNA methylation. The method was fully validated in terms of selectivity, linearity, sensitivity, precision, accuracy, and recovery. Due to nucleobases as target compounds on UHPLC, this technique has the advantage of not only simplifying the derivative process but also being less expensive and reflecting accurate global methylation on account of no enzyme. The analytical procedure showed the determination of target compounds in the low picograms per microgram of DNA. The results obtained from real samples could be considered as the best way to measure differences in the degree of methylation among DNA samples. Considering the sensitivity, accuracy, and rapidity of our HILIC–UHPLC–MS/MS assay, this assay is suitable for application in extensive and routine analysis.
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[28] F.J. Hernandez-Blazquez, M. Habib, J.M. Dumollard, C. Barthelemy, M. Benchaib, A. de Capoa, A. Niveleau, Evaluation of global DNA hypomethylation in human colon cancer tissues by immunohistochemistry and image analysis, Gut 47 (2000) 689–693. [29] F. Lyko, D. Stach, A. Brenner, S. Stilgenbauer, H. Dohner, M. Wirtz, M. Wiessler, O.J. Schmitz, Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients, Electrophoresis 25 (2004) 1530–1535.