Article pubs.acs.org/ac
Formation and Determination of Endogenous Methylated Nucleotides in Mammals by Chemical Labeling Coupled with Mass Spectrometry Analysis Huan Zeng,† Chu-Bo Qi,†,‡ Ting Liu,† Hua-Ming Xiao,† Qing-Yun Cheng,† Han-Peng Jiang,† 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 ‡ Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei 430079, People’s Republic of China S Supporting Information *
ABSTRACT: 5-Methylcytosine (5-mC) is an important epigenetic mark that plays critical roles in a variety of cellular processes. To properly exert physiological functions, the distribution of 5-mC needs to be tightly controlled in both DNA and RNA. In addition to methyltransferase-mediated DNA and RNA methylation, premethylated nucleotides can be potentially incorporated into DNA and RNA during replication and transcription. To exclude the premodified nucleotides into DNA and RNA, endogenous 5-methyl-2′-deoxycytidine monophosphate (5-Me-dCMP) generated from nucleic acids metabolism can be enzymatically deaminated to thymidine monophosphate (TMP). Therefore, previous studies failed to detect 5-Me-dCMP or 5methylcytidine monophosphate (5-Me-CMP) in cells. In the current study, we established a method by chemical labeling coupled with liquid chromatography−electrospray ionization mass spectrometry (LC−ESIMS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP and 5-Me-CMP. As N,Ndimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of nucleotides increased by 88−372fold due to the introduction of a tertiary amino group and a hydrophobic moiety from DMPA. Using this method, we found that endogenous 5-Me-dCMP and 5-Me-CMP widely existed in cultured human cells, human tissues, and human urinary samples. The presence of endogenous 5-Me-dCMP and 5-Me-CMP indicates that deaminases may not fully deaminate these methylated nucleotides. Consequently, the remaining premethylated nucleosides could be converted to nucleoside triphosphates as building blocks for DNA and RNA synthesis. Furthermore, we found that the contents of 5-Me-dCMP and 5-Me-CMP exhibited significant decreases in renal carcinoma tissues and urine samples of lymphoma patients compared to their controls, probably due to more reutilization of methylated nucleotides in DNA and RNA synthesis. This study is, to the best of our knowledge, the first report for detecting endogenous 5-Me-dCMP and 5-Me-CMP in mammals. The detectable endogenous methylated nucleotides indicate the potential deleterious effects of premodified nucleotides on aberrant gene regulation in cancers.
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tRNA, 5-mC has been shown to stabilize tRNA secondary structure.7 Due to the important regulatory roles of DNA and RNA methylation, it is vital to maintain proper DNA and RNA methylation status for the normal functions of cells.8,9 Abnormal DNA and RNA methylation can cause many human diseases, such as diabetes,10,11 neurological disorders,12 and cancers.13−15 In mammals, DNA methylation is mediated by the DNA methyltransferase (DNMT) family of enzymes (DNMT1, DNMT3A, and DNMT3B) that catalyze the transfer of a methyl group from S-adenosyl-L-methionine to DNA.16 On
NA cytosine methylation (5-methylcytosine, 5-mC) is the best-characterized epigenetic mark that plays critical roles in a variety of cellular processes, including regulation of gene expression, genomic imprinting, and X-chromosome inactivation.1 Aberrant DNA methylation is associated with many human diseases.2 In addition to DNA methylation, reversible RNA modification recently has been proposed to represent another realm for biological regulation.3,4 5-mC has long been known to be present in RNA, and recent studies demonstrated that 5-mC is widespread in both coding and noncoding RNA of mammals, implicating that 5-mC in RNA could be critical for regulating gene transcription and protein translation.5 For example, 5-mC modification in rRNA is prevalent and plays critical roles in translational fidelity and tRNA recognition.6 In © 2017 American Chemical Society
Received: January 5, 2017 Accepted: March 8, 2017 Published: March 8, 2017 4153
DOI: 10.1021/acs.analchem.7b00052 Anal. Chem. 2017, 89, 4153−4160
Article
Analytical Chemistry the other hand, 5-mC can be generated in RNA of mammals by enzymes including DNMT2, NSun2, and NSun4.17 In addition to enzymatically methylated DNA and RNA, methylated nucleotides can be incorporated into DNA and RNA during replication and transcription.18 Previous study reported that, in Chinese hamster ovary (CHO) cells treated with 5-methyl-2′-deoxycytidine triphosphate (5-Me-dCTP), certain genes can be silenced by the incorporation of 5-MedCTP into DNA.19 DNA and RNA metabolism can lead to formation of endogenous nucleoside monophosphates, including the modified cytidines of 5-methyl-2′-deoxycytidine monophosphate (5-Me-dCMP) and 5-methylcytidine monophosphate (5-Me-CMP). In this respect, DNA and RNA methylation could be potentially generated by converting 5Me-dCMP and 5-Me-CMP to 5-Me-dCTP and 5-methylcytidine triphosphate (5-Me-CTP), respectively, and the subsequent incorporation into DNA and RNA. To properly exert the biological functions, the distribution of 5-mC in DNA and RNA needs to be tightly controlled.20 Thus, it is essential that these premodified nucleotides are not reincorporated into DNA and RNA. Otherwise, their random positions would alter the normal distribution of 5-mC in DNA and RNA, eventually leading to the dysregulation of gene expression. To prevent them from being introduced into DNA and RNA, it was believed that endogenous 5-Me-dCMP can be enzymatically deaminated to thymidine monophosphate (TMP).21 Supported by this explanation, previous studies have reported that none of these endogenous 5-Me-dCMP and 5-Me-CMP were detected in cells.22 In the current study, we established a chemical labeling method coupled with liquid chromatography−electrospray ionization mass spectrometry (LC−ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5Me-dCMP and 5-Me-CMP. With the method, we found that 5Me-dCMP and 5-Me-CMP widely existed in cultured human cells, human tissues, and human urinary samples. The detectable levels of 5-Me-dCMP and 5-Me-CMP indicated that deaminases may not fully deaminate these methylated nucleotides. These premethylated nucleotides could therefore be potentially converted to nucleoside triphosphates and became building blocks for DNA and RNA synthesis. The utilization of premethylated nucleotides in DNA and RNA synthesis would have deleterious effects on gene regulation.
Figure 1. (A) Chemical structures of 5-Me-dCMP and 5-Me-CMP. (B) Chemical labeling of nucleotides by DMPA. “B” in nucleotides represents nucleobase.
Chromatographic grade methanol and acetonitrile (ACN) were purchased from Tedia Co. Inc. (Fairfield, OH, U.S.A.). The water used throughout the study was purified by a Milli-Q apparatus (Millipore, Bedford, MA). Stock solutions of dAMP, TMP, dCMP, dGMP, AMP, UMP, CMP, GMP, 5-Me-dCMP, and 5-Me-CMP were prepared in water at a concentration of 10 mM. DMPA was prepared in ACN at a concentration of 140 mM. Imidazole was prepared in water at a concentration of 10 mM (pH 6). Biological and Clinical Samples. The first morning urine samples from lymphoma patients and healthy controls were collected from Hubei Cancer Hospital, China. Each group contains 10 urine samples from five males and five females. A total of 18 tissue samples from renal carcinoma patients, including nine pairs of renal carcinoma tissues and matched tumor-adjacent normal tissues, were collected from Hubei Cancer Hospital. Detailed information can be found in Table S1 in Supporting Information. All the patients were diagnosed with cancer for the first time and had not been given any treatment at the time point of urine sample collection. Healthy controls were selected based on medical history and physical examination. The healthy controls and cancer patients were not detected with other diseases. An approval was granted by the Hubei Cancer Hospital Ethics Committee and met the declaration of Helsinki. All the experiments were performed in accordance with Hubei Cancer Hospital Ethics Committee’s guidelines and regulations. Human 293T and HeLa cells were obtained from the China Center for Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Sample Pretreatment. The pretreatment of the urine samples was performed according to a previously described method.23,24 Briefly, the first morning urine samples were collected and then centrifuged immediately at 5000g for 10 min under 4 °C twice. The resulting supernatant was filtered by a PRECLEANTM syringe filter nylon membrane (13 mm × 0.22 μM, ANPEL Scientific Instrument Co., Shanghai, China). At last, the supernatant was collected and stored at −80 °C. The tissue samples were extracted by homogenization in prechilled 80% aqueous methanol (0 °C, 1 mL). After centrifugation at 14 000g for 15 min to remove precipitated protein, supernatants were collected and dried under nitrogen gas and stored at −80 °C.25 The human 293T and HeLa cells, at a density of approximate 7.5 × 105 cells/mL (10 mL), were collected by centrifugation at 2000g under 4 °C for 5 min and then washed twice with icecold phosphate-buffered saline (PBS) to remove the fetal
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EXPERIMENTAL SECTION Chemicals and Reagents. Adenosine 5′-monophosphate (AMP), uridine 5′-monophosphate (UMP), cytidine 5′-monophosphate (CMP), guanosine 5′-monophosphate (GMP), 2′deoxyadenosine 5′-monophosphate (dAMP), thymidine 5′monophosphate (TMP), 2′-deoxycytidine 5′-monophosphate (dCMP), and 2′-deoxyguanosine 5′-monophosphate (dGMP) were purchased from Sigma-Aldrich (Beijing, China). N,NDimethyl-p-phenylenediamine (DMPA), imidazole, and 1ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC) were purchased from Aladdin Reagent Co. (Shanghai, China). 5-Methyl-2′-deoxycytidine 5′-monophosphate (5-Me-dCMP) was purchased from Carbosynth (San Diego, CA, U.S.A.). 5-Methylcytidine 5′-monophosphate (5Me-CMP) was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The structures of 5-Me-dCMP and 5-MeCMP are shown in Figure 1A. The structures of other nucleotides are shown in Figure S1 in Supporting Information. 4154
DOI: 10.1021/acs.analchem.7b00052 Anal. Chem. 2017, 89, 4153−4160
Article
Analytical Chemistry
15−20 min 30% B, 20−22 min from 30% to 70% B, and 22−30 min 70% B was used. The flow rate of the mobile phase was set at 0.2 mL/min. The DMPA-labeled nucleotides were monitored under multiple reaction monitoring (MRM) positive ion mode. The optimal MRM parameters are listed in Table S2 in Supporting Information. Statistical Analysis. Statistical data were processed with Origin 8.0 software (Electronic Arts Inc.). Independent t test was performed to evaluate the differences of nucleotide’s concentrations in urine samples obtained from lymphoma patients and healthy controls. Paired t test was performed to evaluate the concentration differences of nucleotides between renal carcinoma tissues and tumor-adjacent normal tissues. All p values were two-sided, and generally, p values
