生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
4期
537-541
,共5页
张航%胡俊杰%汤瑞华%叶金波%任晓虎%胡韬%黄培武%杨细飞%黄海燕%刘建军
張航%鬍俊傑%湯瑞華%葉金波%任曉虎%鬍韜%黃培武%楊細飛%黃海燕%劉建軍
장항%호준걸%탕서화%협금파%임효호%호도%황배무%양세비%황해연%류건군
高效液相色谱%串联质谱%DNA甲基化
高效液相色譜%串聯質譜%DNA甲基化
고효액상색보%천련질보%DNA갑기화
high performance liquid chromatography%tandem mass spectrometry%DNA methylation
目的：建立高效液相色谱-串联质谱（HPLC-ESI-MS/MS）检测基因组DNA甲基化水平的方法。方法：以5-mdC和dG为标准品，采用全自动高效液相色谱系统进行分离，串联电喷雾质谱检测，选择多反应监测模式（MRM）测定标准品，绘制标准工作曲线。结果：在MRM模式下选取5-mdC（m/z 241.9→126.3）和dG（m/z 268.1→152.3）分别作为定量检测的母子离子对，各化合物能实现良好的基线分离；5-mdC和dG碰撞能均为15 eV，去簇电压分别为40和45 V，最低定量限分别为1.65和2.47 fmol；标准品的响应值比为90%~110%；5-mdC含量的天内相对标准偏差和天间相对标准偏差均小于8%。结论：HPLC-ESI-MS/MS是能应用于检测基因组DNA甲基化的一种高通量、高准确率、高分辨率、高灵敏度且重复性好的方法。
目的：建立高效液相色譜-串聯質譜（HPLC-ESI-MS/MS）檢測基因組DNA甲基化水平的方法。方法：以5-mdC和dG為標準品，採用全自動高效液相色譜繫統進行分離，串聯電噴霧質譜檢測，選擇多反應鑑測模式（MRM）測定標準品，繪製標準工作麯線。結果：在MRM模式下選取5-mdC（m/z 241.9→126.3）和dG（m/z 268.1→152.3）分彆作為定量檢測的母子離子對，各化閤物能實現良好的基線分離；5-mdC和dG踫撞能均為15 eV，去簇電壓分彆為40和45 V，最低定量限分彆為1.65和2.47 fmol；標準品的響應值比為90%~110%；5-mdC含量的天內相對標準偏差和天間相對標準偏差均小于8%。結論：HPLC-ESI-MS/MS是能應用于檢測基因組DNA甲基化的一種高通量、高準確率、高分辨率、高靈敏度且重複性好的方法。
목적：건립고효액상색보-천련질보（HPLC-ESI-MS/MS）검측기인조DNA갑기화수평적방법。방법：이5-mdC화dG위표준품，채용전자동고효액상색보계통진행분리，천련전분무질보검측，선택다반응감측모식（MRM）측정표준품，회제표준공작곡선。결과：재MRM모식하선취5-mdC（m/z 241.9→126.3）화dG（m/z 268.1→152.3）분별작위정량검측적모자리자대，각화합물능실현량호적기선분리；5-mdC화dG팽당능균위15 eV，거족전압분별위40화45 V，최저정량한분별위1.65화2.47 fmol；표준품적향응치비위90%~110%；5-mdC함량적천내상대표준편차화천간상대표준편차균소우8%。결론：HPLC-ESI-MS/MS시능응용우검측기인조DNA갑기화적일충고통량、고준학솔、고분변솔、고령민도차중복성호적방법。
Objective: To estabilish high performance liquid chromatography(HPLC) coupled with tandem mass spectrometry(HPLC-ESI-MS/MS) for genome-wide DNA methylation detection. Methods: 5-mdC and dG were used as the standard. HPLC system and electronic spray were used to separate and detect the standard at multi-ple reaction monitoring(MRM) mode; then the standard working curve was generated by the data obtained. Re-sults: The 5-mdC(m/z 241.9→126.3) and dG(m/z 268.1→152.3) were chosen as parent and child ion pairs for quantitative detection at MRM mode. A good baseline separation can be achieved for the compounds. The collision energy of both 5-mdC and dG was 15 eV, and the cluster voltage was 40 and 45 V, respectively. The DNA meth-ylation rate of 5-mdC and dG was 1% under the minimum quantitative limit. The limit of quantitation of 5-mdC and dG was 1.65 and 2.47 fmol, respectively. The response values of 5-mdC and dG standards range between 90% and 110%. The intra- and inter-day precisions(RSD) of 5-mdC were less than 8%. Conclusion: HPLC-ESI-MS/MS with high-throughput, high sensitivity, high accuracy and good repeatability can be used to measure DNA methylation levels.
