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Sensitive and Simultaneous Determination of 5-Methylcytosine and Its Oxidation Products in Genomic DNA by Chemical Derivatization Coupled with Liquid Chromatography - Tandem Mass Spectrometry Analysis Yang Tang, Shu-Jian Zheng, Chu-Bo Qi, Yu-Qi Feng, and Bi-Feng Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504786r • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Analytical Chemistry
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Sensitive and Simultaneous Determination of
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5-Methylcytosine and Its Oxidation Products in Genomic
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DNA by Chemical Derivatization Coupled with Liquid
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Chromatography - Tandem Mass Spectrometry Analysis
5 Yang Tang,1,3,† Shu-Jian Zheng,1,† Chu-Bo Qi,1,2,† Yu-Qi Feng,1,* and Bi-Feng Yuan1,*
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1
8
Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China
9
2
Department of Pathology, Hubei Cancer Hospital, Wuhan 430079, P.R. China
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3
Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Nanning
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530021, P.R. China
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† These authors contributed equally to this work.
13
*
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+86-27-68755595. E-mail address:
[email protected];
[email protected] Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),
To
whom
correspondence
should
be
addressed.
15
1
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ABSTARCT
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Cytosine methylation (5-methylcytosine, 5-mC) in genomic DNA is an important
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epigenetic mark that has regulatory roles in diverse biological processes. 5-mC can be oxidized
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stepwise by the Ten-eleven translocation (TET) proteins to form 5-hydroxymethylcytosine
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(5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC), which constitutes the
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active DNA demethylation pathway in mammals. Owing to the extremely limited contents of
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endogenous 5-mC oxidation products, no reported method can directly determine all these
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cytosine modifications simultaneously. In the current study, we developed selective
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derivatization of cytosine moieties with 2-bromo-1-(4-dimethylamino-phenyl)-ethanone
25
(BDAPE) coupled with liquid chromatography-electrospray ionization tandem mass
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spectrometry (LC-ESI-MS/MS) for the simultaneous determination of these cytosine
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modifications in genomic DNA. The chemical derivatization notably improved the liquid
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chromatography separation and dramatically increased detection sensitivities of these cytosine
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modifications. The limits of detection (LOD) of the derivatives of 5-mC, 5-hmC, 5-foC and
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5-caC were 0.10, 0.06, 0.11, and 0.23 fmol, respectively. Using this method, we successfully
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quantified 5-mC, 5-hmC, 5-foC and 5-caC in genomic DNA from human colorectal carcinoma
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(CRC) tissues and tumor-adjacent normal tissues. The results demonstrated significant
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depletion of 5-hmC, 5-foC and 5-caC in tumor tissues compared to tumor-adjacent normal
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tissues. And the depletion of 5-hmC, 5-foC and 5-caC may be a general feature of CRC and
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these cytosine modifications could serve as potential biomarkers for the early detection and
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prognosis of CRC. Moreover, the marked depletion of 5-hmC, 5-foC and 5-caC may also have
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profound effects on epigenetic regulation in the development and formation of CRC.
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Keywords:
5-methylcytosine;
5-hydroxymethylcytosine;
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5-carboxylcytosine; derivatization; mass spectrometry; colorectal carcinoma.
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5-formylcytosine;
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INTRODUCTION
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DNA methylation, consisting of the addition of a methyl group at the fifth position of
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cytosine to give 5-methylcytosine (5-mC) at the CpG dinucleotide site, is one of the
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best-characterized epigenetic modifications and highly conserved in most plants and animals.1
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DNA methylation can undergo dynamic changes and is reversible and involved in diverse
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physiological functions, including genome stability, embryogenesis, genomic imprinting, and
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X-chromosome inactivation.2 Maintaining dynamic DNA methylation status by balancing
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methylation and demethylation processes is vital for the normal functions of living cells.3 And
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aberrant DNA methylation may lead to various human diseases, such as heart disease, diabetes,
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neurological disorders and cancers.4,5
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In mammals, DNA methylation is established by de novo DNA methyltransferases,
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DNMT3A and DNMT3B and maintained by maintenance DNA methyltransferase, DNMT1.6
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Recent studies suggested that active DNA demethylation in mammals could be achieved
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through a consecutive oxidation of 5-mC by Ten - eleven translocation (TET) proteins with the
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generation of three intermediates, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine
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(5-foC), and 5-carboxylcytosine (5-caC).7-10 The oxidation products of 5-foC and 5-caC can be
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further recognized and cleaved by thymine-DNA glycosylase (TDG), thereby restoring
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unmethylated cytosine via the base excision repair (BER) pathway.11
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5-mC has been commonly recognized as “the fifth base” for decades, and 5-hmC is now
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viewed to be “the sixth base” of the genome in mammals in addition to adenine, cytosine,
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thymine, guanine, and 5-mC.12 Mapping of 5-hmC in tissues and cell lines demonstrated that 4
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the genomic distribution of 5-hmC is nonrandom and distinct from that of 5-mC,13,14 with
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5-hmC being especially enriched in the gene bodies and enhancers,15 indicating that 5-hmC
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may play important roles on cellular differentiation and epigenetic regulation.16,17 In addition,
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some studies including our recent report demonstrated that 5-hmC significantly decreased in
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various tumors, suggesting that 5-hmC is also associated with tumor formation and
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development.18-20
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5-foC and 5-caC are the further oxidation products of 5-hmC by TET proteins and are
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more transient intermediates in DNA demethylation pathway.10,11 A recent study identified
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many potential proteins that can specifically bind to 5-foC and 5-caC in mouse embryonic stem
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cells, indicating that 5-foC and 5-caC may recruit unique proteins for certain functions beyond
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as the intermediates in DNA demethylation pathway.21 Moreover, we and others recently
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demonstrated that 5-foC and 5-caC can change the fidelity of DNA replication and slow down
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RNA polymerase II transcription, suggesting the possible functional roles of 5-foC and 5-caC
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on DNA replication and transcription.22,23 Whereas, it is still unclear whether 5-foC and 5-caC
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play functional roles in cancer development and formation.24
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5-mC and its oxidation products differ in their abundance within the genome, with 5-hmC
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being present at a frequency around ~ 10- to 100-fold lower than that of 5-mC, and 5-foC and
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5-caC being ~ 40 to 1000-fold lower than that of 5-hmC.10,25-27 Specially, 5-foC occurs at a
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frequency of 1 to 20 per 106 cytosine and 5-caC presents at a much lower level than 5-foC in
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mammals.10,27,28 So far 5-caC is mainly detected in embryonic stem cells that contain relatively
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high amount of 5-caC (~ 3 per 106 cytosine) and has not been identified in the majority of other 5
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tissues.10 Quantifications of these cytosine modifications are challenging due to their low
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in-vivo contents as well as the interference from the highly abundant normal nucleosides.
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In this respect, sensitive and accurate measurements of these cytosine derivatives are
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essential to elucidate their functional roles in living cells. In the past decade, considerable
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advances have been made in the development of analytical methods for the quantification of
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these cytosine modifications. Due to the inherent selectivity and sensitivity, liquid
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chromatography-mass spectrometry (LC-MS) has been frequently used in the analysis of 5-mC
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and its oxidation products.29 LC-ESI-MS/MS with MRM mode method was initially developed
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for
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5-hydroxymethyl-2’-deoxycytidine (5-hmdC).30,31 Later, Ito et al. 10 and Liu et al. 32 established
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LC-ESI-MS/MS for the sensitive quantification of 5-mdC, 5-hmdC, 5-formyl-2’-deoxycytidine
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(5-fodC), and 5-carboxyl-2’-deoxycytidine (5-cadC) in genomic DNA of mammalian tissue
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and cells. In fact, LC-ESI-MS/MS is the only reported method so far used to quantify 5-foC and
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5-caC.27 Whereas, in these methods, either only one or two cytosine modification (s) can be
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directly detected or offline HPLC purification was required prior to MS analysis to remove the
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interference of the bulky normal nucleosides, which is therefore laborious and time-consuming.
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In addition, the ionization efficiencies in electrospray ionization (ESI) of these cytosine
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modifications are usually low. Thus, an easily ionizable moiety can be introduced to these
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cytosine modifications to enhance the ionization efficiency during by LC-ESI-MS/MS
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analysis.
the
quantification
of
5-methyl-2’-deoxycytidine
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(5-mdC)
and
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Analytical Chemistry
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Herein, we developed a chemical derivatization coupled with LC-ESI-MS/MS analysis for
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the sensitive and simultaneous determination of all the four cytosine modifications (5-mdC,
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5-hmdC, 5-fodC and 5-cadC) occurring in DNA demethylation pathway. We evaluated the
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effect
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2-bromo-1-(4-dimethylamino-phenyl)-ethanone (BDAPE). Our results show that good
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derivatization efficiencies of all four cytosine modifications can be achieved with BDAPE
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under mild conditions. Moreover, chemical derivatization dramatically enhanced the detection
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sensitivities (by 35 - 123 folds) and greatly improved the retention behaviors of these cytosine
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modifications in LC-ESI-MS/MS analysis. Upon derivatization, the limits of detection of
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5-mdC, 5-hmdC, 5-fodC and 5-cadC can reach 0.10, 0.06, 0.11, and 0.23 fmol, respectively.
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Using this method, we directly and simultaneously quantified 5-mdC, 5-hmdC, 5-fodC, and
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5-cadC in genomic DNA from human colorectal carcinoma (CRC) and tumor-adjacent normal
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tissues. We demonstrated that, compared to tumor-adjacent normal tissues, the contents of
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5-hmdC, 5-fodC and 5-cadC in tumor tissues significantly decreased (p < 0.01), suggesting that
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5-hmdC, 5-fodC and 5-cadC may play certain functional roles in the regulation of cancer
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development and the marked depletion of 5-hmdC, 5-fodC and 5-cadC may serve as potential
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biomarkers for the early detection and prognosis of CRC.
of
chemical
derivatization
of
these
cytosine
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EXPERIMENTAL SECTION
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Chemicals and Reagents
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modifications
using
Analytical Chemistry
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2’-Deoxycytidine (dC), 2’-deoxyguanosine (dG), 2’-deoxyadenosine (dA), and thymidine
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(T) were purchased from Sigma-Aldrich (Beijing, China). 5-Methyl-2’-deoxycytidine (5-mdC),
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5-hydroxymethyl-2’-deoxycytidine (5-hmdC), 5-formyl-2’-deoxycytidine (5-fodC), and
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5-carboxyl-2’-deoxycytidine (5-cadC) were purchased from Berry & Associates (Dexter, MI).
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2-Bromo-1-(4-dimethylamino-phenyl)-ethanone (BDAPE) was purchased from Sigma-Aldrich
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(Beijing, China).
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Chromatographic-grade methanol and acetonitrile (ACN) were purchased from Tedia Co.
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Inc. (Fairfield, OH). All other solvents and chemicals used were of analytical grade. Formic
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acid, acetic acid, and triethylamine (TEA) were purchased from Sinopharm Chemical Reagent
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Co., Ltd. (Shanghai, China). S1 nuclease and alkaline phosphatase (CIAP) were from Takara
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Biotechnology Co., Ltd. (Dalian, China). Phosphodiesterase I were purchased from
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Sigma-Aldrich (Beijing, China). The water used throughout the study was purified on a Milli-Q
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apparatus (Millipore, Bedford, MA). Stock solutions of dC, dG, dA, and T were prepared in
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Milli-Q water at concentrations of 500 µg/mL. Stock solutions of 5-mdC, 5-hmdC, 5-fodC and
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5-cadC were prepared in Milli-Q water at concentrations of 50, 10, 1, and 1 µg/mL. BDAPE
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and TEA were prepared in ACN at concentrations of 40 and 100 mM, respectively.
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Colorectal Carcinoma Tissues
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This study was approved by the ethics committee of Hubei Cancer Hospital (Wuhan,
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China). A total of 48 formalin-fixed, paraffin-embedded (FFPE) tissue samples from 24
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colorectal carcinoma (CRC) patients, including 24 pairs of colorectal carcinoma tissues and
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matched tumor-adjacent normal tissues were collected in Hubei Cancer Hospital. 8
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Analytical Chemistry
DNA Extraction and Enzymatic Digestion
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FFPE tissues DNA were extracted using E.Z.N.A.® FFPE DNA Kit (Omega Bio-Tek Inc.,
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Norcross, GA) according to the manufacturer’s recommended procedure. Typically, ~ 30 µg of
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genomic DNA can be obtained from 10 mg of FFPE tissue. The concentration of the purified
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genomic DNA was determined on a B-500 spectrophotometer (Metash Instruments Co., Ltd.,
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Shanghai, China).
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As for enzymatic digestion, genomic DNA (10 µg in 16 µL H2O) was first denatured by
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heating at 95 ºC for 5 min and then chilling on ice for 2 min. After adding 1/10 volume (2 µL) of
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S1 nuclease buffer (30 mM CH3COONa, pH 4.6, 280 mM NaCl, 1 mM ZnSO4) and 360 units
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(2 µL) of S1 nuclease, the mixture (20 µL) was then incubated at 37 ºC for 4 h. To the resulting
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solution was subsequently added 10 µL of alkaline phosphatase buffer (50 mM Tris-HCl, 10
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mM MgCl2, pH 9.0), 0.01 units (5 µL) of venom phosphodiesterase I, 30 units (1 µL) of alkaline
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phosphatase and 64 µL H2O. And then the incubation was continued at 37 ºC for an additional
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2 h. The digested DNA was filtered by an ultrafiltration tube (10 kD cut-off, Amicon, Millipore)
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to remove the enzymes; and the obtained solution was further passed through a SPE (solid
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phase extraction) cartridge filled with 50 mg sorbent of graphitized carbon black (Weltech Co.,
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Ltd, Wuhan, China) to remove the salts. The elution was then dried with nitrogen gas at 37 °C
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followed by derivatization.
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The quality control (QC) samples were also prepared to validate the method. Briefly, a
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16-mer oligodeoxynucleotide (5’-GTAGGTCGTGATGAGG-3’) was mixed with a 16-mer
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oligodeoxynucleotide
(5’-GTAGGT(5-mC)GTGATGAGG-3’) 9
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or
a
17-mer
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(5’-CTTAAGCCG(5-hmC)AGGTACC-3’)
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oligodeoxynucleotide
or
a
27-mer
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oligodeoxynucleotide (5’-GAGTCGCTCCCCATGGG(5-foC)ACCGAATTC-3’) or a 27-mer
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oligodeoxynucleotide (5’-GAGTCGCTCCCCATGGG(5-caC)ACCGAATTC-3’) at different
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amounts to make the molar ratios of 5-mdC/102 dG, 5-hmdC/105 dG, 5-fodC/107 dG,
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5-cadC/107 dG range from 1 to 6, 10 to 500, 5 to 50, 1 to 10, respectively. The QC samples were
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then enzymatically digested followed by derivatization using the same procedure as that for
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genomic DNA of CRC tissues.
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Optimization of Chemical Derivatization
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In this study, BDAPE was used to derivatize 5-mdC, 5-hmdC, 5-fodC and 5-cadC. To
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achieve the best derivatization efficiency, we optimized the derivatization conditions, including
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catalyst, reaction temperature and time, and concentration of BDAPE. All the reactions were
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performed in 200 µL ACN with 1 µM of 5-mdC.
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As for the effect of catalyst on the derivatization efficiency, we investigated acetic acid
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and TEA in reaction solution ranging from 0 to 8% (v/v) or 0 to 8 mM, respectively. Generally,
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1 µM 5-mdC and 2.5 mM BDAPE (molar ratio of BDAPE/5-mdC, 2500/1) were dissolved in
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ACN, and then acetic acid or TEA was added followed by incubation at 70 oC for 1 h. The
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reactions were stopped by immediate freezing at -80°C.
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Next, we optimized the reaction temperature ranging from 30 oC to 70 oC. The reactions
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were incubated with 2.5 mM of BDAPE and 4 mM of TEA for 1h. The concentration of
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BDAPE was also optimized. We changed the concentrations of BDAPE ranging from 0.05 to
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10 mM (the molar ratios of BDAPE/5-mdC ranging from 100/1 to 20000/1), and the reactions 10
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Analytical Chemistry
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were incubated at 60 oC with 4 mM of TEA for 1h. Finally, The derivatization time was
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optimized ranging from 1 to 16 hours, and the reactions were performed at 60 °C with 4 mM of
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BDAPE and 4 mM of TEA.
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The derivatization products were examined on a Shimadzu LC-15C HPLC system (Tokyo,
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Japan) equipped with two LC-15C pumps, a CTO-15C thermostated column compartment, a
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SPD-15C UV/vis detector, and a RF-10A fluorescence detector (FLD). The UV/vis detector
193
was connected with FLD in series. The reaction substrates were detected by UV/vis detector
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with a wavelength of 260 nm, and the derivatives of 5-mdC, 5-hmdC, 5-fodC and 5-cadC were
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detected by FLD with 305 nm of excitation wavelength and 370 nm of emission wavelength. A
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Hisep C18-T column (250 mm × 4.6 mm i.d., 5 µ m, Weltech Co., Ltd., Wuhan, China) was
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used for the separation. The column temperature was set at 35 °C. Water containing 1% formic
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acid (v/v, solvent A) and acetonitrile (solvent B) were employed as mobile phase with a flow
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rate of 0.8 mL/min. A gradient of 5% B for 4 min, 5 − 45% B for 40 min was used.
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Simultaneous Analysis of 5-mdC, 5-hmdC, 5-fodC and 5-cadC in Genomic DNA of
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Human CRC Tissues
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The derivatization of all the four cytosine modifications from the digested genomic DNA
203
of human CRC tissues by BDAPE was performed under the optimized conditions. Briefly, the
204
nucleoside mixture of digested tissue genomic DNA (10 µg) was passed through a SPE
205
cartridge filled with 50 mg sorbent of graphitized carbon black (Weltech Co., Ltd, Wuhan,
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China) to remove the salts. The elution was then dried with nitrogen gas at 37 °C followed by
207
derivatization, which was performed in 200 µL ACN and with 4 mM of BDAPE and 4 mM of 11
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TEA. Then the resulting solution was incubated at 60 oC for 6 h with shaking at 1,500 rpm to
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derivatize 5-mdC, 5-hmdC, 5-fodC and 5-cadC. The derivatized products were dried with
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nitrogen gas at 37 °C, and then reconstituted in 100 µL water followed by analysis with
211
LC-ESI-MS/MS.
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The quantification of the derivatives of 5-mdC, 5-hmdC, 5-fodC, and 5-cadC were
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performed on the LC-ESI-MS/MS system consisting of an AB 3200 QTRAP mass
214
spectrometer (Applied Biosystems, Foster City, CA, USA) with an electrospray ionization
215
source (Turbo Ionspray) and a Shimadzu LC-20AD HPLC (Tokyo, Japan) with two LC-20AD
216
pumps, a SIL-20A auto sampler, a CTO-20AC thermostated column compartment and a
217
DGU-20A3 degasser. Data acquisition and processing were performed using AB SCIEX
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Analyst 1.5 Software (Applied Biosystems, Foster City, CA, USA). The HPLC separation was
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performed on a Hisep C18-T column (150 mm × 2.1 mm i.d., 5 µm, Weltech Co., Ltd., Wuhan,
220
China) at 35 oC. Water containing 0.05% formic acid (v/v, solvent A) and ACN (solvent B)
221
were employed as mobile phase. A gradient of 5 – 65% B of 40 min was used. The flow rate of
222
mobile phase was set at 0.2 mL/min.
223
The mass spectrometry detection was performed under positive electrospray ionization
224
(ESI) mode. The nucleosides and derivatives were monitored by multiple reaction monitoring
225
(MRM) mode using the mass transitions (precursor ions product ions) of dC (228.4
226
112.2), T (243.3 127.2), dA (252.4 136.2), dG (268.4 152.4), 5-mdC (242.1 126.1),
227
5-hmdC (258.1 142.1), 5-fodC (256.1 140.0), 5-cadC (272.1 156.0), 5-mdC
228
derivatives (385.2 269.1), 5-hmdC derivatives (401.2 285.1 and 401.2 267.1), 5-fodC 12
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derivatives (399.2 283.1), and 5-cadC derivatives (576.2 460.2 and 576.2 281.1). The
230
MRM parameters of the analytes were optimized to achieve maximal detection sensitivity.
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High resolution mass spectrometry experiments was performed on the LC-QTOF-MS
232
system consisting of a MicrOTOF-Q orthogonal-accelerated TOF mass spectrometer (Bruker
233
Daltonics, Bremen, Germany) with an ESI source (Turbo Ionspray) and a Shimadzu LC-20AB
234
binary pump HPLC (Tokyo, Japan), a SIL-20AC auto sampler, and a DGU-20A3 degasser.
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Data acquisition and processing were performed using Bruker Daltonics Control 3.4 and
236
Bruker Daltonics Data analysis 4.0 software. The HPLC separation was performed on a Hisep
237
C18-T column (150 mm × 2.1 mm i.d., 5 µm, Weltech Co., Ltd., Wuhan, China) at 35 oC. Water
238
containing 0.05% formic acid (v/v, solvent A) and ACN (solvent B) were employed as mobile
239
phase. A gradient of 5 – 65% B of 40 min was used. The flow rate of mobile phase was set at 0.2
240
mL/min.
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Statistical Analysis
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The statistical data were processed with SPSS 19.0 software (SPSS Inc.). The paired t-test
243
was performed to evaluate the differences in cytosine modifications of genomic DNA between
244
tumor tissues and tumor-adjacent normal tissues. All p values were two-sided, and generally, p
245
values < 0.05 were considered to have statistical significance.
246 247
RESULTS AND DISCUSSION
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Chemical Derivatization
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5-mC and its three oxidation products (5-hmC, 5-foC, and 5-caC) are all derived from the
250
modifications of cytosine at the 5th position, which constitutes DNA methylation and
251
demthylation pathway. The accurate and simultaneous quantification of these cytosine
252
modification will facilitate the investigation of their physiological functions. Whereas, direct
253
and simultaneous quantification of these cytosine modification has not been realized due to
254
their low abundance in vivo. And off-line HPLC purification and enrichment is typically used
255
method before LC-MS/MS analysis, which, however is tedious and may cause inaccurate
256
measurements due to the complicated sample pretreatment. And our group recently also
257
established
258
modifications,33-35 but these strategies mainly focus on the labelling of the groups at the 5th
259
position of cytosine, which therefore can only analyze one or two cytosine medication (s) at one
260
time.
chemical derivatization
methods
to
sensitively
detect
these
cytosine
261
In the current study, we used BDAPE which harbors a hydrophobic phenyl group and an
262
easily chargeable tertiary amine group to simultaneously derivatize all the four cytosine
263
modifications (5-mdC, 5-hmdC, 5-fodC and 5-cadC) (Figure 1). The bromoacetonyl group of
264
BDAPE can readily react with the 3-N and 4-N positions of cytosine to form a stable penta
265
cyclic structure. In addition, the introduced hydrophobic phenyl group using BDAPE can
266
dramatically increase the retention and greatly improve the separation of these cytosine
267
modifications on reversed-phase LC, which, therefore can finally increase the detection
268
sensitivities. Moreover, the detection sensitivities of these BDAPE-derivatized products by
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LC-ESI-MS/MS can be further increased due to the introduction of easily chargeable group on
270
BDAPE upon derivatization.
271
Identification of the Derivatives of 5-mdC, 5-hmdC, 5-fodC and 5-cadC
272
After derivatization, we found that the retention of all the four derivatives dramatically
273
increased in reversed-phase LC, with 5-cadC derivative exhibiting the strongest retention than
274
other derivatives (Figure 2). We further examined the derivatives of 5-mdC, 5-hmdC, 5-fodC
275
and 5-cadC using LC-ESI-MS/MS. Figure 3 shows the fragmentation ions of these four
276
derivatives, which clearly demonstrated the desired derivatives of 5-mdC, 5-hmdC and 5-fodC
277
were obtained (Figure 3A, 3B and 3C).
278
It was reported that ester formation between carboxyl and bromoacetonyl are favorable
279
under mildly alkaline conditions.36 We speculated that the carboxyl group at the 5th position of
280
5-cadC may also react with BDAPE besides 3-N and 4-N positions of 5-cadC and thus 5-cadC
281
derivative will harbor two phenyl groups, which could contribute to the strong hydrophobic
282
retention of 5-cadC derivative in reversed-phase LC. In this respect, as for 5-cadC derivative,
283
three ions of m/z 576.2, 460.2, and 281.1, which represent the parent ion of 5-cadC derivative
284
and its two product ions, were observed after BDAPE derivatization (Figure 3D). The results
285
further supported the carboxyl group of the 5th position of 5-cadC also reacted with BDAPE.
286
And no 5-cadC derivative that contains one phenyl group (theoretical m/z 415.2) was found
287
(data no shown). In addition, we also examined the 5-cadC derivatives using neutral loss (NL)
288
scan mode (losing a neutral ribose with m/z of 116) by LC-ESI-MS/MS. The result showed that
289
only the m/z of 576.2 was found and no other obvious signal of m/z was present, further 15
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indicating that the derivatization of 5-cadC mainly produced the derivative carrying two phenyl
291
groups (Figure S1, Supporting Information). And the 5-cadC derivative carrying one phenyl
292
group was barely detected. The results suggested that BDAPE can completely react with
293
5-cadC to form a derivative carrying two phenyl groups, which also benefits the quantitative
294
analysis of 5-cadC.
295
Moreover, the derivatives of 5-mdC, 5-hmdC, 5-fodC and 5-cadC were further confirmed
296
by high-resolution mass spectrometry analysis (Figure S2, Supporting Information).
297
Optimization of Derivatization Conditions
298
To obtain good derivatization efficiencies of 5-mC and its oxidation products, we
299
optimized the derivatization conditions, including catalyst, reaction temperature and time, and
300
concentration of BDAPE.
301
We first investigated the effect of acid (acetic acid) and base (TEA) as the catalyst for the
302
derivatization reaction. The peak area of 5-mdC derivative decreased with the increase of acetic
303
acid, suggesting acetic acid inhibited the reaction (Figure 4A). On the contrary, the addition of
304
TEA can increase the derivatization efficiency and the peak area of 5-mdC derivative reaches a
305
plateau when the concentration of TEA was 4 mM (Figure 4B). Based on these results, we
306
speculated the plausible mechanism of the derivatization reaction of 5-mdC by BDAPE (Figure
307
S3, Supporting Information). In this respect, TEA may assist the deprotonation and the
308
formation of penta cyclic structure (Figure S3, Supporting Information). Therefore, we used
309
TEA (4 mM) as the catalyst in the following experiments. The chemical reaction is based on the
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nucleophilic group (e.g., -NH2) reacting with electrophile to form a cyclic structure. Similar
311
chemical process is also well known in proteins and peptides labeling.37-39
312
We next optimized the reaction temperature for derivatization of 5-mdC by BDAPE. Our
313
results demonstrated that the largest peak areas of 5-mdC derivative can be achieved at 60 °C
314
(Figure 4C). In addition, the optimal concentration of BDAPE for derivatization of 5-mdC was
315
investigated. The results showed that the peak area of 5-mdC derivative reaches a plateau when
316
the concentration of BDAPE was 4 mM (Figure 4D). As for the optimization of derivatization
317
time, the result showed that 6 h was sufficient for the derivatization of 5-mdC by BDAPE
318
(Figure 4E).
319
Taken together, the optimized derivatization conditions for 5-mdC by BDAPE were under
320
60 °C for 6 h with 4 mM of BDAPE using TEA (4 mM) as the catalyst. Under optimized
321
derivatization conditions, more than 99% of 5-mdC, as well as 5-hmdC, 5-fodC and 5-cadC can
322
react with BDAPE to form the corresponding derivatives (Table S1, Supporting Information),
323
suggesting high derivatization efficiencies were achieved.
324
Improvement of Detection Sensitivity and LC Separation of 5-mdC and Its Oxidation
325
Products upon Derivatization
326
The main purpose for derivatization is to improve the LC separation and detection
327
sensitivities of 5-mdC, 5-hmdC, 5-fodC and 5-cadC during LC-ESI-MS/MS analysis. We then
328
examined the of LC separation and detection sensitivities of 5-mdC and its oxidation products
329
upon derivatization with BDAPE. The MRM chromatogram shows that the retention of 5-mdC,
330
5-hmdC, and 5-cadC were relatively weak and they co-eluted on C18 reversed-phase 17
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chromatographic column even under optimized separation conditions (Figure 5A). However,
332
the retention of all their derivatives dramatically increased and their LC separation resolution
333
also notably improved after BDAPE derivatization (Figure 5B). Moreover, the peaks of these
334
derivatives were narrower than the compounds without derivatization (Figure 5B). In addition,
335
we also evaluated the derivatization of other nucleosides with BDAPE. The results showed that
336
dG and T cannot react with BDAPE, and dC and dA can react with BDAPE. While, the
337
separation of the derivatives of dC, dA, 5-mC, 5-hmC, 5-foC and 5-caC is better than their
338
native forms (Figure S4, Supporting Information).
339
Compared to the native 5-mdC, 5-hmdC, 5-fodC and 5-cadC, derivatization by BDAPE
340
could also markedly increase the detection sensitivities by 35-fold for 5-mdC, 93-fold for
341
5-hmdC, 89-fold for 5-fodC, and 123-fold for 5-cadC with the existence of all the bulky normal
342
nucleosides (Table 1). The limits of detection (LOD) of the derivatives of 5-mdC, 5-hmdC,
343
5-fodC and 5-cadC were 0.10, 0.06, 0.11 and 0.23 fmol, respectively (Table 1). The excess
344
amounts of BDAPE present in samples may suppress the ionization of analytes during
345
LC-ESI-MS/MS analysis. In this respect, after 5-cadC derivative was eluted out (~ 32 min), we
346
switched the six-way valve and BDAPE (~ 36 min) (Figure S5, Supporting Information) was
347
then removed to waste line, which can avoid the contamination of mass spectrometer by
348
BDAPE. The notable increase in detection sensitivities achieved by BDAPE derivatization can
349
be attributed to several aspects. Firstly, the derivatization increased the ionization efficiencies
350
of 5-mdC and its oxidation products in ESI. Secondly, the derivatization increased the retention
351
of 5-mdC and its oxidation products on reversed-phase chromatographic column, which 18
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resulted in longer retention time and thus elution within a higher ratio of organic solvent. Thus
353
the analytes could be ionized more effectively in ESI owing to higher spraying and desolvation
354
efficiency under higher ACN content. Thirdly, the improved separation of the derivatives could
355
further minimize the mutual ion suppression of the analytes as well as the ion suppression from
356
the bulky normal nucleosides, which could also contribute to the increased detection
357
sensitivities.
358
Method Validation
359
The calibration curve of 5-mdC, 5-hmdC, 5-fodC and 5-cadC were constructed by plotting
360
the mean peak area ratios of 5-mdC/102 dG, 5-hmdC/105 dG, 5-fodC/107 dG, or 5-cadC/107 dG
361
versus the mean molar ratios of 5-mdC/102 dG, 5-hmdC/105 dG, 5-fodC/107 dG, or 5-cadC/107
362
dG based on data obtained from triplicate measurements using the derivatization strategy
363
combined with LC-ESI-MS/MS analysis. The results showed that good linearities within the
364
range of 0.1-10 5-mdC/102 dG, 2-500 5-hmdC/105 dG, 0.5-100 5-fodC/107 dG, and 0.5-100
365
5-cadC/107 dG were obtained with a correlation coefficient (R) being greater than 0.99 (Table
366
S2, Supporting Information).
367
The accuracy of the proposed method was assessed using the QC samples by comparing
368
the measured 5-mdC, 5-hmdC, 5-fodC and 5-cadC contents to the theoretical 5-mdC, 5-hmdC,
369
5-fodC and 5-cadC contents. Three different molar ratios of 5-mdC/102 dG ranging from 1.0 to
370
6.0, 5-hmdC/105 dG ranging from 10.0 to 500.0, 5-fodC/107 dG ranging from 5.0 to 50.0, and
371
5-cadC/107 dG ranging from 1.0 to 10.0 were measured (Table S3, Supporting Information).
372
The results showed that good accuracy was achieved, which are manifested by the relative 19
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373
errors (RE) being 6.7 – 10.0% for 5-mdC, -10.0 – 7.1% for 5-hmdC, -15.0 – 10.2% for 5-fodC,
374
and -11.0 – 14.0% for 5-cadC, respectively (Table S3, Supporting Information).
375
In addition, the reproducibility of the developed method was evaluated by the
376
measurement of intra- and inter-day precisions. Three parallel treatments of samples over a day
377
gave the intra-day relative standard deviations (RSDs), and the inter-day RSDs were
378
determined by treating samples independently for three consecutive days. The results showed
379
that the intra- and inter-day RSDs were less than 10.2% and 14.0%, respectively (Table S3,
380
Supporting Information), demonstrating that good reproducibility was achieved.
381
Simultaneous Determination of 5-mdC, 5-hmdC, 5-fodC and 5-cadC in Genomic DNA of
382
Human CRC Tissues
383
CRC is one of the most common human cancers and a leading cause of cancer deaths
384
worldwide.40,41 The importance of epigenetic alterations in human CRC, however, has not been
385
rigorously explored despite some reports suggesting changes in global cytosine methylation
386
and hydroxymethylation in cancer cells.18-20,42
387
In the current study, we investigated the amounts of 5-mdC, 5-hmdC, 5-fodC and 5-cadC
388
in genomic DNA of human CRC tissues. A total of 48 tissue samples derived from 24 CRC
389
patients, including 24 paired tumor tissues and tumor-adjacent normal tissues, were analyzed
390
by BDAPE derivatization coupled with LC-ESI-MS/MS analysis (Table S4, Supporting
391
Information). Paired t-test was used to evaluate the differences of 5-mdC and its oxidation
392
products between human CRC tumor tissues and tumor-adjacent normal tissues. The mean
393
contents of 5-mdC in tumor tissues and tumor-adjacent normal tissues were 3.5 ± 0.6/102 dG 20
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and 4.1 ± 1.0/102 dG, respectively, suggesting no significant difference of 5-mdC between
395
tumor tissues and tumor-adjacent normal tissues (p = 0.097, Figure 6A). On the contrary, the
396
mean contents of 5-hmdC, 5-fodC and 5-cadC in human CRC tumor tissues were 27.0 ± 6.4/105
397
dG, 9.2 ± 3.4/107 dG, and 0.9 ± 0.3/107 dG, respectively; and the mean contents of 5-hmdC,
398
5-fodC and 5-cadC in tumor-adjacent normal tissues were 86.8 ± 18.2/105 dG, 26.4 ± 5.2/107
399
dG, and 3.1 ± 1.7/107 dG, respectively. The results suggested significant depletion of 5-hmdC,
400
5-fodC and 5-cadC in human CRC tumor tissues compared to tumor-adjacent normal tissues (p
401
= 1.3×10-14 for 5-hmdC; p = 3.0×10-15 for 5-fodC; p = 1.8×10-6 for 5-cadC, Figure 6B-6D;
402
Table S4, Supporting Information).
403
The discovery that 5-mdC can be oxidized to 5-hmC and further oxidized to 5-fodC and
404
5-cadC by TET enzymes has raised many questions regarding the roles of 5-hmdC, 5-fodC and
405
5-cadC as potential epigenetic marks in diverse biological processes. A role for 5-hmdC,
406
5-fodC and 5-cadC as the intermediates in TET-mediated active DNA demethylation has been
407
biochemically validated by multiple studies.24 Here we found that 5-hmdC dramatically
408
decreased in CRC tissues, which is consistent with previous reports that close correlation
409
existed between decreased 5-hmdC levels and tumor growth and metastasis.42-44 In addition,
410
5-fodC and 5-cadC are also the enzymatic products of TET proteins and the oxidation products
411
derived from 5-hmdC. Therefore, the decreased 5-hmdC may also cause the reduced 5-fodC
412
and 5-cadC. And this study is, to the best of our knowledge, the first quantitative study for the
413
report of significant decreases of all 5-hmdC, 5-fodC and 5-cadC in human CRC tumor tissues
414
compared to normal tissues. The results suggested the depletion of 5-hmdC, 5-fodC and 5-cadC 21
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415
may be a general feature of human CRC, and these cytosine modifications could be the
416
potential biomarkers for the early detection and prognosis of CRC. Moreover, the marked
417
depletion of 5-hmdC, 5-fodC and 5-cadC may also have profound effects on epigenetic
418
regulation in human CRC, which still needs further investigation.
419
We also compared the contents of 5-mdC and 5-hmdC in some tissue samples measured
420
by BDAPE derivatization coupled with LC-ESI-MS/MS analysis with the direct
421
LC-ESI-MS/MS analysis without derivatization. The results showed that the measured 5-mdC
422
and 5-hmdC contents in genomic DNAs were comparable using these two different methods,
423
with REs being -12.4% to 8.8% (Table S5, Supporting Information), indicating the BDAPE
424
derivatization coupled with LC-ESI-MS/MS analysis is reliable for the simultaneous
425
determination of these cytosine modifications in genomic DNA. However, the direct analysis
426
by LC-ESI-MS/MS without derivatization is not able to detect 5-fodC and 5-cadC due to the
427
relatively low detection sensitivity. In addition, stable isotope labeling strategy has been used to
428
discover novel metabolites,45,46 therefore, novel cytosine modifications besides 5-mdC,
429
5-hmdC, 5-fodC and 5-cadC may also be identified using isotope labeled BDAPE coupled with
430
double precursor ion scan mass spectrometry analysis in the future.
431 432
CONCLUSIONS
433
We developed a highly sensitive method to simultaneously determine 5-mC and its
434
oxidation products by BDAPE derivatization coupled with LC-ESI-MS/MS analysis. BDAPE
435
derivatization notably improved the liquid chromatography separation and dramatically 22
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increased detection sensitivities of these cytosine modifications. Using this method, we
437
successfully quantified 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA from human CRC
438
tissues and tumor-adjacent normal tissues. The results demonstrated significant depletion of
439
5-hmC, 5-foC, 5-caC in tumor tissues compared to tumor-adjacent normal tissues and indicated
440
the depletion of 5-hmC, 5-foC and 5-caC may be a general feature of human CRC and these
441
cytosine modifications could be the potential biomarkers for the early detection and prognosis
442
of CRC. This study is the first quantitative study for the report of significant decreases of all
443
5-hmC, 5-foC and 5-caC in human CRC tissues compared to normal controls. And the marked
444
depletion of 5-hmdC, 5-fodC and 5-cadC may also have profound effects on epigenetic
445
regulation in the development and formation of CRC, however, which requires further
446
investigation.
447
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448 449
Notes The authors declare no competing financial interest.
450 451 452
Acknowledgments The authors thank the financial support from the National Basic Research Program of
453
China (973 Program) (2013CB910702, 2012CB720601), the National Natural Science
454
Foundation of China (21205091, 91217309).
455 456 457 458
Supporting Information Additional information as noted in text. This information is available free of charge via the Internet at http://pubs.acs.org/.
459 460
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Table 1. Limits of detection of 5-mdC, 5-hmdC, 5-fodC and 5-cadC with and without BDAPE
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derivatization. LOD (fmol)
Without derivatization After derivatization Increased folds of the detection sensitivity
5-mdC
5-hmdC
5-fodC
5-cadC
3.5
5.6
9.8
28.4
0.10
0.06
0.11
0.23
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89
123
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Figure Legends
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Figure 1. Derivatization of 5-mdC, 5-hmdC, 5-fodC and 5-cadC by BDAPE. “R” represents the
548
different groups, including -CH3, -CH2OH, -CHO, and -COOH.
549 550
Figure 2. (A) HPLC-UV chromatograms for 5-mdC, 5-hmdC, 5-fodC and 5-cadC before and
551
after BDAPE derivatization. (B) HPLC-FLD chromatograms for 5-mdC, 5-hmdC, 5-fodC and
552
5-cadC before and after BDAPE derivatization. Line 1, 3, 5, 7 represent 5-mdC, 5-hmdC,
553
5-fodC and 5-cadC before BDAPE derivatization; and Line 2, 4, 6, 8 represent 5-mdC, 5-hmdC,
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5-fodC and 5-cadC after BDAPE derivatization.
555 556
Figure 3. The product ion spectra of (A) 5-mdC derivative, (B) 5-hmdC derivative, (C) 5-fodC
557
derivative and (D) 5-cadC derivative.
558 559
Figure 4. Optimization of derivatization conditions for 5-mdC by BDAPE. The effects of (A)
560
acetic acid, (B) TEA concentration, (C) reaction temperature, (D) BDAPE concentration and (E)
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reaction time on the derivatization efficiency of 5-mdC.
562 563
Figure 5. MRM chromatograms of 5-mdC, 5-hmdC, 5-fodC and 5-cadC before (A) and after (B)
564
derivatization by BDAPE under optimized conditions.
565
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Figure 6. Quantification and statistical analysis of 5-mdC (A), 5-hmdC (B), 5-fodC (C) and
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5-cadC (D) in human CRC tissues and tumor-adjacent normal tissues. Each point represents the
568
content of each sample. The line represents the mean content of each cytosine modification in
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measured tissue samples.
570
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Analytical Chemistry
Figure 1.
572 573
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Figure 2.
575 576
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Analytical Chemistry
Figure 3.
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Figure 4.
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Analytical Chemistry
Figure 5.
584 585
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Figure 6.
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Analytical Chemistry
For TOC only
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