Formation and Determination of Endogenous Methylated Nucleotides in Mammals by Chemical Labeling Coupled with Mass Spectrometry Analysis

Article abstract of DOI:10.1021/acs.analchem.7b00052

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 5-methylcytidine 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-ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP and 5-Me-CMP. As N,N-dimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of nucleotides increased by 88-372-fold 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.

Full text of DOI:10.1021/acs.analchem.7b00052

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Article
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, Bifeng Yuan, and Yu-Qi Feng
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00052 • Publication Date (Web): 08 Mar 2017
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Page 1 of 10
Analytical Chemistry
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Formation and Determination of Endogenous Methylated Nu-
cleotides in Mammals by Chemical Labeling coupled with
Mass Spectrometry Analysis
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Huan Zeng,1,† Chu-Bo Qi,1,2,† Ting Liu,1 Hua-Ming Xiao,1 Qing-Yun Cheng,1 Han-Peng Jiang,1 Bi-Feng Yuan1,*, Yu-
Qi Feng1
1 Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry,
Wuhan University, Wuhan 430072, P.R. China
2 Department of Pathology, Hubei Cancer Hospital, Wuhan, Hubei 430079, P.R. China
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 needed to be tightly controlled in both
DNA and RNA. In addition to methyltransferase-mediated DNA and RNA methylation, pre-methylated nucleotides
can be potentially incorporated into DNA and RNA during replication and transcription. To exclude the pre-modified
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, pre-
vious studies failed to detect 5-Me-dCMP or 5-methylcytidine 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-ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP
and 5-Me-CMP. As N,N-dimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of
nucleotides increased by 88-372 folds 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 pre-methylated
nucleosides could be converted to nucleosides triphosphate as building blocks for DNA and RNA synthesis. Further-
more, 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 the more reutilization of
methylated nucleotides in DNA and RNA synthesis. This study is, to the best of our knowledge, the first report for de-
tecting endogenous 5-Me-dCMP and 5-Me-CMP in mammals. The detectable endogenous methylated nucleotides in-
dicates the potential deleterious effects of pre-modified nucleotides on aberrant gene regulation in cancers.
Due to the important regulatory roles of DNA and
INTRODUCTION
RNA methylation, it is vital to maintain proper DNA and
DNA cytosine methylation (5-methylcytosine, 5-mC)
RNA methylation status for the normal functions of cells.8,9
is the best characterized epigenetic mark that plays critical
Abnormal DNA and RNA methylation can cause many
roles in a variety of cellular processes, including regulation
human diseases, such as diabetes,10,11 neurological disor-
of gene expression, genomic imprinting and X-
12
ders and cancers.13-15 In mammals, DNA methylation is
chromosome inactivation.1 Aberrant DNA methylation is
associated with many human diseases.2 In addition to DNA
mediated by DNA methyltransferase (DNMT) family of
enzymes (DNMT1, DNMT3A and DNMT3B) that catalyze
methylation, reversible RNA modification recently has
the transfer of a methyl group from S-adenosyl-L-
been proposed to represent another realm for biological
methionine to DNA.16 On the other hand, 5-mC can be
regulation.3,4 5-mC has long been known present in RNA
generated in RNA of mammals by enzymes including
and recent studies demonstrated that 5-mC is widespread in
both coding and noncoding RNA of mammals, implicating
DNMT2, NSun2 and NSun4.17
5-mC in RNA could be critical for regulating gene tran-
In addition to enzymatically methylated DNA and
scription and protein translation.5 For example, 5-mC mod-
RNA, methylated nucleotides can be incorporated into
DNA and RNA during replication and transcription.18 Pre-
ification in rRNA is prevalent and play critical roles in
translational fidelity and tRNA recognition.6 In tRNA, 5-
vious study reported that, in Chinese hamster ovary (CHO)
mC has been shown to stabilize tRNA secondary structure.7
cells treated with 5-methyl-2’-deoxycytidine triphosphate
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(5-Me-dCTP), certain genes can be silenced by the incorpo- 5-Methylcytidine 5’-monophosphate (5-Me-CMP) was
ration of 5-Me-dCTP into DNA.19 DNA and RNA metabo-
lism 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 5-Me-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.
purchased from Takara Biotechnology Co., Ltd. (Dalian,
China). The structures of 5-Me-dCMP and 5-Me-CMP
were shown in Figure 1A. The structures of other nucleo-
tides were shown in Figure S1 in Supporting Information.
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Chromatographic grade methanol and acetonitrile
(ACN) were purchased from Tedia Co. Inc. (Fairfield, OH,
USA). The water used throughout the study was purified by
a Milli-Q apparatus (Millipore, Bedford, MA). Stock solu-
tions of dAMP, TMP, dCMP, dGMP, AMP, UMP, CMP,
GMP, 5-Me-dCMP, and 5-Me-CMP were prepared in wa-
ter at a concentration of 10 mM. DMPA was prepared in
ACN at a concentration of 140 mM. Imidazole was pre-
pared in water at a concentration of 10 mM (pH 6).
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To properly exert the biological functions, the distri-
bution of 5-mC in DNA and RNA needs to be tightly con-
trolled.20 Thus, it is essential that these pre-modified nucle-
otides are not re-incorporated into DNA and RNA. Other-
wise, their random positions would alter the normal distri-
bution 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 none of these
endogenous 5-Me-dCMP and 5-Me-CMP were detected in
cells.22
Figure 1. (A) The chemical structures of 5-Me-dCMP and 5-Me-
CMP. (B) Chemical labeling of nucleotides by DMPA. “B” in
nucleotides represents nucleobase.
In the current study, we established a chemical label-
ing method coupled with liquid chromatography-
electrospray ionization-mass spectrometry (LC-ESI-
MS/MS) for sensitive and simultaneous determination of
10 nucleotides, including 5-Me-dCMP and 5-Me-CMP.
With the method, we found that 5-Me-dCMP and 5-Me-
CMP widely existed in cultured human cells, human tis-
sues, 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 pre-methylated nucleotides could therefore be poten-
tially converted to nucleosides triphosphate and became
building blocks for DNA and RNA synthesis. The utiliza-
tion of pre-methylated nucleotides in DNA and RNA syn-
thesis would have deleterious effects on gene regulation.
Biological and clinical samples
The first morning urine samples from lymphoma pa-
tients and healthy controls were collected from Hubei Can-
cer Hospital, China. Each group contains 10 urine samples
from 5 males and 5 females. A total of 18 tissue samples
from renal carcinoma patients, including 9 pairs of re-
nal carcinoma tissues and matched tumor adjacent normal
tissues, were collected from Hubei Cancer Hospital. De-
tailed 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 exam-
ination. 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 per-
formed in accordance with Hubei Cancer Hospital Ethics
Committee’s guidelines and regulations.
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’-
Human 293T and HeLa cells were obtained from the
China Center for Type Culture Collection and maintained
in Dulbecco’s Modified Eagle 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 37oC.
monophosphate
monophosphate (dCMP) and 2’-deoxyguanosine 5’-
monophosphate (dGMP) were purchased from Sigma-
(TMP),
2’-deoxycytidine
5’-
Aldrich
(Beijing,
China).
N,N-Dimethyl-p-
phenylenediamine (DMPA), imidazole and 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (EDC)
were purchased from Aladdin Reagent Co. (Shanghai, Chi-
na). 5-Methyl-2’-deoxycytidine 5’-monophosphate (5-Me-
dCMP) was purchased from Carbosynth (San Diego, USA).
Sample pretreatment
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Analytical Chemistry
The pretreatment of the urine samples were performed
(ESI) source (Turbo Ionspray) and a Shimadzu LC-20AD
HPLC (Tokyo, Japan). Data acquisition and processing
were performed using AB SCIEX Analyst 1.5 Software
(Applied Biosystems, Foster City, CA, USA). The HPLC
separation was performed on an Inertsil ODS-3 column
(250 mm × 2.0 mm i.d., 5 µm, Tokyo, Japan) at 35N. Wa-
ter containing 2 mM NH4HCO3 (solvent A) and ACN (sol-
vent B) was employed as the mobile phase. A gradient of 0
- 15 min from 5% to 30% B, 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 mul-
tiple reaction monitoring (MRM) positive ion mode. The
optimal MRM parameters were listed in Table S2 in Sup-
porting Information.
according to previously described method.23,24 Briefly, the
first morning urine samples were collected and then centri-
fuged immediately at 5,000 × g for 10 min under 4oC 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 -80oC.
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The tissue samples were extracted by homogenization
in pre-chilled 80% aqueous methanol (0oC, 1 mL). After
centrifugation at 14,000 × g for 15 min to remove precipi-
tated protein, supernatants were collected and dried under
nitrogen gas and stored at -80oC.25
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The human 293T and HeLa cells, at a density of ap-
proximate 7.5 × 105 cells/mL (10 mL), were collected by
centrifugation at 2,000 × g under 4oC for 5 min and then
washed twice with ice-cold phosphate-buffered saline
(PBS) to remove the fetal bovine serum. Cells were rapidly
quenched with pre-chilled 80% aqueous methanol (0oC, 2
mL) and incubated at -20oC for 30 min according to previ-
ous study.25 Then the cell extracts were centrifuged at
14,000 × g for 15 min at 4oC to remove precipitated pro-
tein. Supernatants were collected and then dried under ni-
trogen gas and stored at -80oC.
Statistical analysis
Statistical data were processed with Origin 8.0 soft-
ware (Electronic Arts Inc). Independent t-test was per-
formed to evaluate the differences of nucleotides concen-
trations in urine samples obtained from lymphoma patients
and healthy controls. Paired t-test was performed to evalu-
ate the concentration differences of nucleotides between
renal carcinoma tissues and tumor adjacent normal tissues.
All p values were two-sided, and generally, p values < 0.05
were considered statistically significant.
Enrichment of nucleotides
The nucleotides were isolated from urines, tissues, and
cultured cells by solid phase extraction (SPE) using Clean-
ert NH2 cartridge (200 mg/mL, Weltech Co., Ltd., Wuhan,
China). To achieve good recovery, several extraction con-
ditions were optimized, including the percentage of ACN in
loading solution, the percentage of ACN in desorption solu-
tion and desorption volume.
Chemical Labeling
DMPA was used to label nucleotides (Figure 1B). To
achieve the best labeling efficiency, we optimized the la-
beling conditions, including reaction temperature and time,
the concentration of imidazole, the molar ratio of DMPA
and EDC versus nucleotides. All the reactions were per-
formed in 100 µL imidazole solution (1 mM, pH 6.0).
After DMPA labeling, 300 µL of water and 300 µL of
a dichloromethane-hexane (2:1, v/v) solvent (4oC) were
added to the 100 µL of reaction solution followed by vor-
texing and centrifugation at 13,000 × g for 5 min to remove
excessive DMPA. Then 400 µL of the upper aqueous phase
was collected and mixed with 600 µL of water. Strata X
SPE cartridge (30 mg/mL, Phenomenex, Guangzhou, Chi-
na) was used to remove excessive EDC (Figure 2).
Figure 2. The schematic illustration of the developed method for
the determination of nucleotides by chemical labeling coupled
with LC-ESI-MS/MS analysis. “B” in nucleotides represents nu-
cleobase.
RESULTS AND DISCUSSION
Strategy for determination of methylated nucleotides by
chemical labeling coupled with LC-ESI-MS/MS analysis
Analysis of DMPA-labeled nucleotides by LC-ESI-
MS/MS
DNA and RNA metabolism could lead to the for-
mation of endogenous 5-Me-dCMP and 5-Me-CMP, which
are likely to be converted into 5-Me-dCTP and 5-Me-CTP
and then incorporated into DNA and RNA (Figure S2 in
Supporting Information). Although 5-Me-dCMP can be
enzymatically deaminated to TMP, so far no enzyme has
Analysis of the DMPA-labeled nucleotides was per-
formed on the LC-ESI-MS/MS system consisting of an AB
3200 QTRAP mass spectrometer (Applied Biosystems,
Foster City, CA, USA) with an electrospray ionization
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been reported to be capable of converting 5-Me-CMP, labeled dAMP, TMP, dCMP, dGMP, AMP, UMP, CMP,
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which could be potentially phosphorylated to 5-
methylcytidine diphosphate (5-Me-CDP) and 5-Me-CTP.
Therefore, we suspected that endogenous methylated nu-
cleotides may exist. However, identification and quantifica-
tion of endogenous methylated nucleotides in mammalian
cells has not been realized, possibly due to their low in-
vivo abundance as well as the poor ionization efficiencies
in detecting nucleotides using mass spectrometry.
GMP, 5-Me-dCMP, and 5-Me-CMP were successfully
formed.
DMPA labeling
To obtain good labeling efficiencies by DMPA, we
optimized the labeling conditions, including reaction tem-
perature and time, concentration of imidazole buffer, molar
ratio of DMPA and EDC versus nucleotide. dCMP, UMP
and CMP were used as the analytes for optimization.
9
Introduction of an easily ionizable group to targeted
analytes could enhance the ionization efficiency in mass
spectrometry analysis.26 Our group recently established
chemical labeling methods for sensitive detection of en-
dogenous low-abundant compounds by LC-MS.27-30 Along
this line, here we used DMPA that harbors a hydrophobic
phenyl group and an easily chargeable tertiary amine group
to simultaneously label the phosphate group in 10 nucleo-
tides including 5-Me-dCMP and 5-Me-CMP (Figure 1 and
Figure S1 in Supporting Information). As a consequence,
the detection sensitivities of these DMPA-labeled products
increased on LC-ESI-MS/MS owing to their conjugation to
the pre-charged tertiary amine group on DMPA. In addi-
tion, the introduced hydrophobic phenyl group in DMPA
elongated the retention of these nucleotides on reversed-
phase LC, which further improved the detection sensitivi-
ties. Using the developed method, we were able to distinct-
ly identify and quantify endogenous 5-Me-dCMP and 5-
Me-CMP in mammalian cells.
The reaction temperature was first optimized ranging
from 4oC to 70oC. The reaction was incubated for 1.5 h
with shaking at 1,500 rpm. Our results demonstrated that
the peak area of nucleotide derivatives peaked at 50oC
(Figure 4A). Therefore, 50oC was used for the following
experiments. Next, the reaction time was optimized ranging
from 10 to 120 min, and the reaction was performed at
50oC with shaking at 1,500 rpm. The peak areas of nucleo-
tide derivatives increased with increasing time from 10 to
90 min and slightly decreased over 90 min (Figure 4B).
Thus, 90 min was chosen for the labeling reaction.
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Extraction of nucleotides from biological and clinical
samples
Due to the complex matrix of biological and clinical
samples, it is essential to extract nucleotides and remove
the matrix interferences. Here we used hydrophilic Cleanert
NH2 SPE cartridge to extract nucleotides from pretreated
samples. The extraction conditions were optimized to ob-
tain good extraction efficiencies. The optimized conditions
were as follow: loading solution and washing solution, 3.0
mL of 80% ACN (ACN/0.25% NH4OH, 80/20, v/v); de-
sorption solution, 3.0 mL of 10% ACN in water. More de-
tails could be found in the text and Figure S3 in Supporting
Information.
Under the aforementioned conditions, we evaluated
the recoveries of 10 nucleotides in water and in urine sam-
ples spiked with nucleotides. The results showed that the
recoveries of 10 nucleotides ranged from 90.3% to 101.2%
and 88.3% to 103.3% in water and urine sample, respec-
tively, suggesting good extraction recoveries towards nu-
cleotides (Table S3 in Supporting Information).
Identification of DMPA-labeled nucleotides
We examined the DMPA-labeled nucleotides by LC-
ESI-MS/MS. The labeled products can be produced
through the reaction of the amine group in DMPA with the
phosphate group in nucleotides. The product ion spectra
demonstrated that the fragment ions generated by the de-
rivatives were identical to their corresponding theoretical
values (Figure 3), suggesting that the desired DMPA-
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Analytical Chemistry
Figure 3. Product ions spectra of DMPA-labeled dAMP (A), TMP
(B), dCMP (C), dGMP (D), AMP (E), UMP (F), CMP (G), GMP
(H), 5-Me-dCMP (I), 5-Me-CMP (J).
reaction conditions, the labeling efficiencies of the 10 nu-
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cleotides by DMPA ranged from 84.4% to 100.3% (Table
S4 in Supporting Information). We also evaluated the sta-
bility of DMPA-labeled nucleotides. The results showed
that DMPA-labeled nucleotides were stable at room tem-
perature for at least 24 h (Figure 4F), which is sufficient for
the subsequent processing and analysis. The reaction solu-
tion was then further proceeded to remove excessive
DMPA and EDC and the general recoveries of nucleotide
derivatives ranging from 90.3% to 103.0% were achieved
(Table S5 in Supporting Information).
In the labeling reaction, imidazole was used as buffer
to provide a weak acidic environment (pH 6.0). To assess
the influence of imidazole buffer on labeling, we investi-
gated imidazole buffer concentrations ranging from 0 to 10
mM (pH 6.0). The results showed that the largest peak are-
as of nucleotide derivatives were achieved at 1 mM of im-
idazole (pH 6.0) (Figure 4C). Therefore, 1 mM imidazole
(pH 6.0) in the reaction solution was used for the following
experiments.
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Improvements of detection performance upon DMPA
labeling
In addition, the optimal concentration of DMPA for
the labeling of nucleotides was investigated. The results
showed that the peak areas of nucleotide derivatives
reached plateau when the molar ratio of DMPA/nucleotides
was greater than 40,000 (Figure 4D). Therefore, the molar
ratio of 40,000 of DMPA/nucleotides was used for the la-
beling reaction. Finally, the optimal concentration of acti-
vator EDC was investigated. The results showed that the
largest peak areas of nucleotide derivatives were achieved
when the molar ratio of EDC/nucleotides was greater than
5,000 (Figure 4E). Therefore, the molar ratio of 5,000 of
EDC/nucleotides was used for the labeling reaction.
The main purpose for chemical labeling is to improve
the detection sensitivities of nucleotides during LC-ESI-
MS/MS analysis. Here we investigated the detection sensi-
tivities of 10 nucleotides upon DMPA labeling under their
own optimized mass spectrometry conditions. The extract-
ed-ion chromatograms showed that the retention of 10 na-
tive nucleotides was relatively weak on the reversed-phase
chromatographic column even under optimized separation
conditions (Figure 5A). However, after DMPA labeling,
their retention dramatically increased (Figure 5B), owing to
the increased hydrophobicity of these labeled products en-
dowed by the hydrophobic phenyl group of DMPA.
The limit of detection (LOD) defined as the amount of
analyte at a signal-to-noise ratio (S/N) of 3 was employed
to evaluate the improved detection sensitivities of nucleo-
tides by DMPA labeling. The LODs of 10 nucleotides with
and without DMPA labeling were shown in Table 1. We
found the detection sensitivities of these nucleotides la-
beled by DMPA increased by 88 to 372 folds. We reason
that the tertiary amino group in DMPA can increase ioniza-
tion efficiency during mass spectrometry analysis. In addi-
tion, increased hydrophobicity of these DMPA-labeled
products results in longer retention time on the LC column
and thus delayed in elution with a higher ratio of organic
solvent. In this way, the analytes could be ionized more
effectively during electrospray ionization because of higher
spraying and desolvation efficiency in higher ACN content.
In comparison with previously reported methods, the detec-
tion limits for nucleotides acquired by our method were
reduced by at least 20 folds (Table S6 in Supporting Infor-
mation). Collectively, compared to the native forms of 10
nucleotides, DMPA labeling could dramatically increase
the detection sensitivities.
Figure 4. Optimization of DMPA labeling conditions. (A) Opti-
mization of reaction temperature. (B) Optimization of reaction
time. (C) Optimization of the concentration of imidazole buffer.
(D) Optimization of the molar ratio of DMPA/nucleotides. (E)
Optimization of the molar ratio of EDC/nucleotides. (F) Evalua-
tion of the stabilities of the DMPA-labeled nucleotides.
Taken together, DMPA labeling were conducted at
50oC for 1.5 h in 1 mM imidazole buffer (pH 6.0), and the
molar ratios of DMPA and EDC over nucleotides were set
as 40,000 and 5,000, respectively. Under these optimized
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LODs (fmol)
Detection sensitiv-
ities improved
folds
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Nucleotides
Unlabeled DMPA labeling
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9
dAMP
TMP
25.7
48.4
44.2
33.6
28.7
96.0
56.2
53.0
32.3
34.6
0.13
0.13
0.42
0.38
0.12
0.26
0.47
0.43
0.30
0.36
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dCMP
dGMP
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AMP
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96
UMP
CMP
GMP
5-Me-dCMP
5-Me-CMP
Determination of the methylated nucleotides in mam-
mals
With the established method, we investigated whether
methylated nucleotides (5-Me-dCMP and 5-Me-CMP) ex-
isted in human tissues. Shown in Figure 6 are the typical
extracted-ion chromatograms of 10 nucleotides in human
renal tissue before and after DMPA labeling. The results
demonstrated that, while canonical nucleotides (dAMP,
TMP, dCMP, dGMP, AMP, UMP, CMP and GMP) can be
detected without DMPA labeling, 5-Me-dCMP and 5-Me-
CMP were undetectable (Figure 6A; shown in Figure 6C is
the zoomed-in chromatograms of Figure 6A). On the con-
trary, all the 10 nucleotides, including 5-Me-dCMP and 5-
Me-CMP can be distinctly detected by our developed strat-
egy of DMPA labeling combined LC-ESI-MS/MS analysis
(Figure 6B; shown in Figure 6D is the zoomed-in chroma-
tograms of Figure 6B).
Figure 5. Extracted-ion chromatograms of 10 nucleotides before
(A) and after (B) DMPA labeling.
Method validation
To evaluate the linearity of the method, 10 nucleotide
standards at concentrations of 0.002, 0.005, 0.01, 0.02,
0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500,
800,1000 pg/µl and fixed amount of deuterated DMPA
(DMPA-d4)-labeled nucleotides as internal standards (IS)
were used to construct calibration curves by plotting peak
area ratio (analyte/IS) against the concentrations of DMPA-
labeled nucleotides with triplicate measurements. The re-
sults showed that good linearities within the 3 orders of
magnitude in dynamic range of nucleotides were obtained
with the coefficients of determination (R2) being greater
than 0.9939 (Table S7 in Supporting Information).
To examine whether 5-Me-dCMP and 5-Me-CMP are
widely present in vivo, we further analyzed 5-Me-dCMP
and 5-Me-CMP in various biological samples, including
cultured human 293T cells, HeLa cells, and human urine
samples. As shown in the extracted-ion chromatograms
(Figure 7), DMPA-labeled 5-Me-dCMP and 5-Me-CMP in
human 293T cells, HeLa cells, and human urine samples
had similar retentions as the DMPA-d4-labeled 5-Me-
dCMP and 5-Me-CMP standards, suggesting that the de-
tected compounds from these human samples were ex-
pected methylated nucleotides, i.e., 5-Me-dCMP and 5-Me-
CMP. It is noted that the retention times of DMPA-d4-
labeled 5-Me-dCMP and 5-Me-CMP standards were slight-
ly shorter than those of the corresponding DMPA-labeled
compounds (Figure 7). The phenomenon was frequently
observed in plenty of studies, i.e., the deuterated com-
pounds typically eluted earlier than their corresponding
protiated compounds in C18 stationary phase.31,32 Since C–
H bond has a higher oscillation frequency than the C–D
bond, C–H bond induces greater forces of attraction be-
tween itself and the C18 stationary phase. Therefore, pro-
The accuracy and precision of our method were as-
sessed by the relative errors as well as intra- and inter-day
relative standard deviations (RSDs). Both relative errors
and intra- and inter-day RSDs were calculated by spiking
nucleotide standards in urine samples at three different
concentrations. For each concentration, triplicate measure-
ments were performed. Intra-day variation was evaluated
by repeating the process for three times within one day, and
the inter-day variation was investigated on three successive
days. The accuracy and reproducibility of the developed
method were evaluated with the REs and RSDs being less
than 15.0% and 15.3%, respectively, demonstrating good
accuracy and reproducibility (Table S8 in Supporting In-
formation).
Table 1. Limits of detection of 10 nucleotides with and without
DMPA labeling followed by LC-ESI-MS/MS analysis.
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tiated compounds have stronger binding to the C18 station-
Contents change of nucleotides in tissues and urine
samples of cancer patients
ary phase than deuterated ones.31,32 Collectively, the study
confirmed the wide existence of 5-Me-dCMP and 5-Me-
CMP in mammals.
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Having demonstrated the presence of 5-Me-dCMP and
5-Me-CMP in various human samples, we next asked
whether the levels of these methylated nucleotides differ in
human carcinoma tissues. In this respect, a total of 18 tis-
sue samples derived from renal carcinoma patients, includ-
ing 9 paired tumor tissues and tumor adjacent normal tis-
sues, were analyzed by our method. The results showed
that the mean contents of 5-Me-dCMP in renal carcinoma
tissues and tumor adjacent normal tissues were 0.0039 and
0.0068 pmol/mg protein, respectively; while the mean con-
tents of 5-Me-CMP in renal carcinoma tissues and tumor
adjacent normal tissues were 0.0058 and 0.0084 pmol/mg
protein, respectively. The results revealed significant de-
creases in the levels of 5-Me-dCMP and 5-Me-CMP in
human renal carcinoma tissues compared to tumor-adjacent
normal tissues (p = 0.001 for 5-Me-dCMP; p = 0.001 for 5-
Me-CMP, Figure 8A and 8B; Table S8 in Supporting In-
formation).
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Figure 6. Extracted-ion chromatograms of 10 nucleotides detected
in human renal tissue by DMPA labeling coupled with LC-ESI-
MS/MS analysis. (A) Analysis without DMPA labeling. (B)
Analysis with DMPA labeling. (C) Zoomed-in chromatograms
from (A). (D) Zoomed-in chromatograms from (B).
The mean ratios of 5-Me-dCMP/dCMP and 5-Me-
CMP/CMP in human renal tissues were 0.78% and 0.04%,
respectively, which were only several folds lower than the
typical cytosine methylation in DNA (~ 3%) and RNA (~
0.1%). Therefore, it is very likely that the endogenous 5-
Me-dCMP and 5-Me-CMP could be incorporated into
DNA and RNA after converting to triphosphate nucleo-
sides.
We also compared the contents of 5-Me-dCMP and 5-
Me-CMP in urine samples from human lymphoma patients
and healthy controls. The results showed that the mean
contents of 5-Me-dCMP in urine samples of human lym-
phoma patients and healthy controls were 0.025 pmol/mg
creatinine and 0.048 pmol/mg creatinine, respectively;
while the mean contents of 5-Me-CMP in urine samples of
human lymphoma patients and healthy controls were 0.077
pmol/mg creatinine and 0.176 pmol/mg creatinine, respec-
tively. The results also showed significant decreases in the
levels of 5-Me-dCMP and 5-Me-CMP in urine samples
from human lymphoma patients compared to those from
healthy controls (p = 2.0 × 10-4 for 5-Me-dCMP; p = 2.2 ×
10-4 for 5-Me-CMP, Figure 8C and 8D; Table S10 in Sup-
porting Information). We reason that the significant de-
crease of 5-Me-dCMP and 5-Me-CMP in urine samples of
lymphoma patients may be due to the more reutilization of
methylated nucleotides in DNA and RNA synthesis in
lymphoma patients. On the other hand, global genome hy-
pomethylation is known to be frequently accompanied with
tumor development.33 In this respect, the lower levels of 5-
Me-dCMP and 5-Me-CMP in tumor tissues and urine sam-
ples of cancer patients suggest that they may arise from the
excision repair products of nucleic acids or from the de-
composition of nucleic acids of dead cells. However, the
mechanisms require further investigation.
Figure 7. Extracted-ion chromatograms of DMPA-d4-labeled 5-
Me-dCMP and 5-Me-CMP standards and DMPA-labeled 5-Me-
dCMP and 5-Me-CMP from the human urine, 293T cells and
HeLa cells.
Our quantification data showed that the contents of 5-
Me-dCMP and 5-Me-CMP in human renal tissues ranged
from 0.002 to 0.010 and 0.004 to 0.013 pmol/mg protein,
respectively (Table S9 in Supporting Information); the con-
tents of 5-Me-dCMP and 5-Me-CMP in 293T cells were
0.0025 and 0.010 pmol/mg protein, respectively (Table S10
in Supporting Information); the contents of 5-Me-dCMP
and 5-Me-CMP in HeLa cells were 0.0030 and 0.020
pmol/mg protein, respectively (Table S10 in Supporting
Information); the contents of 5-Me-dCMP and 5-Me-CMP
in human urine samples ranged from 0.01 to 0.08 and 0.03
to 0.29 pmol/mg creatinine, respectively (Table S11 in
Supporting Information).
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This study is, to the best of our knowledge, the first
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report for detecting endogenous 5-Me-CMP and 5-Me-
dCMP in human urine, tissues and cultured cells. 5-Me-
dCMP and 5-Me-CMP can be potentially converted to their
triphosphate forms and became the substrates for DNA and
RNA synthesis. The biological significance of endogenous
5-Me-CMP and 5-Me-dCMP, however, need further inves-
tigation.
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CONCLUSIONS
In the current study, we established a sensitive method
to determine endogenous nucleotides in mammals based on
DMPA labeling coupled with LC-ESI-MS/MS analysis.
The detection sensitivities for nucleotides increased by 88
to 372 folds upon DMPA labeling. Using the developed
method, we were able to identify and quantify endogenous
5-Me-dCMP and 5-Me-CMP in multiple human samples,
including cultured human cells, human tissues, and human
urine samples. The results showed that 5-Me-dCMP and 5-
Me-CMP were widely present in human samples, which,
for the first time, confirmed their existence in vivo. The
quantification results also demonstrated significant deple-
tion of 5-Me-dCMP and 5-Me-CMP in renal carcinoma
tissues and urine samples of lymphoma patients compared
to controls, indicating the potential reutilization of methyl-
ated nucleotides in DNA and RNA synthesis. The biologi-
cal consequence of endogenous 5-Me-dCMP and 5-Me-
CMP still requires further investigation.
Figure 8. Quantification and statistical analysis of methylated
nucleotides in human renal tissues and urine. (A) 5-Me-dCMP in
human renal carcinoma tissues and tumor adjacent normal tissues.
(B) 5-Me-CMP in human renal carcinoma tissues and tumor adja-
cent normal tissues. (C) 5-Me-dCMP in human urines from
healthy controls and lymphoma patients. (D) 5-Me-CMP in hu-
man urines from healthy controls and lymphoma patients.
Deamination of 5-Me-dCMP is an important mecha-
nism to exclude 5-Me-dCMP from incorporation into
DNA. However, deamination of 5-Me-dCMP to TMP may
not be always highly efficient, as demonstrated by our cur-
rent study that endogenous 5-Me-dCMP and 5-Me-CMP
can be unambiguously detected in mammals. The second
mechanism that could prevent 5-Me-dCTP formation and
its incorporation into DNA is the proposed absence of a
kinase that can act on 5-Me-dCMP converting it to 5-
methyl-2’-deoxycytidine diphosphate (5-Me-dCDP).22
However, previous study demonstrated that there was not a
complete absence of kinase activity since the radioactive
labeled 5-[3H]-Me-dC was measurable in DNA upon treat-
ment of CHO cells with 5-[3H]-Me-dC,34 suggesting that
incorporation of 5-Me-dCTP originated from 5-Me-dCMP
is possible.
ASSOCIATED CONTENT
Supporting Information
Supporting Information Available: Optimization of the ex-
traction conditions for nucleotides; Extraction of DMPA-
labeled nucleotides; Table S1 – S11; Figure S1 – S4. This
material is available free of charge via the Internet at
http://pubs.acs.org.
On the other hand, 5-Me-CMP from RNA metabolism
could be phosphorylated to 5-Me-CDP, a substrate for ri-
bonucleotide reductase to produce 5-Me-dCDP. The
formed 5-Me-dCDP can be further phosphorylated to 5-
Me-dCTP since the kinase converting nucleotides diphos-
phates to nucleotides triphosphate is known not to be spe-
cific.35,36 In addition, 5-Me-CDP could be phosphorylated
to 5-Me-CTP that can be used for RNA synthesis. There-
fore, even deaminase may convert 5-Me-dCMP to TMP, 5-
Me-dCTP can be generated from 5-Me-CDP by ribonucleo-
tide reductase and then phosphorylated by nucleoside di-
phosphate kinases. The method developed in our study is
also suitable for the detection of nucleoside triphosphates.
Because nucleoside triphosphates are more hydrophilic
than nucleoside monophosphates, further optimization of
SPE conditions is required to remove the excessive EDC by
Strata X cartridge. Determination of endogenous modified
nucleoside triphosphates will be performed in the future
study.
AUTHOR INFORMATION
Corresponding Author
*Phone: 86-27-68755595. Fax: 86-27-68755595. E-mail:
bfyuan@whu.edu.cn.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final ver-
sion of the manuscript. These authors contributed equally.
†
Notes
The authors declare no competing financial interest.
Acknowledgements
The authors thank the financial support from the Na-
tional Natural Science Foundation of China (21522507,
21672166, 21635006).
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