Fused-core silica column ultra-performance liquid chromatography–ion trap tandem mass spectrometry for determination of global DNA methylation status

Abstract

Epigenetic modifications, such as DNA methylation, play key roles in transcriptional regulation of gene expression. More recently, global DNA methylation levels have been documented to be altered in several diseases, including cancer, and as the result of exposure to environmental toxicants. Based on the potential use of global DNA methylation status as a biomarker of disease status and exposure to environmental toxicants, we sought to develop a rapid, sensitive, and precise analytical method for the quantitative measurement of global DNA methylation status using ultra performance liquid chromatography with detection by ion trap tandem mass spectrometry. Using a fused-core silica column, 2′-deoxyguanosine (2dG) and 5-methyl-2′-deoxycytidine (5mdC) were resolved in less than 1 minute, with detection limits of 0.54 and 1.47 fmol for 5mdC and 2dG respectively. The accuracy of detection was 95% or above and the day-today coefficient of variations was found to be 3.8%. The method was validated by quantification of global DNA methylation status following treatment of cells with the DNA methyltransferase inhibitor 5-aza-2′deoxycytidine, which reduced DNA methylation from 3.1% in control cells to 1.1% in treated cells. The sensitivity and high throughput of this method rend it suitable for large scale analysis of epidemiological or clinical DNA samples.

Application of liquid chromatography/electrospray ionization ion trap tandem mass spectrometry for the evaluation of global nucleic acids: methylation in garden cress under exposure to CuO nanoparticles

“…Results for cytosine‐containing nucleosides in MS 2 and MS 3 mode are presented in Table (Supplementary Table 1S in the Supporting Information summarizes the above parameters evaluated for other compounds in the MS 2 method). Noteworthy, the on‐column detection limits for five compounds of interest were in the range 9–35 fmol using MS 2 conditions and 20–122 fmol using MS 3 ; these values are higher as compared to 0.2–12.8 fmol range reported while using more powerful instrumentation (linear ion trap – quadrupole, orbitrap or triple quadrupole mass spectrometers); however, extremely high detection power is not critical in plant analysis because of the fairly large amount of biomass available and also due to relatively high percentages of DNA methylation as compared to animal genomes. On the other hand, conventional ion trap spectrometers are less expensive, less susceptible to variation in instrumental conditions, easy to operate and allow for several successive ion fragmentations.…”

“…[14] Analytical methodology available for DNA methylation/ hydroxymethylation has been described in a few comprehensive reviews and in the introductory parts of several research articles. [33][34][35][36][37][38][39] The current approach relies on the enzymatic hydrolysis of DNA and separation of the obtained nucleosides by liquid chromatography with tandem mass spectrometry detection. [34,38,40,41] When a conventional lowresolution ion trap instrument is used, multiple reaction monitoring (MRM) helps to achieve high sensitivity and selectivity required when small sample amount is available and when real-world materials with complex chemical matrix are analyzed.…”

Mass spectrometry analysis of nucleosides and nucleotides

“…A different study used a fusedcore silica reverse phase analytical column for separation (to allow for improved resolution without an pressure increase caused by UPLC separations). The separation resolved deoxyguanosine and 5-methyldeoxycytidine in less than 1 min and retained good sensitivity (sub-femtomole for 5-methylcytidine) to demonstrate the value of such HPLC materials compared to similar UPLC systems (Yang et al, 2011). Another study used HILIC HPLC columns to the same analysis of DNA methylation after DNA extraction and acid hydrolysis, to release the methylated base rather than the methylated nucleoside (Zhang et al, 2011a).…”

Simultaneous Determination of Global DNA Methylation and Hydroxymethylation Levels by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

“…Compared with the above methods, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the best and most widely accepted method for quantification. [18][19][20] These techniques rely on the release of nucleosides by enzyme treatment. However, a complete enzymatic reaction cannot be readily ensured, and thus the accuracy might be affected.…”