Formation and Determination of Endogenous Methylated Nucleotides in Mammals by Chemical Labeling Coupled with Mass Spectrometry Analysis

Abstract

5-Methylcytosine (5-mC) is an important epigenetic mark that plays critical roles in a variety of cellular processes. To properly exert physiological functions, the distribution of 5-mC needs to be tightly controlled in both DNA and RNA. In addition to methyltransferase-mediated DNA and RNA methylation, premethylated nucleotides can be potentially incorporated into DNA and RNA during replication and transcription. To exclude the premodified nucleotides into DNA and RNA, endogenous 5-methyl-2'-deoxycytidine monophosphate (5-Me-dCMP) generated from nucleic acids metabolism can be enzymatically deaminated to thymidine monophosphate (TMP). Therefore, previous studies failed to detect 5-Me-dCMP or 5-methylcytidine monophosphate (5-Me-CMP) in cells. In the current study, we established a method by chemical labeling coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS/MS) for sensitive and simultaneous determination of 10 nucleotides, including 5-Me-dCMP and 5-Me-CMP. As N,N-dimethyl-p-phenylenediamine (DMPA) was utilized for labeling, the detection sensitivities of nucleotides increased by 88-372-fold due to the introduction of a tertiary amino group and a hydrophobic moiety from DMPA. Using this method, we found that endogenous 5-Me-dCMP and 5-Me-CMP widely existed in cultured human cells, human tissues, and human urinary samples. The presence of endogenous 5-Me-dCMP and 5-Me-CMP indicates that deaminases may not fully deaminate these methylated nucleotides. Consequently, the remaining premethylated nucleosides could be converted to nucleoside triphosphates as building blocks for DNA and RNA synthesis. Furthermore, we found that the contents of 5-Me-dCMP and 5-Me-CMP exhibited significant decreases in renal carcinoma tissues and urine samples of lymphoma patients compared to their controls, probably due to more reutilization of methylated nucleotides in DNA and RNA synthesis. This study is, to the best of our knowledge, the first report for detecting endogenous 5-Me-dCMP and 5-Me-CMP in mammals. The detectable endogenous methylated nucleotides indicate the potential deleterious effects of premodified nucleotides on aberrant gene regulation in cancers.

Analysis of mononucleotides by tandem mass spectrometry: investigation of fragmentation pathways for phosphate- and ribose-modified nucleotide analogues

“…Tandem mass spectrometry (MS/MS) is a method used for the identification and quantification of nucleotides and their analogues in biological mixtures. MS/MS has been employed to determine the structures 2 , 3 and cellular fates 4 of endogenous nucleotides and to quantify blood or tissue levels of therapeutically relevant nucleotide analogues such as AZT oligophosphates 5 , gemcitabine triphosphate 6 , bisphosphonate (clodronate) metabolites 7 , and others 8 – 12 .…”

Inhibition of CMP-sialic acid transport by endogenous 5-methyl CMP

“…There are two requirements for m 5 CMP to act as a physiological inhibitor of CMP-Sia transport: 1) physiological concentrations of m 5 CMP should align with m 5 CMP's inhibition binding constants, and 2) physiological ratios of m 5 CMP and CMP concentrations should be similar to the observed ratios in binding constants. Until recently, it has not been possible to detect the levels of m 5 CMP inside cells; however, a recent development of a sensitive detection method has provided the first insight into cellular m 5 CMP concentrations (Zeng, Qi et al 2017). In this work, Zeng et al measured the concentration of a panel of nucleotides, including CMP, m 5 CMP, and deoxy-m 5 CMP (m 5 dCMP) for the HEK293T and HeLa cultured cell lines as well as for a number of samples of human renal tissues.…”

“…While it is known that concentrations of NMPs with canonical bases (A, T, C, G, or U) can vary between cell type and fluctuate depending on the metabolic needs of a cell (Traut 1994), there are currently no established links between these fluctuations and regulation of glycosylation. However, it has recently been appreciated that pools of cellular NMPs are comprised of more than just the canonical bases (Zeng, Qi et al 2017). Enzymatic degradation of DNA and RNA that has been modified as part of epigenetic control of gene expression can contribute a vast diversity to cellular pools of free NMPs, since there are hundreds of known ways that DNA and RNA bases may be modified (Liu and Pan 2015, Chen, Zhao et al 2016, Zhao, Roundtree et al 2016.…”

Nucleic Acids Analysis

“…In this respect, chemical labeling in combination with MS analysis has been proposed and proved to be an attractive strategy to increase the detection sensitivities of analytes ( Figure 5) [168,169]. Along this line, a variety of chemical labeling-MS-based methods have been developed for the sensitive and accurate analysis of nucleic acid modifications with the detection sensitivities of nucleosides being down to subfemtomole or even attomole levels [170][171][172][173][174][175].…”