Abbas-Hawks C, Voorhees K J, Hadfield T L
Department of Chemistry, Colorado School of Mines, Golden 80401-1887, USA.
Rapid Commun Mass Spectrom. 1996;10(14):1802-6. doi: 10.1002/(SICI)1097-0231(199611)10:14<1802::AID-RCM761>3.0.CO;2-Y.
Curie-point pyrolysis/triple quadrupole mass spectrometry (Py/MS/MS) has been used with tetramethylammonium hydroxide (TMAH) to conduct in situ methylation of nucleic acid bases. Nitrogen bases in free nucleotides, oligonucleotides, calf thymus DNA and whole bacterial cells reacted in situ during pyrolysis with TMAH to form the methylated bases. Derivatization increased the volatility of the nitrogen bases and the mass of the diagnostic base peaks, thereby removing them from the positions of lower-mass background peaks. The degree of methylation as a function of TMAH concentration for the oligonucleotide, calf thymus DNA, and the whole bacteria samples was determined and found to correlate with the nature of DNA. The methylated bases were identified by their positive-ion electron ionization fragmentation patterns and confirmed with tandem mass spectrometry. The detection of the methylated bases by Py/MS/MS facilitates the goal of identifying the nucleic acids in a complex mixture (i.e. whole bacterial cells) without extraction and prior derivatization.
居里点热解/三重四极杆质谱法（Py/MS/MS）已与氢氧化四甲铵（TMAH）联用，用于对核酸碱基进行原位甲基化。游离核苷酸、寡核苷酸、小牛胸腺DNA和全细菌细胞中的含氮碱基在热解过程中与TMAH发生原位反应，形成甲基化碱基。衍生化增加了含氮碱基的挥发性和诊断性碱基峰的质量，从而将它们从低质量背景峰的位置移除。确定了寡核苷酸、小牛胸腺DNA和全细菌样品中甲基化程度与TMAH浓度的函数关系，发现其与DNA的性质相关。通过正离子电子电离碎裂模式鉴定甲基化碱基，并用串联质谱法进行确认。通过Py/MS/MS检测甲基化碱基有助于在不进行提取和预先衍生化的情况下，鉴定复杂混合物（即全细菌细胞）中的核酸这一目标的实现。
