Method for high performance liquid chromatography(HPLC)-mass spectrometry (MS) detection of DNA oxidation and DNA methylation

Abstract

The invention discloses a method for high performance liquid chromatography (HPLC)-mass spectrometry (MS) detection of DNA oxidation and DNA methylation, and the method comprises the following steps: hydrolyzing DNA, measuring the content of 5-methyl cytosine (5-mdC) and 8-hydroxy deoxyguanosine (8-OHdG) in the DNA hydrolysis product by HPLC-ESI (electrospray ionization)-MS / MS; wherein the high performance liquid chromatography is performed as follows: a mobile phase A is formic acid / methanol with the volume ratio of 0.01-0.3, a mobile phase B is a formic acid water solution with the volume ratio of 0.01 to 0.3%, and under the conditions of the flow velocity of 0.2-0.4ml / min and the column temperature of 15 to 40 DEG C, separation is performed by a liquid chromatography column; ESI (electrospray ionization)-MS is performed as follows: an electrospray ion source is ionized, and mass spectrometric analysis is performed in the positive electrode mode by a multiple reaction monitoring system. The method for high performance liquid chromatography (HPLC)-mass spectrometry (MS) detection of DNA oxidation and DNA methylation can simultaneously detect the content of 8-OHdG and 5-mdC in a DNA sample.

Description

technical field [0001] The invention relates to the field of analytical chemistry, in particular to a method for detecting DNA oxidation and DNA methylation by high performance liquid chromatography mass spectrometry. Background technique [0002] A series of active oxygen free radicals, such as superoxide anion free radicals (O 2- ), singlet oxygen ( 1 o 2 ), hydrogen peroxide (H 2 o 2 ), hydroxyl radicals (-OH), etc. However, these endogenous reactive oxygen species may cause oxidative damage to DNA macromolecules and form a variety of DNA damage molecules. Among them, 8-hydroxydeoxyguanosine (8-OHdG), a ubiquitous oxidized DNA nucleoside, has been used as a biomarker of DNA oxidative damage. Both population survey results and cell experiments indicated that 8-OHdG can induce cancer through two mechanisms: genetic variation and non-genetic variation. Among them, the mechanism of genetic variation mainly involves the base mismatch of 8-OHdG during DNA replication, wh...

AI Technical Summary

Problems solved by technology

Among these methods, thin-layer chromatography requires simple equipment but the accuracy of the results is low; HPCE, HPLC-UV and HPLC-ECD methods can provide high repeatability and sensitivity, but because the detectors of these methods are sensitive to compounds (nucleosides and deoxynucleosides) are not specific, and the accuracy of the results depends on the separation of nucleosides and deoxynucleosides by chromatography

Although GC-MS methods can provide high sensitivity, sample preparation requires a derivatization step, and derivatization can cause some deoxynucleosides and nucleosides to be artificially oxidized

However, these methods are currently only targeting a single compound of 8-OHdG or 5-mdC, but there is no report on the simultaneous detection of 5-mdC and 8-OHdG levels in DNA samples

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