Articles pubs.acs.org/acschemicalbiology
Heavy Metals Induce Decline of Derivatives of 5‑Methycytosine in Both DNA and RNA of Stem Cells Jun Xiong,†,⊥ Xiaona Liu,‡,§,∥,⊥ Qing-Yun Cheng,† Shan Xiao,§ Lai-Xin Xia,*,§ Bi-Feng Yuan,*,† and Yu-Qi Feng† †
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China ‡ School of Life Sciences, University of Science and Technology of China, Hefei 230027, People’s Republic of China § Department of Developmental Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, People’s Republic of China ∥ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China S Supporting Information *
ABSTRACT: Toxic heavy metals have been considered to be harmful environmental contaminations. The molecular mechanisms of heavy-metals-induced cytotoxicity and carcinogenicity are still not well elucidated. Previous reports showed exposures to toxic heavy metals can cause a change of DNA cytosine methylation (5-methylcytosine, 5-mC). However, it is still not clear whether heavy metals have effects on the recently identified new epigenetic marks in both DNA and RNA, i.e., 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5carboxylcytosine (5-caC). Here, we established a chemical labeling strategy in combination with liquid chromatography− electrospray ionization−mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive detection of eight modified cytidines in DNA and RNA. The developed method allowed simultaneous detection of all eight modified cytidines with improved detection sensitivities of 128−443-fold. Using this method, we demonstrated that the levels of 5-hmC, 5-foC, and 5-caC significantly decreased in both the DNA and RNA of mouse embryonic stem (ES) cells while exposed to arsenic (As), cadmium (Cd), chromium (Cr), and antimony (Sb). In addition, we found that treatments by heavy metals induced a decrease of the activities of 10−11 translocation (Tet) proteins. Furthermore, we revealed that a content change of metabolites occurring in the tricarboxylic acid cycle may be responsible for the decline of the derivatives of 5-mC. Our study shed light on the epigenetic effects of heavy metals, especially for the induced decline of the derivatives of 5-mC in both DNA and RNA.
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chronic exposure of cadmium caused increased 5-mC in rat liver cells.12 Therefore, exploration of epigenetic effects of heavy metals will promote understanding of the mechanisms of carcinogenesis induced by heavy metals. Among the known nucleic acid modifications, 5-mC is the most important epigenetic modification in DNA.13 Recent reports demonstrated that 10−11 translocation (Tet) proteins can convert 5-mC in DNA to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5caC).14−17 Similar to 5-mC, these newly identified cytosine modifications are considered to also fulfill the regulation roles in gene expressions.18 Moreover, Wang’s group recently found Tet proteins also can convert 5-mC to 5-hmC in RNA.19 Our group and others further confirmed both 5-foC and 5-caC exist in the RNA of eukaryotes.20−22 However, to the best of our
any toxic heavy metals, including arsenic (As), cadmium (Cd), chromium (Cr), and antimony (Sb), have been considered to be harmful environmental pollutants and noted for their potential toxicity in living organisms.1 As, Cd, and Cr are characterized to be cancer-inducing agents.2 Some heavy metals can produce reactive oxygen species (ROS) that can result in the damage of proteins, nucleic acids, and lipids, eventually leading to dysregulation of physiological functions.3 However, the molecular mechanisms of heavy metals-induced cytotoxicity and carcinogenicity are still not well elucidated.4 Previous studies showed that heavy metals are nonmutagenic or slightly mutagenic,5 indicating the geneticbased carcinogenic mechanism might not play a major role.6,7 Epigenetic modifications of chromatin in higher eukaryotes are essential for regulating gene expression.8 Emerging evidence showed that exposures to heavy metals can induce alteration of DNA cytosine methylation (5-methylcytosine, 5-mC).9 For example, chronic exposure to arsenic caused an increase of 5mC in promoters of p53 and p16 genes;10 short exposure to cadmium led to a decrease of global 5-mC in chick embryos;11 © 2017 American Chemical Society
Received: February 24, 2017 Accepted: April 27, 2017 Published: April 27, 2017 1636
DOI: 10.1021/acschembio.7b00170 ACS Chem. Biol. 2017, 12, 1636−1643
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ACS Chemical Biology
Figure 1. Chemical labeling of cytosine modifications by BDEPE. (A) Reaction between modified cytidines and BDEPE. (B−I) Product-ion spectra of BDEPE labeled 5-mdC, 5-hmdC, 5-fodC, 5-cadC, 5-mrC, 5-hmrC, 5-forC, and 5-carC.
heavy metals on 5-mC as well as its derivatives in both DNA and RNA of mouse ES cells. BDEPE Labeling. The low in vivo abundance makes the determination of 5-mC derivatives in DNA and RNA a challenging task. Some previous studies employed a chemical labeling strategy to increase the detection sensitives of nucleosides by introducing easily ionizable groups to the analytes.27−31 In this respect, here we used BDEPE labeling to improve the detection performance of the derivatives of 5-mC in DNA and RNA (Figure 1A). We first examined the BDEPE-labeled eight standards of the modified cytidines (5-mdC, 5-mrC, 5-hmdC, 5-hmrC, 5-fodC, 5-forC, 5-cadC, and 5-carC). The chemical reaction between BDEPE and modified cytidines is shown in Figure 1A. The product-ion spectra showed that all eight cytosine modifications successfully reacted with BDEPE and formed the desired products (Figure 1B−I). In addition to 3-N and 4-N positions, BDEPE also reacts with the carboxyl group in 5-cadC and 5carC (Figure 1E and I). The products that contain two phenyl groups were the predominant BDEPE-labeled products for 5cadC and 5-carC. Under optimized reaction conditions, the BDEPE labeling efficiency is higher than 95% (data not shown). Improved Detection Performance for Derivatives of 5-mC upon BDEPE Labeling. To achieve good detection performance, we optimized the analytical conditions of mass spectrometry under multiple reaction monitoring (MRM) mode for BDEPE-labeled products. The detailed optimized parameters of mass spectrometry can be found in Supporting Information Table 1.
knowledge, the effects of heavy metals on 5-hmC, 5-foC, and 5caC in either DNA or RNA remain unclear. Considering that heavy metals can have effects on DNA methylation, we speculate that heavy metals may also impose effects on 5-hmC, 5-foC, and 5-caC in DNA and RNA. In this respect, here we aimed to investigate the alteration of 5-mC as well as its derivatives in both DNA and RNA caused by heavy metals. Due to the extremely low abundance of the derivatives of 5-mC, we established a chemical labeling strategy in combination with mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive detection of all eight of these modified cytidines, including 5-mC, 5-hmC, 5-foC, and 5-caC in both DNA and RNA. Using this method, we demonstrated the levels of 5-hmC, 5-foC, and 5-caC significantly decreased in both the DNA and RNA of mouse embryonic stem (ES) cells while exposed to As, Cd, Cr, and Sb. We also found that treatments by heavy metals induced a decrease in the activities of Tet proteins. In addition, we further revealed that the content change of some metabolites in the tricarboxylic acid (TCA) cycle may be responsible for the decline of the derivatives of 5-mC in both DNA and RNA.
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RESULTS AND DISCUSSION Previous reports demonstrated that exposures to toxic heavy metals can possibly lead to a content change of 5-mC. However, it is not clear whether the heavy metals can induce alteration of the newly identified epigenetic marks of cytosine modifications in both DNA and RNA. In this respect, we proposed a chemical labeling strategy in combination with mass spectrometry detection to investigate the effects of exposures to 1637
DOI: 10.1021/acschembio.7b00170 ACS Chem. Biol. 2017, 12, 1636−1643
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Figure 2. Determination of 5-mC and its derivatives in DNA and RNA of mouse ES cells by BDEPE labeling coupled with LC-ESI-MS/MS analysis. (A) Extracted-ion chromatograms of the standards of eight cytosine modifications without BDEPE labeling. (B) Extracted-ion chromatograms of the standards of eight cytosine modifications with BDEPE labeling. (C) Extracted-ion chromatograms of BDEPE-labeled cytosine modifications in DNA and RNA from mouse ES cells.
Figure 3. Confirmation of the cytosine modifications in DNA of mouse ES cells by high-resolution mass spectrometry. (A) Product-ion spectra of BDEPE labeled 5-mdC standard (left) and 5-mdC in DNA of mouse ES cells (right). (B) Product-ion spectra of BDEPE labeled 5-hmdC standard (left) and 5-hmdC in DNA of mouse ES cells (right). (C) Product-ion spectra of BDEPE labeled 5-fodC standard (left) and 5-fodC in DNA of mouse ES cells (right). (D) Product-ion spectra of BDEPE labeled 5-cadC standard (left) and 5-cadC in DNA of mouse ES cells (right).
The results showed that the retention times of the eight native modified cytidines were much shorter than the BDEPElabeled products on the C18 chromatographic column (Figure
2A and B). In addition, BDEPE labeling also effectively improved the LC separation of these modified cytidines. The limits of detection (LODs) were employed to evaluate the 1638
DOI: 10.1021/acschembio.7b00170 ACS Chem. Biol. 2017, 12, 1636−1643
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ACS Chemical Biology improved detection sensitivities of nucleosides by BDEPE labeling (Supporting Information Table 2). The LODs of BDEPE-labeled 5-mdC, 5-hmdC, 5-fodC, 5-cadC, 5-mrC, 5hmrC, 5-forC, and 5-carC were 0.04, 0.06, 0.05, 0.07, 0.04, 0.06, 0.05, and 0.08 fmol, respectively (Supporting Information Table 2). Compared to the native nucleosides, BDEPE labeling dramatically increased the detection sensitivities of modified cytidines by 128−443-fold (Supporting Information Table 2). The tertiary amino group in these labeled products endowed by BDEPE can enhance the ionization efficiency of the analytes during mass spectrometry analysis, which will eventually lead to increased detection sensitivities. This phenomenon is consistent with our previous reports.25,28 Under optimized conditions, method validation demonstrated that the developed method exhibited good accuracy and reproducibility (the detailed validation can be found in the Supporting Data). Heavy Metals Induce Cell Apoptosis. To study the effects of exposures to As, Cd, Cr, and Sb on cytosine modifications of DNA and RNA in mouse ES cells, we first examined whether these heavy metals induced cell apoptosis by evaluating the activity of Caspase 3, an executioner of cell apoptosis. The detailed analytical procedure for the assay of activity of Caspase 3 can be found in the Supporting Data. As shown in Supporting Information Figure 1, exposures to As, Cd, and Sb led to a significant increase in the activity of Caspase 3, suggesting heavy metals can induce the apoptosis of mouse ES cells. Heavy Metals Induce Decline of Derivatives of 5-mC in DNA and RNA. We next then examined the content change of the derivatives of 5-mC in both DNA and RNA of mouse ES cells upon exposure to heavy metals. The results showed that all eight of these cytosine modifications can be simultaneously and clearly detected (Figure 2C). In addition, the results obtained by high resolution mass spectrometry demonstrated that the parent ions and the product ions of BDEPE-labeled cytosine modifications from mouse ES cells were equal to the standard nucleosides (Figure 3 and Supporting Information Figure 2), further confirming the detected modified cytidines from mouse ES cells. The quantitative results demonstrated that the levels of 5mdC were similar between control and heavy-metal-treated mouse ES cells (Figure 4A). However, the contents of 5-hmdC, 5-fodC, and 5-cadC significantly decreased under As, Cd, Cr, and Sb treatments (Figure 4B, C, and D). Particularly, treatments of mouse ES cells by As, Cd, Cr, and Sb led to a 41.7%, 44.7%, 47.9%, and 29.8% decrease of 5-hmdC; a 49.1%, 53.1%, 43.4%, and 49.0% decrease of 5-fodC; and a 76.8%, 75.3%, 54.9%, and 69.3% decrease of 5-cadC, respectively (Supporting Information Table 3). RNA cytosine modifications showed similar alteration trends upon treatments by heavy metals to those of DNA. The contents of 5-mrC were similar between control and heavymetal-treated cells (Figure 4E). However, the contents of 5hmrC, 5-forC, and 5-carC significantly decreased under As, Cd, Cr, or Sb treatments (Figure 4F, G, and H). Treatments of cells by these heavy metals led to an approximately 54.6%, 44.9%, 66.6%, and 46.1% decrease of 5-hmrC; 42.1%, 71.0%, 70.8%, and 44.6% decrease of 5-forC; and 41.3%, 34.8%, 43.3%, and 57.9% decrease of 5-carC, respectively (Supporting Information Table 3). Heavy Metals Induce Decrease of Activities of Tet Proteins. Since Tet proteins can potentially convert 5-mC to the corresponding derivatives,19,31,32 we speculate the decline of
Figure 4. Quantification and statistical analysis of (A) 5-mdC, (B) 5hmdC, (C) 5-fodC, (D) 5-cadC, (E) 5-mrC, (F) 5-hmrC, (G) 5-forC, and (H) 5-carC in mouse ES cells treated by heavy metals. *p < 0.05; **p < 0.01; ***p < 0.001. Data are represented as mean ± SEM with triplicate measurements.
the derivatives of 5-mC may be due to the decreased activities or expressions of Tet proteins. In this respect, here we then evaluated the activities of Tet proteins as well as the expression of TET genes. The result showed that the activities of Tet proteins significantly decreased under treatments by heavy metals (Figure 5). Specifically, treatments of mouse ES cells by As, Cd, Cr, and Sb led to a 42.1%, 43.1%, 62.1%, and 53.9% decrease of activities of Tet proteins, respectively (Figure 5). However, exposures to heavy metals did not induce significant
Figure 5. Relative activities of Tet proteins from nuclear extracts of mouse ES cells exposed to heavy metals. The activities of the Tet proteins were normalized to total protein contents. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SEM with triplicate measurements. 1639
DOI: 10.1021/acschembio.7b00170 ACS Chem. Biol. 2017, 12, 1636−1643
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Figure 6. Determination of the metabolites in the TCA cycle from mouse ES cells by high resolution mass spectrometry analysis. (A) Extracted-ion chromatograms of metabolite standards in TCA cycle. (B) Extracted-ion chromatograms of metabolites in the TCA cycle from mouse ES cells.
Figure 7. Quantification of the content changes of metabolites in the TCA cycle from mouse ES cells treated by heavy metals. Tet protein, 10−11 translocation protein; IDH, isocitrate dehydrogenase. The concentrations of metabolites are represented as pmol μg−1 protein. *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SEM with triplicate measurements.
regulation of epigenetics.33,34 Some endogenous metabolites are essential cofactors for the activities of enzymes that are responsible for epigenetic modifications; therefore cellular metabolism can play certain roles in gene regulation by altering the activities of enzymes that are involved in epigenetic modifications.35 Here, we further investigated the possible mechanism of the heavy metals induced decline of the
changes of the expressions of TET1, TET2, and TET3 genes by quantitative real-time PCR (qRT-PCR) analysis (Supporting Information Figure 3), revealing that the decline of the derivatives of 5-mC was mainly due to the attenuated activities of Tet proteins. Contents Change of the Metabolites in Tricarboxylic Acid Cycle upon Heavy Metals Treatments. Recent studies indicated the metabolic state of cells is correlated with the 1640
DOI: 10.1021/acschembio.7b00170 ACS Chem. Biol. 2017, 12, 1636−1643
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ACS Chemical Biology derivatives of 5-mC by analyzing the changes of certain endogenous metabolites. Previous reports demonstrated that 5-mdC in DNA and 5mrC in RNA can be converted to 5-hmdC and 5-hmrC, respectively, by Tet proteins with α-ketoglutarate (2-KG) as the essential cofactor. On the other hand, 2-hydroxyglutaric acid (2-HG) is considered to be the inhibitor of Tet proteins.36,37 Therefore, the content changes of these endogenous 2-KG and 2-HG may alter the activities of Tet proteins, which can further change the contents of cytosine modifications in DNA and RNA. In this respect, here we quantified the metabolites in the TCA cycle, including 2-KG, isocitrate, citrate, oxaloacetate, malate, fumarate, succinate, and 2-HG. Some other chemicals, such as quinones38 and ascorbic acid,39 have also been reported to be able to affect the activities of Tet proteins. But quinones typically are not added in the culturing medium. The effect of ascorbic acid can be deducted since the control sample also contains the same medium. The detailed optimized mass spectrometry parameters and the calibration curves for the quantification of these metabolites are listed in Supporting Information Tables 4 and 5. Figure 6A and B showed the extracted-ion chromatograms of metabolite standards and metabolites in the TCA cycle from mouse ES cells, respectively. The similar retention times between the metabolite standards and the compounds from mouse ES cells confirmed the detected metabolites. It was worth noting that the isomers of citrate and isocitrate can be well separated under the optimized chromatographic separation conditions. The quantitative results showed that treatments of mouse ES cells by these heavy metals induced an overall decrease of the metabolites in the TCA cycle (Figure 7 and Supporting Information Table 3). Cd and Sb treatments led to 52.6% and 45.2% decreases of 2-KG (Figure 7), while As and Cr treatments did not significantly alter the level of 2-KG. However, the content of 2-HG showed noticeable elevation, with 177.6%, 488.0%, and 118.5% increases under Cd, Cr, and Sb treatments, respectively, while As treatment induced no significant change (Figure 7). The measured content of 2-KG decreased upon heavy metal treatments, which will compromise the activities of Tet proteins and eventually lead to the decline of the derivatives of 5-mC. Here, we also found that exposures to heavy metals could noticeably increase the 2-HG level, which may also attenuate the activities of Tet proteins and therefore cause the decrease of the derivatives of 5-mC. We observed that Cr treatment did not induce the decrease of 2-KG, but Cr treatment dramatically induced the increase of 2-HG. Therefore, the decline of the derivatives of 5-mC by Cr treatment may be due to the combined effect of 2-KG and 2-HG on the activities of Tet proteins. Shown in Figure 8 are the overall changes of the derivatives of 5-mC as well as the metabolites in the TCA cycle upon treatments by heavy metals, clearly suggesting the correlation between epigenetic modifications and metabolites. Heavy metals can induce ROS in cells.40 In addition to the Tet-protein-induced formation of the derivatives of 5-mC, it is possible that ROS may also participate in the formation of derivatives of 5-mC. However, the decline of these cytosine modifications upon treatments by heavy metals suggested ROS may not play dominant roles. The cytotoxicity and carcinogenicity mechanisms induced by heavy metals are complex. Nevertheless, the induced decline of the derivatives of 5-mC in both DNA and RNA indicated that heavy metals may jeopardize cell growth and development through causing
Figure 8. Summary of the effects of heavy metals on (A) cytosine modifications and (B) metabolites occurring in the TCA cycle. The color represents the logarithm values of the ratio heavy metal treatment/control. Blue color represents decreased contents under heavy metal treatments; red color represents increased contents under heavy metal treatments.
the disorder of DNA and RNA modifications. Our study shed light on the epigenetic effects of heavy metals. Future studies on the site-specific alteration of these cytosine modifications may further reveal the mechanism of the cytotoxicity and carcinogenicity of heavy metals from the view of an epigenetic angle. In conclusion, a chemical labeling strategy in combination with mass spectrometry analysis was established for the detection of cytosine modifications in DNA and RNA. With this method, we observed that the levels of 5-hmC, 5-foC, and 5-caC significantly decreased in both DNA and RNA of mouse ES cells under exposure to As, Cd, Cr, and Sb. We further demonstrated that treatments by heavy metals induced the decrease of the activities of Tet proteins but did not cause the decreased expressions of TET genes. The decreased activities of Tet proteins may be due to the combined effect of the content changes of 2-KG and 2-HG. Together, these findings suggested heavy metals may exert the cellular toxicity through compromising the epigenetic modifications of DNA and RNA.
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METHODS
Materials. 5-Methyl-2′-deoxycytidine (5-mdC), 5-methylcytidine (5-mrC), 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA), 2′deoxycytidine (dC), thymidine (T), guanosine (rG), adenosine (rA), cytidine (rC), uridine (rU), phosphodiesterase I, 2-bromo-1-(4diethylaminophenyl)-ethanone (BDEPE), sodium arsenite (NaAsO2), α-ketoglutarate were from Sigma-Aldrich. 5-Hydroxymethyl-2′-deoxycytidine (5-hmdC), 5-formyl-2′-deoxycytidine (5-fodC), 5carboxyl-2′-deoxycytidine (5-cadC), 5-hydroxymethylcytidine (5hmrC), 5-formylcytidine (5-forC), and 5-carboxylcytidine (5-carC) were from Berry and Associates. S1 nuclease and alkaline phosphatase were purchased from Takara Biotechnology Co. Ltd. Cadmium chloride (CdCl2), sodium dichromate (Na 2Cr2 O7), antimony trichloride (SbCl3), citrate, succinate, fumarate, DL-malate, and oxaloacetate were from Aladdin Reagent Co. Ltd. D-2-Hydroxyglutaric acid and DL-isocitrate were purchased from TCI Co., Ltd. Cell Culture and Heavy Metals Treatments. The CGR8 mouse embryonic stem (ES) cells were cultured in Glasgow’s MEM medium containing 10% fetal bovine serum, 2 mM GlutaMAX TM-1, 1000 U mL−1 leukemia inhibitory factor, 1% MEM nonessential amino acids, 0.055 mM β-mercaptoethanol, and 1% penicillin-streptomycin on gelatin-coated dishes, in a 5% CO2 incubator under 37 °C. For heavy metal treatments, mouse ES cells were treated with 5 μM of various heavy metals including As, Cd, Cr, and Sb for 24 h. 1641
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ACS Chemical Biology Nucleic Acids Isolation and Enzymatic Digestion. Genomic DNA of the cultured cells was isolated using an E.Z.N.A. Tissue DNA Kit (Omega Bio-Tek Inc.). Total RNA was isolated using an E.Z.N.A. Total RNA Kit (Omega Bio-Tek Inc.). The isolation of genomic DNA and total RNA was done according to the protocol provided by the manufacturer. Measurement of the concentrations of extracted DNA and RNA was according to previously reported method.23 Isolated DNA and RNA were enzymatically digested according to a previously reported method.24 Typically, the mixture of 10 μg of DNA and 10 μg of RNA in 18 μL of H2O was denatured under 95 °C for 2 min followed by immediate chilling in ice water. Then, 2 μL of S1 nuclease buffer (Takara Biotechnology Co. Ltd.) and 100 units (0.5 μL) of S1 nuclease were added. The resulting mixture was incubated for 2 h under 37 °C. Then, 66 μL of H2O, 10 μL of alkaline phosphatase buffer (Takara Biotechnology Co. Ltd.), 30 units (1 μL) of alkaline phosphatase, and 0.005 units (3 μL) of venom phosphodiesterase were added. The solution was further incubated for 2 h under 37 °C. After adding 200 μL of H2O, the resulting solution was extracted with 300 μL of chloroform three times. BDEPE Labeling. Here, we used BDEPE as the labeling reagent to simultaneously detect all the targeted analytes, i.e., 5-mdC, 5-hmdC, 5fodC, and 5-cadC from DNA, 5-mrC, 5-hmrC, 5-forC, and 5-carC from RNA. The chemical labeling reaction conditions were set according to our previous study.25 The BDEPE-labeled products were dried under nitrogen gas and redissolved in 100 μL of water for the subsequent analysis. Simultaneous Analysis of Eight Modified Cytidines from DNA and RNA. Analysis of the BDEPE-labeled nucleosides was carried out on the Shimadzu LC-20AD HPLC coupled with AB 3200 QTRAP mass spectrometer (Applied Biosystems) under positive ESI mode. Multiple reaction monitoring (MRM) mode was used for the detection. The MRM mass transitions and optimized mass spectrometry parameters are listed in Supporting Information Table 1. A VP-ODS column (250 mm × 2.1 mm i.d., 5 μm, Shimadzu) was used for the LC separation under 35 °C. The flow rate was set at 0.2 mL min−1. Solvent A (0.01% formic acid in water) and solvent B (ACN) were used as the mobile phase. The gradient for the LC separation was as follows: 0−5 min 5% B, 18−35 min 65% B, and 36− 50 min 5% B. An LTQ-Orbitrap Elite high resolution mass spectrometer (Thermo Fisher Scientific) coupled to an UltiMate 3000 UHPLC (ThermoDionex) was used for the further qualitative determination of target analytes. The procedure and conditions for LC separation were the same as those used in the AB 3200 QTRAP mass spectrometer. MS/ MS spectra were obtained using CID activation mode by a collision energy of 35 V and an acquisition time of 10 ms at a resolution of 60 000. Analysis of Metabolites in Tricarboxylic Acid Cycle. The metabolites of mouse ES cells were extracted according to a previous study.26 Briefly, 0.8 mL of 80% methanol in water (0 °C) spiked with internal standards (biphthalate) was added to a mouse ES cell pellet (∼2 × 106 cells). After vortexing for 1 min, the resulting mixture was incubated for 2 h under −80 °C followed by centrifugation at 13 000g under 4 °C for 10 min. Insoluble pellets were re-extracted with 0.3 mL of 80% methanol. Supernatants were combined and dried in a nitrogen stream, and then resuspended in 100 μL of 90% ACN in water for LCMS analysis. Extracted metabolites were analyzed on an LTQ-Orbitrap Elite mass spectrometer coupled to an UltiMate 3000 UHPLC under negative ion mode. A Thermo Syncronis HILIC column (150 mm × 2.1 mm i.d., 5 μm, Thermo Scientific) was used for LC separation under 35 °C. The flow rate was set at 0.2 mL min−1. Ammonium formate (10 mM, pH 4.6) in water (solvent A) and ACN (solvent B) were used as mobile phases. The gradient for the LC separation was as follows: 0−5 min 85% B, 15−25 min 15% B, and 26−40 min 85% B. MS spectra were obtained with full scan detection mode from m/z 50 to 200 at a resolution of 60 000. Assay of Activities of Tet Proteins. Mouse ES cell nuclear lysates were prepared using a Nucleoprotein Extraction Kit (Sangon). The BCA Protein Assay Kit (Beyotime) was used to measure the
concentrations of total protein according to the manufacturer’s recommended procedure. The activities of Tet proteins were measured using an Epigentek Epigenase 5-mC hydroxylase Tet Activity/Inhibition kit according to the supplier’s protocol. The activities of the Tet proteins were normalized to total protein contents.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00170. Additional tables and figures as mentioned in the text (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *Tel.: +86-27-68755595. Fax: +86-27-68755595. E-mail:
[email protected]. ORCID
Bi-Feng Yuan: 0000-0001-5223-4659 Author Contributions ⊥
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the financial support from the National Natural Science Foundation of China (21672166, 21522507, 21635006).
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REFERENCES
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ACS Chemical Biology
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DOI: 10.1021/acschembio.7b00170 ACS Chem. Biol. 2017, 12, 1636−1643
