液相色谱-串联质谱分析组织中基因组脱氧核糖核酸羟甲基胞嘧啶水平

5-羟甲基胞嘧啶通过阻止脱氧核糖核酸(DNA)甲基化转移酶1(DMNT1)甲基化胞嘧啶来影响DNA甲基化的程度。本文建立了液相色谱-串联质谱(LC-MS/MS)测定组织中全基因组5-羟甲基胞嘧啶水平的方法。采用苯酚-氯仿提取组织DNA,提取的DNA用88%甲酸在140 ℃下裂解,DNA裂解液加入同位素胞嘧啶作内标,经N2吹干后,加乙腈-水(9:1, v/v)溶解,用LC-MS/MS检测5-羟甲基胞嘧啶的含量,并计算全基因组中5-羟甲基胞嘧啶的水平。结果表明,5-羟甲基胞嘧啶的线性范围为0.1～30 ng/mL,相关系数为0.9969,检出限(信噪比为3计)和定量限(信噪比为10计)分别为0.057 ng/mL和0.090 ng/mL;日内相对标准偏差和日间相对标准偏差分别为5.13%和6.24%;加标回收率为90.24%～97.53%。用该方法检测了大鼠大脑组织DNA羟甲基化水平,平均结果为0.66%。该方法简便,重现性好,灵敏度较高,能满足全基因组5-羟甲基胞嘧啶定量检测的要求。     

液相色谱-串联质谱法测定生物样本全基因组DNA甲基化

建立了基于液相色谱-电喷雾串联质谱的分析方法,对生物样本中全基因组DNA甲基化水平进行定量测定.首先将DNA从生物样本中提取出来,将DNA片段酶解为单核苷,利用液相色谱-串联质谱测定胞嘧啶核苷和5-甲基胞嘧啶核苷的含量,从而计算出其全基因组DNA甲基化率.利用该法研究了暴露于全氟辛烷磺酸的L-02细胞、10例原发性肝癌病例血浆样本和10例对照血浆样本的全基因组DNA甲基化水平,得出了它们的总甲基化率变化的初步结果.本方法操作简单,具有很高的灵敏度和稳定性,为研究生物样本,尤其是临床上易得但DNA含量极低的血浆样本的总甲基化水平提供了思路.     

白菜DNA甲基化水平的反相离子对高效液相色谱法研究

建立了反相离子对液相色谱测定白菜的DNA甲基化水平的方法。采用的色谱柱为HypersilBDSC18柱(200mm×4.0mm,5μm),以pH4.0的甲醇-5mmol·L-1庚烷磺酸钠-三乙胺(10:90:0.2,体积比)的混合液为流动相,UV检测器波长为273nm。通过测定DNA水样所产生的胞嘧啶和甲基胞嘧啶的含量,即可得知DNA甲基化程度。胞嘧啶和甲基胞嘧啶回收率分别为99.6%及103.3%,RSD为3.7%(日内)及5.2%(日间)。     

DNA胞嘧啶的甲基化与去甲基化进展

DNA胞嘧啶甲基化调控基因的表达以及多种生物功能,如细胞分化.概念上,DNA去甲基化是将已甲基化DNA核苷转化为未修饰核苷,是DNA甲基化的逆向过程.但在生物体内,这是一个涉及多步反应的非常复杂的过程.本综述简要介绍了动植物中DNA胞嘧啶的甲基化与去甲基化的研究现状,并讨论了恶性肿瘤(癌症)、阿尔茨海默氏病、心血管疾病...     

老鼠Tet1蛋白能氧化单链脱氧核糖核酸上的5-甲基胞嘧啶到5-羟甲基胞嘧啶和5-羧基胞嘧啶(英文)

5-甲基胞嘧啶在哺乳动物细胞中具有广泛的作用.而它被双脱氧家族Tet蛋白氧化所得的产物5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶也被证明在细胞发育和5-甲基胞嘧啶动态平衡调控中具有关键的作用.已有的研究结果表明,Tet蛋白能够识别双链DNA上的5-甲基胞嘧啶,并将其氧化成5-羟甲基胞嘧啶、5-醛基胞嘧啶和5-羧基胞嘧啶.我们通过质谱仪检测发现,老鼠Tet1蛋白的DNA结合结构域还能识别和氧化单链DNA上的5-甲基胞嘧啶.这一发现暗示我们,Tet蛋白家族不但具有已经发现的氧化双链DNA上5-甲基胞嘧啶的功能,还有可能作用于DNA的复制及转录,甚至具有氧化单链RNA上对应的甲基修饰碱基的能力.     

亲水作用色谱法测定组织中全基因组DNA甲基化水平

建立了亲水作用色谱(HILIC)测定组织中全基因组DNA甲基化水平的方法。采用苯酚-氯仿提取组织中的DNA,提取的DNA用88%甲酸在140 ℃下裂解,经N2吹干后,加乙腈-水(9:1, v/v)溶解,用Waters BEH HILIC柱进行分离,在277 nm波长下检测胞嘧啶(Cyt)及5-甲基胞嘧啶(5-mCyt)含量。结果表明,以乙腈-10 mmol/L甲酸铵溶液(94:6, v/v)为流动相,流速为0.5 mL/min, Cyt与5-mCyt分离较好,保留时间分别为2.6与3.1 min。胞嘧啶的线性范围为1～900 μmol/L,相关系数为0.9999; 5-甲基胞嘧啶的线性范围为1～64 μmol/L,相关系数为0.9998。胞嘧啶和5-甲基胞嘧啶的检出限为54 nmol/L(柱中为0.54 pmol),定量限为250 nmol/L(柱中为2.5 pmol);在5～900 μmol/L的添加水平下,胞嘧啶和5-甲基胞嘧啶的平均加标回收率为94.7%～100.5%,相对标准偏差小于1.48%。用该方法检测了结肠癌组织中DNA甲基化水平,结果显示该癌组织中全基因组的DNA甲基化均值为4.0%。该方法快速、简单,稳定性好,灵敏度较高,能满足全基因组DNA甲基化的检测要求。     

DNA甲基化检测研究进展

DNA甲基化可以在不改变DNA碱基序列的情况下改变基因活动和功能,影响基因印迹、细胞分化、细胞增殖、染色质重塑、胚胎发育等重要生命活动,是表观遗传的研究重点和热点。为了研究DNA甲基化的分布、状态以及调控机制,越来越多的DNA甲基化分析和检测技术已被开发。本文归纳了近年来的DNA甲基化检测方法,讨论了不同方法的优缺点,为未来深入研究DNA甲基化提供参考。     

表观遗传修饰——5-羟甲基胞嘧啶检测的研究进展

表观遗传修饰是指在不改变DNA序列的情况下,基因表达发生的可遗传变化.5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)是继5-甲基胞嘧啶(5-methylcytosine, 5mC)后发现的表观遗传修饰,被称为"第六种碱基". 5hmC广泛分布于哺乳动物的组织和细胞中,它的异常表达与肿瘤发生、发育性疾病和神经系统疾病密切相关.由于5hm C的结构与5m C相似,并且其丰度远远低于5m C,传统方法难以实现对5hm C的准确和灵敏检测.近年来,科学家们结合新的修饰方法和信号放大策略,发展了一系列超灵敏检测5hm C的新方法,包括液相色谱串联质谱法、荧光法、电化学法、光电化学法、单碱基分辨率的测序法和单分子检测技术.这些方法各自具有其独特优势,有力推动了表观遗传学的发展.本综述总结了5hmC检测方法的最新研究进展,并对其面临的挑战和发展趋势做了展望.     

人体结肠癌组织样品中基因组DNA甲基化水平的HPLC—ESI MS／MS检测

利用多重监测反应模式(MRM),通过对液相色谱及质谱条件的优化,建立了人体结肠癌组织样品中基因组DNA甲基化的高效液相色谱-电喷雾质谱(HPLC-ESI MS/MS)检测方法,对结肠癌组织及癌旁组织中基因组DNA甲基化进行了检测.结果表明,5-甲基脱氧胞苷(5mdC)的检出限是1 pg,线性范围为0.037 5～0.5 mg/L,工作曲线相关系数大于0.999 0,该方法的相对标准偏差为2.32%～9.21%.结肠癌病人样品检测结果表明,结肠癌组织基因组DNA甲基化水平低于相应的癌旁组织(P<0.05).     

基于高效液相色谱的喹啉类药物对刺参DNA甲基化水平的影响研究

建立了高效液相色谱/紫外法测定刺参组织全基因组DNA甲基化水平的方法,并运用此方法对喹噁啉药物处理过的刺参样品DNA甲基化水平进行分析。色谱条件为:色谱柱为ZORBAX SB-Aq(4.6 mm×250mm;5μm);柱温为30℃;检测波长为280 nm;进样量20μL;流动相为甲醇-7 mmol/L乙酸铵(7∶93),流速为1.0 mL/min。5-甲基脱氧胞苷和脱氧胞苷的质量浓度在0.05~1.0 mg/L范围内时,线性关系良好,相关系数(r)均为0.999 9。5-甲基脱氧胞苷和脱氧胞苷的检出限均为0.05 mg/L,在加标浓度为0.05,0.25,1.0 mg/L时,回收率为90.4%~100.6%,批内、批间相对标准偏差均小于4%。采用CTAB法提取刺参组织DNA,酶解后进行HPLC测定,结果表明:喹噁啉药物处理过的组织样品DNA甲基化水平低于对照组,该类药物通过改变DNA甲基化水平而影响基因的正常表达,可能是其产生遗传毒性的一种机制,建立的方法可以很好地用于全基因组DNA总甲基化水平的检测。     

Detection of 5-methylcytosine and 5-hydroxymethylcytosine in DNA via host–guest interactions inside α-hemolysin nanopores

Cytosine methylation and hydroxymethylation are both important epigenetic modifications of DNA in mammalian cells. Therefore, profiling DNA (hydroxy)methylation across the genome is vital for understanding their roles in gene regulation. Here, we report a nanopore-based approach for quick and reliable detection of 5-methylcytosine and 5-hydroxymethylcytosine in DNA at the single-molecule level. The single-stranded DNA containing 5-methylcytosine or 5-hydroxymethylcytosine was first selectively modified on the epigenetic base to attach a host–guest complex. Threading of the modified DNA molecules through α-hemolysin nanopores causes unbinding of the host–guest complex and generates highly characteristic current signatures. Statistical analysis of the signature events affords quantitative information about 5-methylcytosine and 5-hydroxymethylcytosine in DNA. Our results suggest that other DNA modifications could also be detected with the developed method. Furthermore, we anticipate our nanopore sensing strategy to be generally useful in biochemical analysis and to find applications in the early diagnosis of diseases.     

Determination of DNA Methylation and Hydroxymethylation Levels in Biological Samples by Field-Amplified Sample Injection-Capillary Zone Electrophoresis with UV Detection

In this study, a method of field-amplified sample injection coupled with capillary zone electrophoresis with ultraviolet detection was established for evaluation of DNA methylation and hydroxymethylation levels in biological materials. By modifying an existing method, the separation of cytosine (C), 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) was performed on an uncoated capillary column (40 cm × 75 μm I.D.) using 300 mmol L⁻¹ tris solution (pH 2.90) as running buffer and detected at 280 nm. The detection limits (S/N = 3) of the method were 0.004 ng mL⁻¹ for cytosine (C), 0.01 ng mL⁻¹ for 5-methylcytosine (5-mC), and 0.02 ng mL⁻¹ for 5-hydroxymethylcytosine (5-hmC). The proposed method has been successfully applied to the evaluation of DNA methylation and hydroxymethylation levels of blood samples from 15 hepatocellular carcinoma patients and 5 liver cirrhosis patients and liver tissues from 50 pairs of tumor and matched tumor-adjacent samples.

     

Quantitation of 8-hydroxydeoxyguanosine in DNA by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry

A methodology has been developed and validated for quantifying 8-hydroxydeoxyguanosine (8-OHdG) in both commercial DNA and DNA isolated from livers of male Sprague-Dawley rats by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry. The analytical method conditions, including conditions for stabilizing 8-OHdG during complex nuclease P1 enzymatic digestion, were also evaluated. The limit of detection for 8-OHdG was 1.0 ng/mL (17.6 fmol on-column), and the linearity of the calibration curve was greater than 0.998 from 1.0 to 500 ng/mL. The intraday assay precision relative standard deviation (RSD) value for quality control (QC) samples was < or =5.59% with accuracies ranging from 91.84 to 117.61%. The interday assay precision (RSD) value was < or =1.76% with accuracies ranging from 91.84 to 116.67%. This method, combined with the LC/UV analysis of deoxyguanosine (dG), was used for determination of the levels of 8-OHdG/10(6) dG in DNA nuclease P1 enzymatic hydrolysates from both commercial DNA and rat liver DNA.     

Rapid and sensitive quantification of urinary N7-methylguanine by isotope-dilution liquid chromatography/electrospray ionization tandem mass spectrometry with on-line solid-phase extraction

A rapid, highly specific and sensitive isotope-dilution liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with an on-line solid-phase extraction (SPE) system was developed to measure N7-methylguanine (N7-MeG) in urine. 15N5-Labeled N7-MeG was synthesized to serve as an internal standard, and an on-line SPE cartridge was used for on-line sample cleanup and enrichment. The urine sample can be directly analyzed within 15 min without prior sample purification. The detection limit for this method was estimated as 8.0 pg/mL (4.8 pmol) on-column. This method was further applied to study exposure to methylating agents arising from cigarette smoke. Sixty-seven volunteers were recruited, including 32 regular smokers and 35 nonsmokers. Urinary cotinine, a major metabolite of nicotine, was also determined using an isotope-dilution LC/MS/MS method. The results showed that urinary levels of N7-MeG observed in smokers (4215 +/- 1739 ng/mg creatinine) were significantly (P < 0.01) higher than those in nonsmokers (3035 +/- 720 ng/mg creatinine). It was further noted that the urinary level of N7-MeG was found to be correlated with that of cotinine for smokers, implying that cigarette smoking resulted in increased DNA methylation, followed by depurination and excretion of N7-MeG in urine. As a result of the on-line extraction system, this method is capable of routine high-throughput analysis and accurate quantitation of N7-MeG, and could be a useful tool for health surveillance of methylating agent exposure.     

On-line solid-phase extraction LC-MS/MS for the determination of Ac-SDKP peptide in human plasma from hemodialysis patients

We developed a high-throughput method based on on-line solid-phase extraction liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) to determine N-terminal thymosin-β fragment peptide (N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP) in human plasma samples. Quantification of Ac-SDKP was performed using direct injection for on-line SPE based on C(18), reversed-phase LC separation and stable isotope dilution electrospray ionization-MS/MS in multiple reaction-monitoring (MRM) mode. The Ac-SDKP-(13)C(6), (15)N(2) (m/z 496 → 137) was synthesized for the internal standard. The MRM ion for Ac-SDKP was m/z 488 → 129 (quantitative ion)/226. The limit of detection and lower limit of quantitation were 0.05 and 0.1 ng/mL in standard solution, respectively. Recovery values were 98.3-100.4% with inter-day (relative standard deviation, RSD, 0.4-14.1%) and intra-day (RSD, 0.8-19.7%) assays. This method was applied to the measurement of Ac-SDKP levels in plasma from hemodialyzed subjects. Concentrations were 0.59 ± 0.23 ng/mL (pre-hemodialyzed subjects, n = 9) and 0.44 ± 0.19 ng/mL (post-hemodialyzed subjects, n = 9). All plasma Ac-SDKP levels were decreased by dialysis. Thus, plasma Ac-SDKP was decreased through dialysis in chronic kidney disease. The findings in this study will be useful for the treatment of anemia in chronic kidney disease with dialysis.     

高效液相色谱-电喷雾离子阱串联质谱法检测芥子气经皮肤染毒SD大鼠肺中的DNA加合物

运用高效液相色谱-电喷雾离子阱串联质谱(HPLC-ESI-MS/MS)技术,建立了快速、简单、灵敏的SD大鼠肺中N7-(2-羟乙基硫代乙基)鸟嘌呤(N7-HETEG)的检测方法。以N7-苯甲基鸟嘌呤为内标,用甲醇和水为流动相进行梯度洗脱,正离子模式检测,方法的检出限(信噪比(S/N)≥10)为300 pg/mL,定量限(S/N≥20)为850 pg/mL。在300 pg/mL～1.28 μg/mL的质量浓度范围内,N7-HETEG浓度与N7-HETEG和内标的峰面积比呈良好的线性关系(线性相关系数为0.9929)。高、中、低3个添加水平的日内测定精密度(以相对标准偏差(RSD)计)和日间测定精密度均小于10%(n=7),回收率为100%～132%。对SD大鼠背部皮肤染芥子气,剂量分别为5.5、11、22和45 mg/kg,染毒4 d后检测大鼠肺脏中N7-HETEG的含量。各个不同染毒剂量下,每克组织中分别检测到（0.56±0.16）、（0.67±0.12）、（1.36±0.68）和(5.14±0.92) ng N7-HETEG, N7-HETEG的含量随着染毒剂量的增大而增大,表明N7-HETEG可用作芥子气暴露的体内生物标志物。     

Simultaneous determination of trimethylamine and trimethylamine N‐oxide in mouse plasma samples by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry

A method was developed that applies hydrophilic interaction liquid chromatography with tandem mass spectrometry in the multiple reaction monitoring mode to separate and accurately quantify trimethylamine and trimethylamine N‐oxide in a single chromatographic run. This was achieved by converting trimethylamine to ethyl betaine, which is less volatile and hence results in greatly improved quantitation. Ethyl betaine also gives a similar response to trimethylamine N‐oxide using positive‐ion electrospray ionization mass spectrometry. It is readily separated from trimethylamine N‐oxide by hydrophilic liquid chromatography in a 5 min run and with improved peak shape compared to underivatized trimethylamine. Validation of the method yielded a limit of detection (S/N ≥ 3) of 0.5 ng/mL for trimethylamine and 0.25 ng/mL for trimethylamine N‐oxide. Method accuracies of 91.4–105.3% with precisions of 0.4–5.5% were obtained for standard mixtures over the range of 2.5–500 ng/mL. Recoveries measured for the extraction of trimethylamine and trimethylamine N‐oxide spikes into mouse plasma were both >90%. The method, which simultaneously measures trimethylamine and trimethylamine N‐oxide, was successfully applied to mouse plasma samples and could be adapted for use with other biological fluids.     

Enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma and urine by liquid chromatography/tandem mass spectrometry

A sensitive method using enantiospecific liquid chromatography/tandem mass spectrometry detection for the quantitation of S- and R-mephenytoin as well as its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma and urine has been developed and validated. Plasma samples were prepared by protein precipitation with acetonitrile, while urine samples were diluted twice with the mobile phase before injection. The analytes were then separated on a chiral alpha(1)-acid glycoprotein (AGP) column and thereafter detected, using electrospray ionization tandem mass spectrometry. In plasma, the lower limit of quantification (LLOQ) was 1 ng/mL for S- and R-4'-hydroxymephenytoin and S-nirvanol and 3 ng/mL for R-nirvanol and S- and R-mephenytoin. In urine, the LLOQ was 3 ng/mL for all compounds. Resulting plasma and urine intra-day precision values (CV) were <12.4% and <6.4%, respectively, while plasma and urine accuracy values were 87.2-108.3% and 98.9-104.8% of the nominal values, respectively. The method was validated for plasma in the concentration ranges 1-500 ng/mL for S- and R-4'-hydroxymephenytoin, 1-1000 ng/mL for S-nirvanol, and 3-1500 ng/mL for R-nirvanol and S- and R-mephenytoin. The validated concentration range in urine was 3-5000 ng/mL for all compounds. By using this method, the metabolic activities of two human drug-metabolizing enzymes, cytochrome P450 (CYP) 2C19 and CYP2B6, were simultaneously characterized.