Sensitive and Simultaneous Determination of 5-Methylcytosine and Its

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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

1

Sensitive and Simultaneous Determination of

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5-Methylcytosine and Its Oxidation Products in Genomic

3

DNA by Chemical Derivatization Coupled with Liquid

4

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,*

6 7

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

10

3

Guangxi Zhuang Autonomous Region Center for Disease Prevention and Control, Nanning

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530021, P.R. China

12

† These authors contributed equally to this work.

13

*

14

+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

ACS Paragon Plus Environment

Tel.:+86-27-68755595;

fax:


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ABSTARCT

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Cytosine methylation (5-methylcytosine, 5-mC) in genomic DNA is an important

18

epigenetic mark that has regulatory roles in diverse biological processes. 5-mC can be oxidized

19

stepwise by the Ten-eleven translocation (TET) proteins to form 5-hydroxymethylcytosine

20

(5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC), which constitutes the

21

active DNA demethylation pathway in mammals. Owing to the extremely limited contents of

22

endogenous 5-mC oxidation products, no reported method can directly determine all these

23

cytosine modifications simultaneously. In the current study, we developed selective

24

derivatization of cytosine moieties with 2-bromo-1-(4-dimethylamino-phenyl)-ethanone

25

(BDAPE) coupled with liquid chromatography-electrospray ionization tandem mass

26

spectrometry (LC-ESI-MS/MS) for the simultaneous determination of these cytosine

27

modifications in genomic DNA. The chemical derivatization notably improved the liquid

28

chromatography separation and dramatically increased detection sensitivities of these cytosine

29

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

31

quantified 5-mC, 5-hmC, 5-foC and 5-caC in genomic DNA from human colorectal carcinoma

32

(CRC) tissues and tumor-adjacent normal tissues. The results demonstrated significant

33

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

2


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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;

40

5-carboxylcytosine; derivatization; mass spectrometry; colorectal carcinoma.

41

3


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

44

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,

51

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

101

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

103

analysis.

the

quantification

of

5-methyl-2’-deoxycytidine

6


(5-mdC)

and

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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

121 122

EXPERIMENTAL SECTION

123

Chemicals and Reagents

7


modifications

using


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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

129

(Beijing, China).

130

Chromatographic-grade methanol and acetonitrile (ACN) were purchased from Tedia Co.

131

Inc. (Fairfield, OH). All other solvents and chemicals used were of analytical grade. Formic

132

acid, acetic acid, and triethylamine (TEA) were purchased from Sinopharm Chemical Reagent

133

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

135

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

137

Milli-Q water at concentrations of 500 µg/mL. Stock solutions of 5-mdC, 5-hmdC, 5-fodC and

138

5-cadC were prepared in Milli-Q water at concentrations of 50, 10, 1, and 1 µg/mL. BDAPE

139

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,

142

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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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.,

147

Norcross, GA) according to the manufacturer’s recommended procedure. Typically, ~ 30 µg of

148

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.,

150

Shanghai, China).

151

As for enzymatic digestion, genomic DNA (10 µg in 16 µL H2O) was first denatured by

152

heating at 95 ºC for 5 min and then chilling on ice for 2 min. After adding 1/10 volume (2 µL) of

153

S1 nuclease buffer (30 mM CH3COONa, pH 4.6, 280 mM NaCl, 1 mM ZnSO4) and 360 units

154

(2 µL) of S1 nuclease, the mixture (20 µL) was then incubated at 37 ºC for 4 h. To the resulting

155

solution was subsequently added 10 µL of alkaline phosphatase buffer (50 mM Tris-HCl, 10

156

mM MgCl2, pH 9.0), 0.01 units (5 µL) of venom phosphodiesterase I, 30 units (1 µL) of alkaline

157

phosphatase and 64 µL H2O. And then the incubation was continued at 37 ºC for an additional

158

2 h. The digested DNA was filtered by an ultrafiltration tube (10 kD cut-off, Amicon, Millipore)

159

to remove the enzymes; and the obtained solution was further passed through a SPE (solid

160

phase extraction) cartridge filled with 50 mg sorbent of graphitized carbon black (Weltech Co.,

161

Ltd, Wuhan, China) to remove the salts. The elution was then dried with nitrogen gas at 37 °C

162

followed by derivatization.

163

The quality control (QC) samples were also prepared to validate the method. Briefly, a

164

16-mer oligodeoxynucleotide (5’-GTAGGTCGTGATGAGG-3’) was mixed with a 16-mer

165

oligodeoxynucleotide

(5’-GTAGGT(5-mC)GTGATGAGG-3’) 9


or

a

17-mer


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(5’-CTTAAGCCG(5-hmC)AGGTACC-3’)

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oligodeoxynucleotide

or

a

27-mer

167

oligodeoxynucleotide (5’-GAGTCGCTCCCCATGGG(5-foC)ACCGAATTC-3’) or a 27-mer

168

oligodeoxynucleotide (5’-GAGTCGCTCCCCATGGG(5-caC)ACCGAATTC-3’) at different

169

amounts to make the molar ratios of 5-mdC/102 dG, 5-hmdC/105 dG, 5-fodC/107 dG,

170

5-cadC/107 dG range from 1 to 6, 10 to 500, 5 to 50, 1 to 10, respectively. The QC samples were

171

then enzymatically digested followed by derivatization using the same procedure as that for

172

genomic DNA of CRC tissues.

173

Optimization of Chemical Derivatization

174

In this study, BDAPE was used to derivatize 5-mdC, 5-hmdC, 5-fodC and 5-cadC. To

175

achieve the best derivatization efficiency, we optimized the derivatization conditions, including

176

catalyst, reaction temperature and time, and concentration of BDAPE. All the reactions were

177

performed in 200 µL ACN with 1 µM of 5-mdC.

178

As for the effect of catalyst on the derivatization efficiency, we investigated acetic acid

179

and TEA in reaction solution ranging from 0 to 8% (v/v) or 0 to 8 mM, respectively. Generally,

180

1 µM 5-mdC and 2.5 mM BDAPE (molar ratio of BDAPE/5-mdC, 2500/1) were dissolved in

181

ACN, and then acetic acid or TEA was added followed by incubation at 70 oC for 1 h. The

182

reactions were stopped by immediate freezing at -80°C.

183

Next, we optimized the reaction temperature ranging from 30 oC to 70 oC. The reactions

184

were incubated with 2.5 mM of BDAPE and 4 mM of TEA for 1h. The concentration of

185

BDAPE was also optimized. We changed the concentrations of BDAPE ranging from 0.05 to

186

10 mM (the molar ratios of BDAPE/5-mdC ranging from 100/1 to 20000/1), and the reactions 10


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187

were incubated at 60 oC with 4 mM of TEA for 1h. Finally, The derivatization time was

188

optimized ranging from 1 to 16 hours, and the reactions were performed at 60 °C with 4 mM of

189

BDAPE and 4 mM of TEA.

190

The derivatization products were examined on a Shimadzu LC-15C HPLC system (Tokyo,

191

Japan) equipped with two LC-15C pumps, a CTO-15C thermostated column compartment, a

192

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

194

with a wavelength of 260 nm, and the derivatives of 5-mdC, 5-hmdC, 5-fodC and 5-cadC were

195

detected by FLD with 305 nm of excitation wavelength and 370 nm of emission wavelength. A

196

Hisep C18-T column (250 mm × 4.6 mm i.d., 5 µ m, Weltech Co., Ltd., Wuhan, China) was

197

used for the separation. The column temperature was set at 35 °C. Water containing 1% formic

198

acid (v/v, solvent A) and acetonitrile (solvent B) were employed as mobile phase with a flow

199

rate of 0.8 mL/min. A gradient of 5% B for 4 min, 5 − 45% B for 40 min was used.

200

Simultaneous Analysis of 5-mdC, 5-hmdC, 5-fodC and 5-cadC in Genomic DNA of

201

Human CRC Tissues

202

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,

206

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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208

TEA. Then the resulting solution was incubated at 60 oC for 6 h with shaking at 1,500 rpm to

209

derivatize 5-mdC, 5-hmdC, 5-fodC and 5-cadC. The derivatized products were dried with

210

nitrogen gas at 37 °C, and then reconstituted in 100 µL water followed by analysis with

211

LC-ESI-MS/MS.

212

The quantification of the derivatives of 5-mdC, 5-hmdC, 5-fodC, and 5-cadC were

213

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

218

Analyst 1.5 Software (Applied Biosystems, Foster City, CA, USA). The HPLC separation was

219

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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229

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.

231

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.

235

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.

241

Statistical Analysis

242

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

248

Chemical Derivatization

13



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Page 14 of 37

249

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

14


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269

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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290

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

16


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310

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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331

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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352

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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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394

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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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436

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

23



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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

24


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(41) Brenner, H.; Kloor, M.; Pox, C. P. Lancet 2014, 383, 1490-502.

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(42) Lian, C. G.; Xu, Y.; Ceol, C.; Wu, F.; Larson, A.; Dresser, K.; Xu, W.; Tan, L.; Hu, Y.;

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Zhan, Q.; Lee, C. W.; Hu, D.; Lian, B. Q.; Kleffel, S.; Yang, Y.; Neiswender, J.; Khorasani, A.

530

J.; Fang, R.; Lezcano, C.; Duncan, L. M.; Scolyer, R. A.; Thompson, J. F.; Kakavand, H.;

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Houvras, Y.; Zon, L. I.; Mihm, M. C., Jr.; Kaiser, U. B.; Schatton, T.; Woda, B. A.; Murphy, G.

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F.; Shi, Y. G. Cell 2012, 150, 1135-46.

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(43) Song, S. J.; Poliseno, L.; Song, M. S.; Ala, U.; Webster, K.; Ng, C.; Beringer, G.; Brikbak,

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N. J.; Yuan, X.; Cantley, L. C.; Richardson, A. L.; Pandolfi, P. P. Cell 2013, 154, 311-24.

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Tang, F. M.; Lee, S. B.; Hsu, H. M.; Yu, J. C.; Hsiao, P. W.; Juan, L. J. Cell Rep 2012, 2,

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539

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540

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Page 28 of 37

542

Table 1. Limits of detection of 5-mdC, 5-hmdC, 5-fodC and 5-cadC with and without BDAPE

543

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

35

93

89

123

544 545

28


Page 29 of 37

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546

Figure Legends

547

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,

554

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)

561

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

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

566

Figure 6. Quantification and statistical analysis of 5-mdC (A), 5-hmdC (B), 5-fodC (C) and

567

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

569

measured tissue samples.

570

30


Page 30 of 37

Page 31 of 37

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571


Figure 1.

572 573

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

574

Figure 2.

575 576

32


Page 32 of 37

Page 33 of 37

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577


Figure 3.

578 579

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

580

Figure 4.

581 582

34


Page 34 of 37

Page 35 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

583


Figure 5.

584 585

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

586

Figure 6.

587 588

36


Page 36 of 37

Page 37 of 37

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589


For TOC only

590

37


Sensitive and Simultaneous Determination of 5-Methylcytosine and Its

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