質(zhì)譜法定量生態(tài)物種基因組DNA低甲基化

胡俊杰,吳毅聰,陳桂連,高圓圓,趙 桐,余 廣,蘭善紅,呂小梅*

質(zhì)譜法定量生態(tài)物種基因組DNA低甲基化

胡俊杰1,吳毅聰1,陳桂連1,高圓圓2,趙 桐3,余 廣1,蘭善紅1,呂小梅1*

(1.東莞理工學(xué)院生態(tài)環(huán)境與建筑工程學(xué)院,廣東 東莞 523808；2.東莞市生態(tài)環(huán)境技術(shù)中心,廣東 東莞 523009；3.長(zhǎng)江大學(xué)資源與環(huán)境學(xué)院,湖北 武漢 434023)

為了定量生態(tài)相關(guān)物種基因組中低豐度的5-甲基-2-脫氧胞苷(5-mdC),建立了一種采用高效液相色譜串聯(lián)質(zhì)譜(HPLC-MS/MS)的無標(biāo)記方法.全基因組DNA甲基化計(jì)算公式5-mdC(mole)/(5-mdC(mole)+dC(mole)可以轉(zhuǎn)化為1/(1+dC(mole)/5-mdC(mole)),然后通過HPLC-MS/MS得到DNA樣品中5-mdC和dC的物質(zhì)的量比(dC(mole)/5-mdC(mole))即可得到基因組DNA甲基化值.此外,還對(duì)HPLC條件進(jìn)行了優(yōu)化使正常核苷和修飾核苷基線分離,消除分析物的交叉干擾.本方法避免了使用昂貴的穩(wěn)定同位素標(biāo)記內(nèi)標(biāo),在等度高效液相色譜條件下實(shí)現(xiàn)了正常和修飾核苷基線分離,5-mdC和dC的保留時(shí)間分別為5.50和3.06min.5-mdC和dC的定量限分別為14.2和19.1pg/mL.日內(nèi)和日間偏差和準(zhǔn)確性(相對(duì)誤差)都≤10%.該方法測(cè)定的小牛胸腺DNA和大型蚤及秀麗線蟲的全基因組DNA甲基化值分別為(6.44 ± 0.25)%,(0.097 ± 0.010)%和(0.025 ± 0.002)%.采用HPLC-MS/MS技術(shù)建立并驗(yàn)證了一種快速、高精密度和靈敏度的DNA樣品全基因組DNA甲基化測(cè)定方法,適用于評(píng)估環(huán)境污染物對(duì)生態(tài)學(xué)相關(guān)物種的潛在表觀遺傳風(fēng)險(xiǎn).

全基因組DNA甲基化；無標(biāo)記；電噴霧質(zhì)譜；表觀遺傳效應(yīng)；生態(tài)物種

DNA甲基化是表觀遺傳學(xué)的重要內(nèi)容,涉及DNA的構(gòu)象和染色體結(jié)構(gòu)的改變,以及DNA和轉(zhuǎn)錄因子間相互作用.DNA甲基化在基因表達(dá)調(diào)節(jié)和基因組印記中發(fā)揮著重要作用[1].不良健康效應(yīng),如致癌發(fā)生、生殖異常和糖尿病等都與DNA甲基化圖譜的變化密切相關(guān)[2-5].已有研究發(fā)現(xiàn),金屬和有機(jī)污染物可導(dǎo)致環(huán)境物種中DNA甲基化狀態(tài)異常[6].因此,DNA甲基化被認(rèn)為是一個(gè)有前途的生物監(jiān)測(cè)標(biāo)志物,用于評(píng)估環(huán)境因素的不良表觀遺傳效應(yīng)[7].在一些環(huán)境物種中,如海洋生物和大型蚤,這些物種的全基因組中5-甲基-2-脫氧胞苷(5-mdC)的豐度很低[8],對(duì)全基因組DNA甲基化精密定量提出了挑戰(zhàn).因此,建立測(cè)量低豐度基因組DNA甲基化的方法是表觀遺傳學(xué)在毒害效應(yīng)評(píng)估研究中的先決條件.

到目前為止,現(xiàn)有的DNA甲基化檢測(cè)方法包括高效液相色譜(HPLC)[9]、氣相色譜串聯(lián)質(zhì)譜(GC-MS/MS)[10]、液相色譜串聯(lián)質(zhì)譜(HPLC-MS/ MS)[11-12]、毛細(xì)管電泳[13-14]、免疫定量[15]、LINE1測(cè)序[16]和熒光甲基化檢測(cè)[17].這些方法可以提供具有高準(zhǔn)確性和可重復(fù)性的全基因組DNA甲基化值,但有存在不足[18].例如,GC-MS/MS具有高靈敏度,但需要用衍生劑對(duì)核苷進(jìn)行繁瑣而耗時(shí)的衍生[10].熒光法甲基化檢測(cè)適用于沒有參考基因組序列信息的任何物種,而它只能檢測(cè)CCGG序列內(nèi)的甲基化狀態(tài),這導(dǎo)致基因組中其他CpG位點(diǎn)的信息丟失.目前的大多數(shù)方法適用于測(cè)定具有高豐度DNA甲基化的樣品.因此,需要開發(fā)一種高靈敏度、省時(shí)簡(jiǎn)便的方法來評(píng)估低豐度全基因組DNA甲基化的生態(tài)物種的表觀遺傳風(fēng)險(xiǎn).

HPLC-MS/MS技術(shù)可以高靈敏度和選擇性地檢測(cè)修飾核苷,近年來已經(jīng)開發(fā)了多種該類方法.然而,大多數(shù)方法均缺乏合適的5-mdC和胞苷(dC)的內(nèi)標(biāo)來校正質(zhì)譜信號(hào)的偏差.一些研究使用了同位素標(biāo)記的化合物,如2H4-5-甲基2-脫氧胞苷[12]、Cyt13C15N2[19]和15N3-2'-脫氧胞苷[20],作為內(nèi)標(biāo)或替代內(nèi)標(biāo).然而,這些同位素標(biāo)記的化學(xué)品需要定制合成,而且往往價(jià)格昂貴.因此,一些研究人員使用DNA中5-mdC與dG的物質(zhì)的量比作為全基因組DNA甲基化值[21].此外,修飾核苷與正常核苷的共同洗脫會(huì)干擾目標(biāo)物的電離效果,特別是那些低含量的目標(biāo)物[22].因此,優(yōu)良的色譜條件,將5-mdC與正常核苷進(jìn)行基線分離,對(duì)于低豐度5-mdC的定量至關(guān)重要.正常核苷和修飾核苷的梯度分離需要更多的時(shí)間對(duì)色譜柱進(jìn)行修飾.選擇合適的分離和檢測(cè)條件以達(dá)到所需的選擇性和靈敏度非常重要.

因此,本文開發(fā)一種新的低成本和高準(zhǔn)確性的方法,利用無標(biāo)記HPLC-MS/MS對(duì)生態(tài)相關(guān)物種的DNA樣品中的全基因組DNA甲基化進(jìn)行定量分析.研究通過HPLC-MS/MS測(cè)定DNA樣品中5-mdC (mole)/dC(mole)的摩爾比,然后通過Methylation%= 1/(1+dC(mole)/5-mdC(mole))×100%的公式計(jì)算出全基因組DNA甲基化值,以期與現(xiàn)有方法相比.

1 材料與方法

1.1 材料和試劑

HPLC級(jí)乙腈、甲醇和甲酸(德國(guó)默克公司).乙酸銨和碳酸氫銨(美國(guó)杜邦公司).核酸酶P1(來自檸檬青霉菌)、磷酸二酯酶I(來自Crotalus adamanteus)和小牛腸道堿性磷酸酶與10×孵化緩沖液(500mM乙酸鉀;200mM乙酸三鈉;100mM乙酸鎂;1mg/mL BSA)(美國(guó)Sigma公司).小牛胸腺DNA(北京鼎國(guó)生物技術(shù)有限公司).5-mdC、2-脫氧腺苷(dA)、2-脫氧鳥苷(dG)、2-脫氧胞苷(dC)、A、G、C、T和U(美國(guó)Amresco公司).去離子水采用純水機(jī)制備(美國(guó)Millipore公司).其他化學(xué)試劑均為分析純.

兩種21堿基DNA寡核苷酸,其序列為5-ACT CAC TCA CGC AGT GAG AGA-3(7A:6C:5G:3T)和5-A(5mC)T (5mC)A(5mC) T(5mC)A(5mC) AGT GAG AGA GAG-3(7A:6mC:5G:3T)從上海生工生物技術(shù)有限公司采購(gòu).

5-mdC和dC溶于水制備1mg/mL濃度的儲(chǔ)備液.5-mdC(1、10、100和1000ng/mL)和dC(50、100、500和1000μg/mL)的標(biāo)準(zhǔn)工作溶液通過去離子水進(jìn)一步稀釋儲(chǔ)備液制備.

1.2 DNA的酶促水解

基因組DNA和寡核苷酸酶解成核苷的分析方法如前所述[21].將約1μg DNA溶解在3μL去離子水中,然后100℃下孵育3min,并立即在冰中冷卻以變性.加入1μL(2U)核酸酶P1和1μL 0.1mol/L乙酸銨(CH3COONH4)(pH 5.3)后,將混合物在45℃孵育3h.繼續(xù)加入1μL(0.002U)蛇毒磷酸二酯酶I和1μL 1M碳酸氫銨(NH4HCO3),混合物在37℃孵育2h.5μL (0.5U)堿性磷酸酶,1μL 10×孵育緩沖液和1.5μL去離子水,37℃孵育1h.最終的DNA水解物在Microcon 10kDa離心過濾裝置(Microcon YM-10; Millipore, MW cut-off 10000)上以12000×g4℃離心30min進(jìn)行純化.

1.3 HPLC-MS/MS測(cè)定

儀器分析采用島津LC20A系列HPLC系統(tǒng)(日本島津公司)(配備真空脫氣機(jī)、自動(dòng)進(jìn)樣器、四級(jí)泵和紫外檢測(cè)器)和TurbolonSpray源與API 4000Q-trap質(zhì)譜儀(美國(guó)Applied Biosystems).色譜分離使用Hypersil-HyPURITY C18反相柱(150mm× 2.1mm, 5μm 粒徑)(美國(guó)Thermo Fisher 公司),由C18保護(hù)柱(20mm×2.1mm, 5μm粒徑)(美國(guó)Phenomenex公司)保護(hù).流動(dòng)相為甲酸、甲醇和水(0.1:5:95,//),流速為0.3mL/min.樣品進(jìn)樣量為10μL.

使用電噴霧電離(ESI)三重四極桿獲得5-mdC和dC的MS/MS特征.溶解在去離子水中的20ng/mL的分析物通過注射泵注入ESI探針,流速為10μL/min,以優(yōu)化采集參數(shù).質(zhì)譜儀在ESI+MS/MS模式下運(yùn)行.優(yōu)化后參數(shù)為:噴霧電壓4000V;渦輪氣體溫度430℃;解聚電位45V;入口電壓7V;碰撞能量18V;碰撞池出口電位8V;氣體1(設(shè)置32)和氣體2(設(shè)置55);氣簾氣(設(shè)置20).5-mdC和dC的多反應(yīng)監(jiān)測(cè)(MRM)離子對(duì)分別是242.2>126.1和228.0>112.2.此外,C、dA、A、G、dG、T和U的離子對(duì)為/244.1>112.2,/252.3>135.9,/268. 1>136.3,/284.1>152.3,/268.1>152.3,/243.3>127.2,/245.3>112.9.每個(gè)離子對(duì)駐留時(shí)間為200ms.四極桿Q1和Q3分辨率被設(shè)定為單位質(zhì)量分辨率. Analyst?軟件1.3版(Applied Biosystems)用于數(shù)據(jù)采集和處理.

1.4 環(huán)境應(yīng)用

實(shí)驗(yàn)是按照經(jīng)濟(jì)合作與發(fā)展組織(OECD)的標(biāo)準(zhǔn)進(jìn)行的[23].為了更好地觀察各種參數(shù)的變化,將大型蚤暴露在NaAsO2(0–10μmol/L)中,每個(gè)濃度設(shè)置5個(gè)平行樣品(包括對(duì)照組).溫度控制在(25±1)°C,光照強(qiáng)度為(1500±100)lx,光照與黑暗時(shí)間循環(huán)為16h:8h.采用半靜態(tài)試驗(yàn)方法,用新鮮的網(wǎng)格喂養(yǎng)大型蚤,每天更換一次培養(yǎng)液.

野生型N2秀麗線蟲采用固體生長(zhǎng)培養(yǎng)基,20℃的孵化器中黑暗中培養(yǎng)(除了操作過程中)[24].將L4秀麗線蟲轉(zhuǎn)移到K培養(yǎng)基中,并分成24孔板,每孔約50個(gè)秀麗線蟲.在每個(gè)孔中加入不同濃度的NaAsO2溶液(=5).在空白對(duì)照組中加入等量K培養(yǎng)基.在暴露期間,每天重復(fù)喂毒計(jì)劃.

暴露后,使用天根基因組DNA提取試劑盒(北京,中國(guó))從大型蚤和秀麗線蟲中分離出基因組DNA.使用Nanodrop測(cè)定基因組DNA的濃度和純度,用去離子水將DNA樣品調(diào)整到500μg/mL的最終濃度,-80℃保存.

1.5 統(tǒng)計(jì)學(xué)分析

數(shù)據(jù)以平均值±標(biāo)準(zhǔn)差表示. 統(tǒng)計(jì)分析采用SPSS19,統(tǒng)計(jì)學(xué)顯著性水平設(shè)定為<0.05.

2 結(jié)果和討論

2.1 公式推導(dǎo)和測(cè)量誤差分析

全基因組DNA甲基化值被定義為5-mdC與總dC的摩爾比,通過公式(1)計(jì)算.

甲基化率= 5-mdC(mole)/(5-mdC(mole) +

dC(mole)) × 100% (1)

式中:5-mdC(mole)是5-mdC核苷的物質(zhì)的量量; dC (mole)是DNA樣品中dC核苷的物質(zhì)的量量.

根據(jù)式1提出一個(gè)變型方程,如式(2)所示,用于計(jì)算全基因組DNA甲基化率.

甲基化率=1/(1+dC(mole)/5-mdC(mole))×100% (2)

因此,只要通過HPLC-MS/MS準(zhǔn)確地確定DNA樣品中5-mdC(mole)/dC(mole)的摩爾比值,即可利用公式(2)獲得全基因組DNA甲基化值.該方法避免了對(duì)DNA樣品中5-mdC和dC含量的分別獨(dú)立測(cè)量.減少了分析誤差,提高了準(zhǔn)確性.

2.2 HPLC-MS/MS條件的優(yōu)化

合適的流動(dòng)相和固定相是實(shí)現(xiàn)正常和修飾核苷基線分離的先決條件.首先以甲醇和水(30:70,/)的混合物作為流動(dòng)相進(jìn)行分離測(cè)試, 結(jié)果顯示正常核苷和修飾核苷的保留時(shí)間很短, 且存在部分分析物的共洗脫.流動(dòng)相中水的比例增加到80%(/),盡管保留時(shí)間增加, C和U與目標(biāo)分析物共同洗脫.因此,流動(dòng)相的水溶液比例進(jìn)一步增加到95%(/),盡管所有的核苷酸都相互分離,但dC的峰不對(duì)稱.為了糾正這種情況,流動(dòng)相中加入0.1%(/)的甲酸.結(jié)果表明在0.3mL/min的流速下,所有目標(biāo)物質(zhì)都實(shí)現(xiàn)了基線分離,典型的色譜峰如圖1所示.正常和修飾核苷間實(shí)現(xiàn)基線分離,5-mdC和dC保留時(shí)間分別為5.50和3.06min(圖2).可見,在優(yōu)化條件下5-mdC、dC和其他分析物的測(cè)定不會(huì)交叉干擾.

已經(jīng)有文獻(xiàn)報(bào)道正常和修飾核苷的HPLC分離方法[25-26].其中,Song等[25]和Magana等[26]討論了DNA樣品被RNA污染時(shí)的色譜性能.Song等[25]提出的總離子色譜圖中,5-mdC和U之間的峰有重疊現(xiàn)象.Magana等[26]開發(fā)的分離方法實(shí)現(xiàn)了所有核苷酸的基線分離,但在色譜條件中使用了三種流動(dòng)相,其中一個(gè)是磷酸鹽緩沖液.

此外,流動(dòng)相的梯度很復(fù)雜,需要更多的時(shí)間實(shí)現(xiàn)柱子的重新平衡.Zhang等[20]使用2.5mmol/L甲酸銨和乙腈(:=7:93)作為流動(dòng)相.該方法線性關(guān)系好,但由于使用不易揮發(fā)的鹽溶液作為流動(dòng)相,容易堵塞LC-MS/MS管路和色譜柱.與以往報(bào)道的方法相比,本文提供的色譜分析時(shí)間短,采用含有0.1%甲酸的水和甲醇作為流動(dòng)相有利于ESI電離.本研究方法避免了RNA和其他物質(zhì)的污染,且使用方便,節(jié)省時(shí)間.

圖1 正常核苷和修飾核苷的典型總離子色譜

DNA提取過程中沒有加入RNase以消除RNA

圖2 水解基因組DNA樣品產(chǎn)生的典型MRM色譜

2.3 校準(zhǔn)曲線和靈敏度

將不同濃度的5-mdC(0.1,0.5,2.5,5,10,15,20,25, 30,35,40,45,50和55ng/mL)加入500ng/mL dC中,制備新鮮校準(zhǔn)標(biāo)準(zhǔn)溶液,5-mdC/dC的物質(zhì)的量比范圍為0.02%～10.31%.通過繪制5-mdC與dC的MRM/MS信號(hào)比和5-mdC與dC的物質(zhì)的量比來獲得校準(zhǔn)曲線.該方法的線性方程為=3.482,連續(xù)5d的測(cè)試均獲得了良好線性關(guān)系,相關(guān)系數(shù)2= 0.9998(圖3).結(jié)果表明所開發(fā)的方法在濃度范圍內(nèi)具有良好線性關(guān)系.

5-mdC和dC(/=3)的檢測(cè)限(LOD)分別為3.5和7.4pg/mL.5-mdC和dC(/=10)的定量限(LOQ)分別為14.2和19.1pg/mL.

圖3 DNA甲基化校準(zhǔn)曲線

2.4 基質(zhì)效應(yīng)評(píng)估

將兩個(gè)目標(biāo)物溶解在水和酶制劑緩沖液中以評(píng)估酶解緩沖液對(duì)5-mdC和dC MS/MS信號(hào)的影響.酶解緩沖液含有0.1mol/L CH3COONH4(pH 5.3)、1mol/L NH4HCO3、10×孵化緩沖液和水,體積比為1 : 1 : 1 : 7,5-mdC的最終濃度為0.5,5,50和100ng/mL, dC的最終濃度為5,10,50和100ng/mL.采用優(yōu)化的HPLC-MS/MS條件對(duì)其進(jìn)行分析,并通過比較加在酶解緩沖液中的分析物與溶解在水中的分析物的峰面積來評(píng)估基質(zhì)效應(yīng).酶解緩沖液對(duì)5-mdC和dC的MS/MS信號(hào)沒有明顯的基質(zhì)效應(yīng)(數(shù)據(jù)未提供).

2.5 準(zhǔn)確度和精密度

制備質(zhì)量控制(QC)樣品評(píng)估方法的準(zhǔn)確性和精密性,QC樣品5-mdC/dC物質(zhì)的量比為0.5%、1%、5%和10%.日內(nèi)精密度通過在同一天的不同時(shí)間測(cè)試QC樣品來評(píng)估(=3).日間精度通過連續(xù)五d每天3次測(cè)試質(zhì)控樣品來評(píng)估(=15).日內(nèi)精密度為98.4%～107.5%(=3),而日間精密度為97.2%～ 109.6%(表1).在所有樣品中,使用所開發(fā)的方法得到的變異系數(shù)<10%,表明使用本方法測(cè)定DNA甲基化值是可靠的.

使用兩種21堿基DNA寡核苷酸,其序列為5-ACT CAC TCA CGC AGT GAG AGA-3(7A:6C: 5G:3T)和5-A(5mC)T (5mC)A(5mC) T(5mC)A(5mC) AGT GAG AGA GAG-3(7A:6mC:5G:3T)制備校準(zhǔn)溶液,分別含有0,5,2.5,5和10%的DNA甲基化率.寡核苷酸消化后,樣品用Microcon超濾裝置(Microcon YM-10;Millipore,截留分子量10000)進(jìn)行超濾.濾液用HPLC-MS/MS進(jìn)行分析,以估計(jì)本方法的準(zhǔn)確性和精密度.日間測(cè)量的分析精度為94.0%～101.4%(=5),而日間測(cè)量的分析精度為92.0%～102.0%(=5).標(biāo)準(zhǔn)偏差<10%,表明該方法具有良好的準(zhǔn)確性(表2).

表1 HPLC-MS/MS測(cè)定流動(dòng)相中添加的5-mdC和dC百分比的準(zhǔn)確度和精密度

表2 HPLC-MS/MS測(cè)定合成寡核苷酸混合物中5-mdC和dC百分比的準(zhǔn)確性和精密度

表3 HPLC-MS/MS測(cè)定小牛胸腺DNA的甲基化值與文獻(xiàn)值比較

通過檢測(cè)小牛胸腺DNA基因組DNA甲基化值驗(yàn)證本方法.根據(jù)Zinellu等[27]方法水解小牛胸腺DNA.采用本方法測(cè)得小牛胸腺DNA基因組DNA甲基化值為(6.44±0.25)%(=5),這與其他報(bào)道的數(shù)據(jù)一致(表3).為了探索DNA量對(duì)全基因組DNA甲基化檢測(cè)的影響,評(píng)估了25,50,100,250和500ng采用小牛胸腺DNA的基因組DNA甲基化值,結(jié)果分別為(6.43±0.21)%、(6.47±0.19)%、(6.46± 0.13)%、(6.41±0.25)%和(6.49±0.27)%.可見,本方法在亞微克DNA量時(shí)可以提供準(zhǔn)確的基因組DNA甲基化值(圖4).結(jié)果表明,本方法具有良好的準(zhǔn)確性和良好的方法可替代性前景.

圖4 不同數(shù)量小牛胸腺DNA樣品中的全基因組DNA甲基化(n = 3)

2.6 生物樣品分析

大型蚤和秀麗線蟲是DNA甲基化程度低的物種.采用本方法檢測(cè)了大型蚤和秀麗線蟲的全基因組DNA甲基化值,分別為(0.097±0.010)%、(0.025± 0.002)%.與文獻(xiàn)中大型蚤(0.146±0.0126)%和秀麗線蟲(0.035±0.003)%的數(shù)據(jù)要低(～30%)[8,31].由于沒有標(biāo)準(zhǔn)物質(zhì)來確證這種差異的來源,有可能與大型蚤和秀麗線蟲的培養(yǎng)環(huán)境有關(guān).

環(huán)境中的一些有機(jī)污染物和金屬可誘發(fā)全基因組DNA甲基化.本文分析了NaAsO2對(duì)大型蚤和秀麗線蟲基因組DNA甲基化的影響(圖5).暴露于NaAsO2會(huì)誘發(fā)大型蚤和秀麗線蟲的DNA去甲基化.大型蚤的全基因組DNA甲基化值為0.071%～ 0.104%,甲基化值隨著NaAsO2濃度的增加而明顯下降.同樣,秀麗線蟲的全基因組DNA甲基化值隨著NaAsO2濃度的增加而下降,其含量為0.020%～ 0.027%,低于大型蚤的水平.總之,本研究提供了一個(gè)節(jié)省成本,易于使用且可以為低豐度DNA甲基化生物物種提供高精密度結(jié)果的方法,可以用于評(píng)估環(huán)境污染物對(duì)生態(tài)物種表觀遺傳毒性風(fēng)險(xiǎn).

3 結(jié)語

本文提出了一種用于全基因組DNA甲基化檢測(cè)的無標(biāo)記且準(zhǔn)確的HPLC-MS/MS方法.該方法通過HPLC-MS/MS檢測(cè)樣品中5-mdC(mole)/dC (mole)的物質(zhì)的量比,然后利用甲基化率公式計(jì)算出全基因組DNA甲基化值.5-mdC和dC的LOQ分別為14.2和19.1pg/mL.5-mdC和dC的日內(nèi)和日間相對(duì)標(biāo)準(zhǔn)偏差均小于10%.通過檢測(cè)大型蚤和秀麗線蟲的全基因組DNA甲基化值,驗(yàn)證了本方法的可行性.

[1] Schulz H, Ruppert A K, Herms S, et al. Genome-wide mapping of genetic determinants influencing DNA methylation and gene expression in human hippocampus [J]. Nature Communications, 2017, 8:1511–1521.

[2] Hu J, Yang Y, Lv X, et al. Dichlorodiphenyltrichloroethane metabolites inhibit DNMT1activity which confers methylation-specific modulation of the sex determination pathway [J]. Environmental Pollution, 2021,279:116828.

[3] Yang Z, Qi W, Sun L, et al. DNA methylation analysis of selected genes for the detection of early-stage lung cancer using circulating cell-free DNA [J]. Advances in Clinical and Experimental Medicine, 2019,28:355–360.

[4] 周新文,朱國(guó)念,Jilisa Mwalilino, et al. Cu、Zn、Pb、Cd及其混合重金屬離子對(duì)鯽魚()DNA甲基化水平的影響[J]. 中國(guó)環(huán)境科學(xué), 2001,21(6):549–552.

Zhou Xinwen, Zhu guonian, Jilisa Mwalilino, et al. Influence of Cu、Zn、Pb、Cd and their heavy metalion mixture on the DNA methylation level of fish () [J]., 2001,21(6):549-552.

[5] 吳思寒,吳雨濛,王雙杰,等.全氟辛烷磺酰胺在小麥和蚯蚓中的富集與轉(zhuǎn)化[J]. 中國(guó)環(huán)境科學(xué), 2021,41(5):2434–2440.

Wu S H, Wu Y M, Wanf S J, et al. Bioaccumulation and biotransformation of perfluorooctane sulfonamide in wheat and earthworms [J]. China Environmental Science, 2021,41(5):2434–2440.

[6] Hu J, Liu J, Li J, et al. Metal contamination, bioaccumulation, ROS generation, and epigenotoxicity influences on zebrafish exposed to river water polluted by mining activities [J]. Journal of Hazardous Materials, 2021,405:124150.

[7] De Paoli-Iseppi R, Deagle B E, Polanowski A M, et al. Age estimation in a long-lived seabird (Ardenna tenuirostris) using DNA methylation-based biomarkers [J]. Molecular Ecology Resources, 2019,19:411–425.

[8] Hearn J, Plenderleith F, Little TJ. DNA methylation differs extensively between strains of the same geographical origin and changes with age in Daphnia magna [J]. Epigenetics Chromatin, 2021,14:4.

[9] Rastegar L, Mighani H, Ghassempour A. A comparison and column selection of hydrophilic interaction liquid chromatography and reversed-phase high-performance liquid chromatography for detection of DNA methylation [J]. Analytical Biochemistry, 2018,557:123–130.

[10] Tang Y, Gao X D, Wang Y, et al. Widespread existence of cytosine methylation in yeast DNA measured by gas chromatography/mass spectrometry [J]. Analytical Chemistry, 2012,84:7249.

[11] Nakagawa T, Wakui M, Hayashida T, et al. Intensive optimization and evaluation of global DNA methylation quantification using LC- MS/MS [J]. Analytical and Bioanalytical Chemistry, 2019,411:7221– 7231.

[12] Hu C W, Lee H, Chen J L, et al. Optimization of global DNA methylation measurement by LC-MS/MS and its application in lung cancer patients [J]. Analytical and Bioanalytical Chemistry, 2013, 405:8859–8869.

[13] Zinellu A, Sotgiu E, Sotgia S, et al. A capillary electrophoresis UV detection-based method for global genomic DNA methylation Assessment in human whole blood [J]. Methods in Molecular Biology, 2019,1972:213–219.

[14] Zinellu A, Sotgia S, De Murtas V, et al. Evaluation of methylation degree from formalin-fixed paraffin-embedded DNA extract by field- amplified sample injection capillary electrophoresis with UV detection [J]. Analytical and Bioanalytical Chemistry, 2011,399:1181–1186.

[15] Bhattacharjee R, Moriam S, Nguyen NT, et al. A bisulfite treatment and PCR-free global DNA methylation detection method using electrochemical enzymatic signal engagement [J]. Biosensors and Bioelectronics, 2019,126:102–107.

[16] Childebayeva A, Jones TR, Goodrich JM, et al. LINE-1and EPAS1DNA methylation associations with high-altitude exposure [J]. Epigenetics, 2019,14(1):1–15.

[17] Luttropp K, Sj?holm L K, Ekstr?m T J. Global analysis of DNA 5-methylcytosine using the luminometric methylation assay, LUMA [J]. Methods in Molecular Biology, 2015,1315:209.

[18] Chowdhury B, Cho I H, Irudayaraj J. Technical advances in global DNA methylation analysis in human cancers [J]. Journal of Biological Engineering, 2017,11:10.

[19] Hu J J, Zhang W B, Ma H M, et al. Simultaneous determination of 8-hydroxy-2'-deoxyguanosine and 5-methyl-2'-deoxycytidine in DNA sample by high performance liquid chromatography/positive electrospray ionization tandem mass spectrometry [J]. Journal of Chromatography B, 2010,878:2765–2769.

[20] Zhang J J, Zhang L, Zhou K, et al. Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry [J]. Analytical Biochemistry, 2011,413:164–170.

[21] Hu J, Liu J, Lv X, et al. Assessment of epigenotoxic profiles of Dongjiang River: A comprehensive of chemical analysis, in vitro bioassay and in silico approach [J]. Environmental Pollution, 2021,282: 116961.

[22] Sun Y, Stransky S, Aguilan J, et al. High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry [J]. Analytica Chimica Acta, 2021,1180:338880.

[23] 楊曉凡,陸光華,劉建超,等.環(huán)境相關(guān)濃度下的藥物對(duì)大型蚤的多代慢性毒性[J]. 中國(guó)環(huán)境科學(xué), 2013,33(3):538–545.

Yang X F, Lu G H, Liu J C, et al. Multigenerational chronic effects of pharmaceuticals onat environmentally relevant concentrations [J]. China Environmental Science, 2013,33(3):538-545.

[24] 張緒超,陳 懿,胡 蝶,等.生物炭中持久性自由基對(duì)秀麗隱桿線蟲的毒性[J]. 中國(guó)環(huán)境科學(xué), 2019,39(6):2644–2651.

Chen Y, Hu D, Zhao L, et al. Neurotoxic effect of environmental persistent free radicals in rice biochar to Caenorhabditis elegans [J]. China Environmental Science, 2019,39(6):2644–2651.

[25] Song K, James S R, Kazim L, et al. Specific method for the determination of genomic DNA methylation by liquid chromatography-electrospray ionization tandem mass spectrometry [J]. Analytical Chemistry, 2005,77:504–510.

[26] Maga?a A A, Wrobel K, Caudillo Y A, et al. High-performance liquid chromatography determination of 5-methyl-2'-deoxycytidine, 2'- deoxycytidine, and other deoxynucleosides and nucleosides in DNA digest [J]. Analytical Biochemistry, 2008,374:378–385.

[27] Zinellu A, Sotgiu E, Assaretti S, et al. Evaluation of global genomic DNA methylation in human whole blood by capillary electrophoresis UV detection [J]. Journal of Analytical Methods in Chemistry, 2017,2017:4065892.

[28] Kok R M, Smith D E C, Barto R, et al. Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects [J]. Clinical Chemistry and Laboratory Medicine, 2007,45: 903–911.

[29] Rocha M S, Castro R, Rivera I, et al. Global DNA methylation: comparison of enzymatic- and non-enzymatic-based methods [J]. Clinical Chemistry and Laboratory Medicine, 2010,48:1793–1798.

[30] Jatinderpal S, Balvinder K, Christine A, et al. Determination of 5-methyl-2’-deoxycytidine in genomic DNA using high performance liquid chromatography-ultraviolet detection [J]. Journal of Chromatography B, 2009,877:1957–1961.

[31] Thaulow J, Song Y, Lindeman LC, et al. Epigenetic, transcriptional and phenotypic responses in Daphnia magna exposed to low-level ionizing radiation [J]. Environmental Research, 2020,190:109930.

HPLC-MS-MS method for low level DNA methylation value determination in ecological species.

HU Jun-jie1, WU Yi-chong1, CHEN Gui-lian1, GAO Yuan-yuan2, ZHAO Tong3, YU Guang1, LAN Shan-hong1, Lü Xiao-mei1*

(1.School of Environment and Civil Engineering, Dongguan University of Technology, Dongguan 523808, China；2.Environmental Technology Center of Dongguan, Dongguan 523009, China；3.College of Resources and Environment, Yangtzeu University, Wuhan 434023, China)., 2022,42(7)：3385～3391

DNA methylation patterns which associated with normal tissue development and disease initiation could be affected by environmental contaminants and related toxicity. For the requirement of quantification low abundant of 5-methyl-2’-deoxycytidine (5-mdC) in the genome of ecologically relevant species, a label-free and accurate method using high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was developed and validated. The global DNA methylation calculation formula of 5-mdC(mole)/(5-mdC(mole)+dC(mole)) could be transformed as 1/(1+dC(mole)/5-mdC(mole)), then the molar ratio of 5-mdC and dC (dC(mole)/5-mdC(mole)) in DNA samples was obtained by HPLC-MS/MS. The important features of the provided method are as follows: (1) avoiding the usage of expensive stable isotope labeled internal standards; (2) isocratic separating normal and modified nucleosides and eliminating cross-interferences of analytes; (3) providing data with high accuracy even detecting low levels of methylated DNA samples. The limits of quantification for 5-mdC and dC were 40 and 60pg/mL, respectively. The intra-day and inter-day relative standard deviations (￡10%) and accuracy (relative errors) were satisfactory. The results obtained from the analysis of calf thymus DNA and genomic DNA from Daphnia magna and elegans demonstrated the feasibility of the new developed method in evaluation of the potential epigenetic risk of environmental pollutions on ecologically relevant species.

global DNA methylation；label-free；electrospray tandem mass spectrometry；epigenetic risk；ecological species

X172

A

1000-6923(2022)07-3385-07

胡俊杰(1984-),男,湖北武漢人,特聘副教授,博士,主要從事環(huán)境毒理學(xué)和環(huán)境監(jiān)測(cè)方面研究.發(fā)表論文10余篇.

2022-12-20

廣東省自然科學(xué)基金資助項(xiàng)目(2019A1515110750);東莞市社會(huì)科技發(fā)展重點(diǎn)項(xiàng)目(20211800905532,20211800904652,2020507140150)

* 責(zé)任作者, 講師, 2017158@dgut.edu.cn