Determination of Oxidation Products of 5-Methylcytosine in Plants by

Jun 26, 2014 - Bi-Feng YuanQuan-Fei ZhuNing GuoShu-Jian ZhengYa-Lan WangJie ... of 5-Methylcytosine and Its Oxidation Products in Genomic DNA by ...
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Determination of Oxidation Products of 5‑Methylcytosine in Plants by Chemical Derivatization Coupled with Liquid Chromatography/ Tandem Mass Spectrometry Analysis Yang Tang, Jun Xiong, Han-Peng Jiang, Shu-Jian Zheng, Yu-Qi Feng,* and Bi-Feng Yuan* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China S Supporting Information *

ABSTRACT: Cytosine methylation (5-methylcytosine, 5-mC) in DNA is an important epigenetic mark that has regulatory roles in various biological processes. In plants, active DNA demethylation can be achieved through direct cleavage by DNA glycosylases, followed by replacement of 5-mC with cytosine by base excision repair (BER) machinery. Recent studies in mammals have demonstrated 5-mC can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5foC), and 5-carboxylcytosine (5-caC) by Ten−eleven translocation (TET) proteins. The consecutive oxidations of 5-mC constitute the active DNA demethylation pathway in mammals, which raised the possible presence of oxidation products of 5mC (5-hmC, 5-foC, and 5-caC) in plant genomes. However, there is no definitive evidence supporting the presence of these modified bases in plant genomic DNA, especially for 5-foC and 5-caC. Here we developed a chemical derivatization strategy combined with liquid chromatography−electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method to determine 5-formyl-2′-deoxycytidine (5-fodC) and 5-carboxyl-2′-deoxycytidine (5-cadC). Derivatization of 5-fodC and 5-cadC by Girard’s reagents (GirD, GirT, and GirP) significantly increased the detection sensitivities of 5-fodC and 5-cadC by 52−260fold. Using this method, we demonstrated the widespread existence of 5-fodC and 5-cadC in genomic DNA of various plant tissues, indicating that active DNA demethylation in plants may go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with BER. Moreover, we found that environmental stresses of drought and salinity can change the contents of 5-fodC and 5-cadC in plant genomes, suggesting the functional roles of 5-fodC and 5-cadC in response to environmental stresses.

T

DNA demethylation can be accomplished through active removal of 5-mC from DNA, which is achieved by DNA glycosylases (Demter, DME; repressor of silencing 1, ROS1) in combination with the base excision repair (BER) pathway.5,6 Generally, the glycosylases DME/ROS1 remove the 5-mC base and then cleave the DNA backbone at the resulting abasic site, followed by replacement of 5-mC with cytosine by BER machinery (Figure 1). In mammalian cells, however, there is no reported glycosylase that can excise 5-mC specifically. Nevertheless,

he process of DNA methylation, consisting of addition of a methyl group at the fifth position of cytosine to give 5methylcytosine (5-mC) at the CpG dinucleotide site, is one of the best-characterized epigenetic modifications and plays a crucial role in a variety of cellular processes.1 Properly established and maintained DNA methylation patterns are crucial for the normal functions of living cells.2 Although the levels of 5-mC are relatively low in mammalian genomes (3−8% of total cytosine), 5-mC is much more abundant in plant genomes (5−25% of total cytosine).3 DNA methylation is dynamic and reversible. In plants, de novo DNA methylation is established by domains rearranged methyltransferase 2 (DRM2) and maintained by DNA methyltransferase 1 (MET1), chromomethylase 3 (CMT3), and DRM2.4 © XXXX American Chemical Society

Received: May 6, 2014 Accepted: June 26, 2014

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Chemical derivatization has been widely used to increase the detection sensitivity of target compounds in LC/ESI-MS analysis.14−16 Girard’s reagents (GirD, GirT, and GirP), which harbor an easily chargeable moiety, can readily react with aldehydes to form hydrazones under mild conditions. In addition, Girard’s reagents harbor a hydrazide moiety, which may attack the carbonyl group of carboxylic acid as a nucleophile to form a stable hydrazide. Therefore, derivatization of 5-fodC and 5-cadC by Girard’s reagents to increase the detection sensitivity by LC/ESI-MS could be a promising strategy to determine 5-fodC and 5-cadC in biological samples. Herein, we evaluate chemical derivatization of 5-fodC and 5cadC by Girard’s reagents. Our results show that good derivatization efficiencies of 5-fodC and 5-cadC can be achieved with all three Girard’s reagents under mild conditions. Moreover, chemical derivatization significantly increased the detection sensitivities of 5-fodC and 5-cadC by 52−260-fold with LC/ESI-MS/MS analysis. Limits of detection of 5-fodC and 5-cadC can reach 0.03 and 0.42 fmol, respectively, upon derivatization. Using this method, we demonstrated the widespread existence of 5-fodC and 5-cadC in genomic DNA of various plant tissues, which indicates that active DNA demethylation in plants may also go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with base excision repair. Furthermore, we found that environmental stresses of drought and salinity can change the contents of 5-fodC and 5-cadC in plants, suggesting the functional roles of 5-fodC and 5-cadC in response to environmental stresses.

Figure 1. Structures of cytosine derivatives. 5-mC modification is catalyzed by DNA methyltransferases. In plants, glycosylases can remove the 5-mC base and then cleave the DNA backbone at the resulting abasic site, followed by replacement of 5-mC with cytosine by BER machinery. In mammals, 5-mC can be demethylated through the oxidation of 5-mC by TET proteins to produce 5-hmC, 5-foC, and 5caC. BER, base excision repair; TDG, thymine-DNA glycosylase; TET, Ten−eleven translocation.

recent studies suggested that active DNA demethylation in mammals could be achieved through a sequential oxidation of 5-mC by Ten−eleven translocation (TET) proteins with the generation of three intermediates: 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5caC).7−10 The oxidation products of 5-foC and 5-caC can be further recognized and cleaved by thymine-DNA glycosylase (TDG), thereby restoring unmethylated cytosine via the BER pathway (Figure 1).11 Discovery of the novel active DNA demethylation mechanism by TET protein-catalyzed oxidation of 5-mC in mammals raised the possible presence of consecutive oxidations of 5-mC to 5-hmC, 5-foC, and 5-caC in plants. One preliminary study reported the presence of 5-hmC in Arabidopsis thaliana genomic DNA by immunostaining assay,12 which encouraged us to look for 5-foC and 5-caC in plant genomes. These novel identified cytosine modifications differ in their abundance, with 5-foC occurring at a frequency of (1−20)/106 cytosines and 5-caC being present at a much lower level than 5foC in mammals.9,13 So far, 5-caC has been detected mainly in embryonic stem cells, which contain relatively high amounts of 5-caC (∼3/106 cytosines), and has not been identified in the majority of other tissues.9 Quantification of these cytosine modifications is challenging due to their low in vivo content as well as interference from the highly abundant normal nucleosides. Therefore, to achieve quantitative analysis of 5foC and 5-caC content in plant genomes, a highly sensitive detection method is required. Owing to the inherent sensitivity and selectivity, liquid chromatography−electrospray ionization mass spectrometry (LC/ESI-MS) has been used in the determination of 5-formyl-2′-deoxycytidine (5-fodC) and 5carboxyl-2′-deoxycytidine (5-cadC).9 However, the ionization efficiencies in electrospray ionization (ESI) of aldehydes or carboxylic acids are usually low. In this respect, an easily ionizable moiety can be introduced to enhance the ionization efficiency for sensitive detection by LC/ESI-MS analysis.

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EXPERIMENTAL SECTION Chemicals and Reagents. 2′-Deoxycytidine (dC), 2′deoxyguanosine (dG), 2′-deoxyadenosine (dA), and thymidine (dT) were purchased from Sigma−Aldrich (Beijing, China). 5Formyl-2′-deoxycytidine (5-fodC) and 5-carboxyl-2′-deoxycytidine (5-cadC) were purchased from Berry & Associates (Dexter, MI). Chromatographic-grade methanol and acetonitrile (ACN) were purchased from Tedia Co. Inc. (Fairfield, OH). All other solvents and chemicals used were of analytical grade. Aqueous ammonia (25−28% by weight) and triethylamine (TEA) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Girard’s reagents D, T, and P (GirD, GirT, and GirP) were purchased from TCI Co., Ltd. (Shanghai, China). S1 nuclease and alkaline phosphatase (CIAP) were from Takara Biotechnology Co., Ltd. (Dalian, China). 1Chloro-4-methylpyridinium iodide (CMPI) and phosphodiesterase I were purchased from Sigma−Aldrich (Beijing, China). The water used throughout the study was purified on a Milli-Q apparatus (Millipore, Bedford, MA). Stock solutions of dC, dG, dA, and dT were prepared in Milli-Q water at concentrations of 200 μg/mL. Stock solutions of 5-fodC and 5-cadC were prepared in Milli-Q water at concentrations of 10 μg/mL. CMPI and TEA were prepared at concentrations of 20 and 40 mM, respectively, in ACN. Plant Materials and Environmental Stresses Treatment. Plant samples, including Arabidopsis thaliana, Lycopersicon esculentum, Ginkgo biloba, Platycladus orientalis, Zea mays, and Oryza sativa were used in this study. A. thaliana was cultured according to a previously reported procedure.17 L. esculentum was grown in peat pots and maintained under a day−night cycle of 16 h of light (30 μE·m−2·s−1) at 30 °C and 8 h of darkness at 25 °C, and the L. esculentum leaves were B

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°C. For 5-fodC, the dried samples were added to 100 μL of ACN and then incubated at different temperatures ranging from 30 to 70 °C for 30 min with shaking at 1500 rpm. For 5cadC, the dried samples were added to 100 μL of ACN containing 40 nmol of CMPI and 100 nmol of TEA and then incubated at different temperatures ranging from 30 to 70 °C for 30 min with shaking at 1500 rpm. The reactions were stopped by immediate freezing at −80 °C. The amount of GirD was also optimized. We changed the molar ratios ranging from 5/1 to 800/1 (GirD/5-fodC, with 5fodC being fixed at 400 pmol) or from 30/1 to 1200/1 (GirD/ 5-cadC, with 5-cadC being fixed at 400 pmol). For derivatization of 5-cadC, CMPI was added in equal amount with GirD. All the reactions were incubated at 40 °C for 30 min with shaking at 1500 rpm. The derivatization time was optimized ranging from 5 to 60 min, and the reactions were performed at 40 °C with molar ratios of 50/1 (GirD/5-fodC) or 150/1 (GirD/5-cadC). The derivatization reactions were examined on a Shimadzu LC-15C HPLC system (Tokyo, Japan) equipped with two LC15C pumps, a CTO-15C thermostated column compartment, and a SPD-15C UV/vis detector. A Hisep ODS-A column (250 mm × 4.6 mm i.d., 5 μm, Weltech Co., Ltd., Wuhan, China) was used for the separation. The column temperature was set at 35 °C. Ammonium formate (20 mM, solvent A) and acetonitrile (solvent B) were employed as mobile phase with a flow rate of 0.80 mL/min. A gradient of 5−10% B over 20 min was used. Optimization of derivatization conditions of 5-fodC and 5cadC by GirT and GirP is described in Supporting Information. Analysis of 5-fodC and 5-cadC in Genomic DNA of Plant Samples. Derivatization of 5-fodC and 5-cadC from the digested plant genomic DNA by GirD was performed under optimized conditions. Briefly, 50 nmol of GirD was added into the nucleoside mixture of digested plant genomic DNA (20 μg), followed by drying with nitrogen gas at 37 °C and then redissolving in 100 μL of ACN. The resulting solution was incubated at 40 °C for 5 min to derivatize 5-fodC. Then 2.5 μL of CMPI (20 mM in ACN) and 2.5 μL of TEA (40 mM in ACN) were added, and the reaction mixture was incubated for an additional 40 min at 40 °C to derivatize 5-cadC. The derivatized products were dried with nitrogen gas at 37 °C and then reconstituted in 100 μL of water, followed by LC/ESIMS/MS analysis. Quantification of 5-fodC and 5-cadC derivatives was performed on the LC/ESI-MS/MS system consisting of an AB 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA) with an electrospray ionization source (Turbo Ionspray) and a Shimadzu LC-20AD HPLC (Tokyo, Japan) with two LC-20AD pumps, a SIL-20A auto sampler, a CTO20AC thermostated column compartment, and a DGU-20A3 degasser. Data acquisition and processing were performed with AB Sciex Analyst 1.5 software (Applied Biosystems, Foster City, CA). The HPLC separation was performed on a Shimadzu VP-ODS column (150 mm × 2.0 mm i.d., 5 μm, Tokyo, Japan) at 35 °C. Water (solvent A) and ACN (solvent B) were employed as mobile phase. A gradient of 5% B for 5 min, 5−50% B over 25 min, 50% B for 3 min, and 5% B for 10 min was used. The flow rate of mobile phase was set at 0.2 mL/ min. Mass spectrometric detection was performed in positive electrospray ionization mode. The nucleosides and derivatives were monitored by multiple reaction monitoring (MRM) of the

harvested after 3 weeks of growth. G. biloba and P. orientalis were obtained from Wuhan University (China). Z. mays was purchased in a local market in Wuhan, China. O. sativa seeds were germinated at 30 °C in an incubation room, and then the germinated seeds were planted in a seedling nursery and grown under a day−night cycle of 16 h of light (30 μE·m−2·s−1) at 30 °C and 8 h of darkness at 25 °C. O. sativa was harvested after 15 days of growth. Drought stress was applied for 48 h by draining water and stopping irrigation of O. sativa seedling growth at 15 days according to a previously described method.18 Salt stress was applied according to a previously reported procedure by irrigating seedlings at 15 days with 300 mM NaCl solution, and then the O. sativa were harvested after 24 h of salt stress.19 All the harvested plant samples were weighted and immediately frozen in liquid nitrogen and then stored at −80 °C. DNA Extraction and Enzymatic Digestion. Plant genomic DNA was extracted by use of an E.Z.N.A. Plant DNA Kit (Omega Bio-Tek Inc., Norcross, GA) according to the manufacturer’s recommended procedure. Typically, ∼25 μg of genomic DNA can be obtained from 100 mg of plant tissue. The concentration of the purified plant genomic DNA was determined on a B-500 spectrophotometer (Metash Instruments Co., Ltd., Shanghai, China). The enzymatic digestion of genomic DNA was performed according to the previously described method.20 Briefly, genomic DNA (20 μg in 16 μL of H2O) was first denatured by heating at 95 °C for 5 min and then chilling on ice for 2 min. After addition of 1/10 volume (2 μL) of S1 nuclease buffer (30 mM CH3COONa, pH 4.6, 280 mM NaCl, and 1 mM ZnSO4) and 360 units (2 μL) of S1 nuclease, the mixture (20 μL) was then incubated at 37 °C for 16 h. To the resulting solution were subsequently added 10 μL of alkaline phosphatase buffer (50 mM Tris-HCl and 10 mM MgCl2, pH 9.0), 0.01 unit (5 μL) of venom phosphodiesterase I, 30 units (1 μL) of alkaline phosphatase, and 64 μL of H2O. Then the incubation was continued at 37 °C for an additional 4 h. The digested DNA was filtered by an ultrafiltration tube (10 kDa cutoff, Amicon, Millipore) to remove the enzymes, and the obtained solution was further passed through a SPE (solid-phase extraction) cartridge filled with 50 mg of graphitized carbon black sorbent (Weltech Co., Ltd., Wuhan, China) to remove the salts. The eluate was then dried with nitrogen gas at 37 °C, followed by derivatization. Quality control (QC) samples were also prepared to validate the method. Briefly, a 17-mer oligodeoxynucleotide (5′GAATTCGGCGCCCATGG-3′) was mixed with a 27-mer oligodeoxynucleotide (5′-GAGTCGCTCCCCATGGGXACCGAATTC-3′, where X indicates 5-fodC or 5-cadC) at different amounts to make the molar ratios of 5-fodC/106 dG or 5cadC/106 dG range from 0.20 to 5.00 or from 0.08 to 2.00, respectively. The QC samples were then enzymatically digested, followed by derivatization by the same procedure as that for plant genomic DNA. Chemical Derivatization. In this study, Girard’s reagents GirD, GirT, and GirP were all used to derivatize 5-fodC and 5cadC. To achieve the best derivatization efficiency, we optimized the derivatization conditions, including reaction temperature, amounts of Girard’s reagent, and reaction time. For optimization of derivatization conditions by GirD, we first optimized the reaction temperature. Generally, 20 nmol of GirD (dissolved in water) was added to 5-fodC (400 pmol) or 5-cadC (400 pmol), followed by drying with nitrogen gas at 37 C

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Figure 2. Schematic diagram for determination of 5-fodC and 5-cadC in genomic DNA of plant samples by chemical derivatization by Girard’s reagents coupled with LC/ESI-MS/MS analysis.

Figure 3. Derivatization reactions between (A) 5-fodC and GirD and (B) 5-cadC and GirD, and product ion spectra of (C) 5-fodC derivative and (D) 5-cadC derivative.

mass transitions (precursor ions → product ions) of dC (228.4 → 112.2), dT (243.3 → 127.2), dA (252.4 → 136.2), dG (268.4 → 152.4), 5-fodC (256.0 → 140.3), 5-cadC (272.2 → 156.2), 5-fodC derivatives (355.2 → 239.1 for GirD-derivatized 5-fodC, 369.2 → 253.1 for GirT-derivatized 5-fodC, and 389.2 → 273.1 for GirP-derivatized 5-fodC), and 5-cadC derivatives (371.2 → 255.1 for GirD-derivatized 5-cadC, 385.2 → 269.1 for GirT-derivatized 5-cadC, and 405.2 → 289.1 for GirPderivatized 5-cadC). The MRM parameters of the analytes were optimized to achieve maximal detection sensitivity. High-resolution mass spectrometric experiments were performed on the LC/QTOF-MS system consisting of a MicrOTOF-Q orthogonal-accelerated time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) with an ESI source (Turbo Ionspray) and a Shimadzu LC20AB binary pump HPLC (Tokyo, Japan), a SIL-20AC auto sampler, and a DGU-20A3 degasser. Data acquisition and processing were performed with Bruker Daltonics Control 3.4 and Bruker Daltonics Data Analysis 4.0 software. The HPLC separation was performed on a Shimadzu VP-ODS column (150 mm × 2.0 mm i.d., 5 μm, Tokyo, Japan) at 35 °C. Water (solvent A) and ACN (solvent B) were employed as mobile

phase. A gradient of 5% B for 5 min, 5−50% B over 15 min, 50% B for 3 min, and 5% B for 10 min was used. The flow rate of mobile phase was set at 0.2 mL/min.

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RESULTS AND DISCUSSION

Recently identified novel DNA modifications (5-hmC, 5-foC, and 5-caC) were considered to have potential regulatory roles and may add additional layers of complexity to gene expression regulation.21 We and others also demonstrated that 5-foC and 5-caC can change the fidelity of DNA replication and slow down RNA polymerase II transcription, which indicated the possible functional roles of 5-foC and 5-caC on replication and transcription.22,23 However, there is no definitive evidence supporting the presence of 5-foC and 5-caC in plants due to the low contents of these cytosine derivatives and the poor ionization efficiency when analyzed by mass spectrometry. Here we developed a chemical derivatization strategy to modify 5fodC and 5-cadC with an easily chargeable moiety, which can significantly increase the mass spectrometry response (Figure 2). Using the developed method, we successfully identified and quantified 5-fodC and 5-cadC in genomic DNA of various plant samples. D

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Figure 4. Optimization of derivatization conditions for 5-fodC and 5-cadC by GirD: optimization of reaction temperature for (A) 5-fodC and (B) 5cadC; optimization of molar ratios (C) GirD/5-fodC and (D) GirD/5-cadC; and optimization of reaction time for (E) 5-fodC and (F) 5-cadC. Also shown are HPLC chromatograms under optimized conditions for (G) 5-fodC and 5-fodC derivative and (H) 5-cadC and 5-cadC derivative.

Chemical Derivatization. Girard’s reagents harbor a hydrazide moiety that can readily react with aldehydes to give hydrazone derivatives24 with easily chargeable moieties (quaternary ammonium/pyridinium/tertiary ammonium) (Figure 2). In addition, the hydrazide group can conjugate to carboxylic acids with CMPI as an activator, which can activate carboxyl groups to form a reactive ester for direct and rapid reaction with primary amines via amide bond formation in slightly alkaline conditions.25 Therefore, we chose Girard’s reagents to derivatize 5-fodC and 5-cadC.

In a preliminary experiment, GirD was added to 5-fodC or 5cadC for derivatization at a molar ratio of 100/1. The reactions were performed at 40 °C for 2 h, and then the resulting solutions were subjected to LC/ESI-MS/MS analysis to examine the derivatization products. As expected, two ions of m/z 355.2 and 239.2, which represent the parent ion of 5-fodC derivative and its product ion, were observed after GirD treatment (Figure 3A,C). Similarly, two ions of m/z 371.2 and 255.2, which represent the parent ion of 5-cadC derivative and its product ion, were also observed after GirD treatment E

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With the use of appropriate derivatization reagents, the derivatized products could also be selectively enriched from complex matrix, which therefore can increase the detection sensitivity. Optimization of Derivatization Conditions. We first optimized the reaction temperature for derivatization of 5-fodC and 5-cadC by GirD. Our results demonstrated that the largest peak areas of both 5-fodC and 5-cadC derivatives can be achieved at 40 °C (Figure 4A,B). We next investigated the optimal amount of GirD for derivatization of 5-fodC and 5-cadC. The results showed that the peak area of 5-fodC derivative reaches a plateau when the molar ratio of GirD/5-fodC is 50/1 (Figure 4C). Likewise, the peak area of 5-cadC derivative reaches a plateau when the molar ratio of GirD/5-cadC is 150/1 (Figure 4D). For optimization of derivatization time, the result showed that derivatization of 5-fodC by GirD was very fast and 5 min was enough for efficient reaction (Figure 4E). The best derivatization of 5-cadC by GirD can be achieved with 40 min (Figure 4F). Taken together, the optimized derivatization conditions for 5-fodC by GirD were 40 °C for 5 min with a 50/1 molar ratio of GirD/5-fodC; and the optimized derivatization conditions for 5-cadC by GirD were 40 °C for 40 min with a 150/1 molar ratio of GirD/5-cadC. Under optimized derivatization conditions, more than 99% of 5-fodC and more than 95% of 5cadC can react with GirD to form the corresponding derivatives (Figure 4G,H), suggesting high derivatization efficiencies were achieved. It was reported that hydrazone formation between carbonyls and hydrazides are typically favorable under mildly acidic conditions.29 Because the derivatization of 5-cadC requires slightly alkaline condition, we performed the derivatization of 5fodC in ACN, which then can be easily adjusted to alkaline by addition of TEA to obtain appropriate conditions for derivatization of 5-cadC. Since high derivatization efficiencies of 5-fodC and 5-cadC by GirD (99% and 95%) can be achieved in a relatively short time, we therefore chose ACN as the reaction solution. In addition, optimization of the derivatization conditions of 5-fodC and 5-cadC by GirT and GirP can be seen (Figures S4 and S5 in Supporting Information). The results suggested that good derivatization efficiencies (≥95%) can be achieved by GirT and GirP under their own optimized derivatization conditions (data not shown). Enhancement of Detection Sensitivities of 5-fodC and 5-cadC upon Derivatization. The main purpose for derivatization is to increase the detection sensitivities of 5fodC and 5-cadC during mass spectrometric analysis. We then further examined the enhancement of detection sensitivities of 5-fodC and 5-cadC in LC/ESI-MS/MS analysis by derivatization with three Girard’s reagents under their own optimized conditions. Compared to the underivatized 5-fodC and 5-cadC, three Girard’s reagents could significantly increase the detection sensitivities by 52−260-fold for 5-fodC and 135−247-fold for 5cadC (Table 1). Shown in Figure S6 in Supporting Information are the MRM chromatograms of 5-fodC and 5cadC before and after derivatization by GirD. The limit of detection (LOD) of 5-fodC derivative is 0.03 fmol by GirP derivatization, and the LOD of 5-cadC derivative is 0.42 fmol by GirD derivatization (Table 1), which are, to the best of our knowledge, the lowest LODs reported for 5-fodC and 5-cadC by direct LC/ESI-MS/MS analysis. The significant increase in

Table 1. Limits of Detection of 5-fodC and 5-cadC with and without Derivatization by Girard’s Reagents 5-fodC

5-cadC

Girard’s reagenta

LOD (fmol)

detection sensitivity increase (x-fold)

LOD (fmol)

detection sensitivity increase (x-fold)

none GirD GirT GirP

7.8 0.15 0.09 0.03

52 87 260

103.9 0.42 0.77 0.75

247 135 139

a

Derivatizations were performed under their own optimized conditions.

Table 2. Accuracy and Precision (Intra- and Interday) of Methods for Determination of 5-fodC and 5-cadC QC

theor value (/106 dG)

low medium high

0.20 1.00 5.00

low medium high

0.08 0.40 2.00

measd value (/106 dG)

rel error (%)

Determination 0.22 1.09 5.27 Determination 0.09 0.44 2.14

of 5-fodC 10.0 9.0 5.4 of 5-cadC 12.5 10.0 7.0

intraday (RSD %, n = 3)

interday (RSD %, n = 3)

6.9 9.4 10.1

8.2 10.8 12.0

3.4 8.7 5.5

7.5 10.8 11.0

Table 3. 5-fodC and 5-cadC Content in Genomic DNA of Various Plant Samplesa plant sample A. thaliana leaves G. biloba leaves P. orientalis leaves O. sativa roots O. sativa stems O. sativa leaves Z. mays leaves Z. mays kernels Z. mays silks L. esculentum stems L. esculentum leaves

no. of 5-fodC/106 dG 4.2 2.8 4.7 3.7 2.2 2.6 3.0 3.1 4.0 4.4 2.1

± ± ± ± ± ± ± ± ± ± ±

1.1 1.4 0.3 0.6 0.4 0.4 0.3 1.8 0.4 0.5 0.2

no. of 5-cadC/106 dG 3.4 1.9 3.6 0.9 0.3 0.2 0.2 0.6 0.3 0.4 0.4

± ± ± ± ± ± ± ± ± ± ±

0.6 0.2 0.2 0.03 0.04 0.02 0.02 0.1 0.1 0.1 0.1

a

Data represent the mean and standard deviation of results from three independent measurements.

(Figure 3B,D). The derivatization products of 5-fodC and 5cadC were also confirmed by high-resolution mass spectrometric analysis (Figure S1A,B, Supporting Information). In addition, we also examined the derivatization by two other Girard’s reagents (GirT and GirP) of 5-fodC or 5-cadC. The results demonstrated GirT and GirP could also react with 5fodC and 5-cadC to form hydrazones and amides conjugates (Figures S2 and S3, Supporting Information). Next, we attempted to derivatize 5-fodC and 5-cadC simultaneously with Girard’s reagents. Because chemical derivatization of 5-cadC by Girard’s reagents requires the activator CMPI, we then added CMPI in the reaction mixture. Unfortunately, 5-fodC was unable to react with Girard’s reagents in the presence of CMPI. Therefore, we chose to sequentially derivatize 5-fodC and 5-cadC. It is worth noting that the aldehyde group is relatively active and can readily react with many other nucleophiles,15 such as aminothiols,26,27 hydroxylamine,28 hydrazine,29 hydrazide,30 etc. F

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Figure 5. Representative MRM chromatograms for quantification of (A) 5-fodC and (B) 5-cadC in genomic DNA of A. thaliana leaves and derivatization products of (C) 5-fodC standard and (D) 5-cadC standard.

dG and 5-cadC/106 dG were obtained with correlation coefficients (R) being greater than 0.99 (Table S1, Supporting Information). The accuracy of the proposed method was assessed in QC samples by comparing the measured 5-fodC and 5-cadC contents to the theoretical values. Three different molar ratios of 5-fodC/106 dG, ranging from 0.20 to 5.00, and of 5-fodC/ 106 dG, ranging from 0.08 to 2.00, were measured (Table 2). In addition, the reproducibility of the developed method was evaluated by measurement of intra- and interday precision. The intra- and interday relative standard deviations (RSDs) were calculated with different QCs. Three parallel treatments of samples over a day gave the intraday RSDs, and the interday RSDs were determined by treating samples independently for three consecutive days. The results showed that good accuracy was achieved, which is manifested by the relative errors (RE) being less than 10.0% for 5-fodC and 12.5% for 5-cadC (Table 2). The results also showed that the intra- and interday RSDs were less than 10.1% and 12.0%, respectively (Table 2), demonstrating that good reproducibility was achieved. Determination of 5-fodC and 5-cadC in Genomic DNA of Plant Tissues. After establishing the highly sensitive detection method, we explored the existence and amounts of 5fodC and 5-cadC in plant genomes. Using this GirD derivatization-based LC/ESI-MS/MS method, we successfully identified and quantified 5-fodC and 5-cadC in genomic DNA of various plant samples, including Arabidopsis thaliana, Lycopersicon esculentum, Ginkgo biloba, Platycladus orientalis, Zea mays, and Oryza sativa (Table 3). We also employed highresolution mass spectrometry to analyze the 5-fodC and 5-cadC derivatives. The results showed that product ion spectra of the derivatives of 5-fodC and 5-cadC from plant samples (Figure

Figure 6. Content change of 5-fodC and 5-cadC in genomic DNA of O. sativa under drought or salt stress (300 mM NaCl).

detection sensitivities achieved by Girard’s reagents is mainly due to the formation of easily ionizable derivatives. Considering the relatively low detection limit of 5-cadC achieved by derivatization with GirD, we chose GirD as the derivatization reagent for determination of 5-fodC and 5-cadC in plant genomes. Method Validation. The calibration curves of 5-fodC and 5-cadC were constructed by plotting the mean peak area ratios of 5-fodC/106 dG or 5-cadC/106 dG versus the mean molar ratios of 5-fodC/106 dG or 5-cadC/106 dG based on data obtained from triplicate measurements of derivatization strategy combined with LC/ESI-MS/MS analysis. The results showed that good linearities within the range 0.05−10 for 5-fodC/106 G

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combination with LC/ESI-MS/MS analysis. The key advantage of the method is that derivatization with Girard’s reagents can dramatically enhance the detection sensitivity, making it feasible to detect low-abundance 5-fodC and 5-cadC in biological samples. Using this method, we were able to identify and quantify 5-fodC and 5-cadC in genomic DNA of various plant samples, which indicates that active DNA demethylation in plants may also go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with base excision repair.

S1C,D, Supporting Information) were identical with those of derivatives of 5-fodC and 5-cadC standards (Figure S1A,B, Supporting Information), supporting the expected existence of 5-fodC and 5-cadC in plants. Shown in Figure 5 panels A and B, respectively, are typical chromatograms of 5-fodC and 5cadC derivatives in A. thaliana. Figure 5 panels C and D show typical chromatograms of derivatives of 5-fodC and 5-cadC standards, respectively. The quantitative results demonstrated that 5-fodC and 5-cadC can be found in all the examined plant samples, with contents of 5-fodC ranging from 2.1 to 4.7 modifications per 106 dG and the contents of 5-cadC ranging from 0.2 to 3.6 modifications per 106 dG (Table 3). The measured contents of 5-fodC and 5-cadC in the genomes of plant samples are similar to the levels in mammalian cells or tissues.9 The existence of 5-fodC and 5-cadC in various plant samples, together with previously identified 5-hmdC, suggested the oxidation of 5-mdC in plants. Therefore, the active DNA demethylation in plants may go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with base excision repair (Figure 1). Effects of Environmental Stresses on Content of 5fodC and 5-cadC. Since 5-fodC and 5-cadC may have functional roles in plants, we further examined the levels of 5fodC and 5-cadC in genomic DNA of O. sativa under drought and salt stress. Compared to the control samples, the content of 5-fodC and 5-cadC in genomic DNAs of O. sativa roots significantly decreased under either drought or salt stress (Figure 6). The content of 5-fodC in roots decreased from 3.7 modifications/106 dG to 0.9 and 1.2 modifications/106 dG under drought or salinity stress, respectively. Likewise, the content of 5-cadC in roots decreased from 0.9 modifications/ 106 dG to 0.2 and 0.6 modifications/106 dG under drought or salt stress, respectively. In addition, 5-fodC content slightly decreased in genomic DNA of O. sativa stems and leaves under either drought or salinity stress. However, 5-cadC content slightly decreased under drought stress but increased under salinity stress in genomic DNA of O. sativa stems and leaves. The results demonstrated that environmental stresses showed a significant level of tissue specificity in the content alteration of 5-fodC and 5-cadC. Drought and salinity are major environmental factors that limit productivity of crop plants, including rice, in which a wide range of natural variability exists. Recent evidence implicates epigenetic mechanisms in modulating gene expression in plants under drought or salt stress, where DNA methylation normally decreased.18,19 The content of 5-fodC and 5-cadC can be affected by the content change of 5-mdC from two sides. On one hand, since 5-mdC may be oxidized to 5-fodC and 5-cadC, a decrease of 5-mdC could contribute to the accumulation of 5fodC and 5-cadC; on the other hand, decreased 5-mdC could also cause the reduced 5-fodC and 5-cadC since they are derived from 5-mdC. We can see 5-fodC and 5-cadC decreased in all the plant tissues under either drought or salinity stress except that 5-cadC slightly increased in stems and leaves under salinity stress. The specific reason for the content change of 5fodC and 5-cadC under environmental stresses still needs further investigation.

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ASSOCIATED CONTENT

S Supporting Information *

Additional text describing optimization of derivatization conditions by GirT and GirP; one table listing linearities of 5-fodC and 5-cadC; and six figures showing product ion spectra obtained by HR-MS, derivatization reactions and product ion spectra, optimization of derivatization conditions, and MRM chromatograms of 5-fodC and 5-cadC before and after derivatization by GirD. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone +86-27-68755595; fax +86-27-68755595; e-mail [email protected]. *Telephone +86-27-68755595; fax +86-27-68755595; e-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support from the National Basic Research Program of China (973 Program) (2013CB910702, 2012CB720601), the National Natural Science Foundation of China (91217309, 91017013, 21205091, 21228501), Ph.D. Programs Foundation of Ministry of Education of China (20120141120037), and Fundamental Research Funds for the Central Universities.

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CONCLUSION In this study, we developed a highly sensitive method to determine 5-fodC and 5-cadC by chemical derivatization in H

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