2 research outputs found
Sensitive and Simultaneous Determination of 5‑Methylcytosine and Its Oxidation Products in Genomic DNA by Chemical Derivatization Coupled with Liquid Chromatography-Tandem Mass Spectrometry Analysis
Cytosine
methylation (5-methylcytosine, 5-mC) in genomic DNA is
an important epigenetic mark that has regulatory roles in diverse
biological processes. 5-mC can be oxidized stepwise by the ten–eleven
translocation (TET) proteins to form 5-hydroxymethylcytosine (5-hmC),
5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC), which constitutes
the active DNA demethylation pathway in mammals. Owing to the extremely
limited contents of endogenous 5-mC oxidation products, no reported
method can directly determine all these cytosine modifications simultaneously.
In the current study, we developed selective derivatization of cytosine
moieties with 2-bromo-1-(4-dimethylamino-phenyl)-ethanone (BDAPE)
coupled with liquid chromatography-electrospray ionization tandem
mass spectrometry (LC-ESI-MS/MS) for the simultaneous determination
of these cytosine modifications in genomic DNA. The chemical derivatization
notably improved the liquid chromatography separation and dramatically
increased detection sensitivities of these cytosine modifications.
The limits of detection (LODs) of the derivatives of 5-mC, 5-hmC,
5-foC, and 5-caC were 0.10, 0.06, 0.11, and 0.23 fmol, respectively.
Using this method, we successfully quantified 5-mC, 5-hmC, 5-foC,
and 5-caC in genomic DNA from human colorectal carcinoma (CRC) tissues
and tumor-adjacent normal tissues. The results demonstrated significant
depletion of 5-hmC, 5-foC, and 5-caC in tumor tissues compared to
tumor-adjacent normal tissues, and the depletion of 5-hmC, 5-foC,
and 5-caC may be a general feature of CRC; these cytosine modifications
could serve as potential biomarkers for the early detection and prognosis
of CRC. Moreover, the marked depletion of 5-hmC, 5-foC, and 5-caC
may also have profound effects on epigenetic regulation in the development
and formation of CRC
Determination of Oxidation Products of 5‑Methylcytosine in Plants by Chemical Derivatization Coupled with Liquid Chromatography/Tandem Mass Spectrometry Analysis
Cytosine
methylation (5-methylcytosine, 5-mC) in DNA is an important
epigenetic mark that has regulatory roles in various biological processes.
In plants, active DNA demethylation can be achieved through direct
cleavage by DNA glycosylases, followed by replacement of 5-mC with
cytosine by base excision repair (BER) machinery. Recent studies in
mammals have demonstrated 5-mC can be sequentially oxidized to 5-hydroxymethylcytosine
(5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC)
by Ten–eleven translocation (TET) proteins. The consecutive
oxidations of 5-mC constitute the active DNA demethylation pathway
in mammals, which raised the possible presence of oxidation products
of 5-mC (5-hmC, 5-foC, and 5-caC) in plant genomes. However, there
is no definitive evidence supporting the presence of these modified
bases in plant genomic DNA, especially for 5-foC and 5-caC. Here we
developed a chemical derivatization strategy combined with liquid
chromatography–electrospray ionization tandem mass spectrometry
(LC/ESI-MS/MS) method to determine 5-formyl-2′-deoxycytidine
(5-fodC) and 5-carboxyl-2′-deoxycytidine (5-cadC). Derivatization
of 5-fodC and 5-cadC by Girard’s reagents (GirD, GirT, and
GirP) significantly increased the detection sensitivities of 5-fodC
and 5-cadC by 52–260-fold. Using this method, we demonstrated
the widespread existence of 5-fodC and 5-cadC in genomic DNA of various
plant tissues, indicating that active DNA demethylation in plants
may go through an alternative pathway similar to mammals besides the
pathway of direct DNA glycosylases cleavage combined with BER. Moreover,
we found that environmental stresses of drought and salinity can change
the contents of 5-fodC and 5-cadC in plant genomes, suggesting the
functional roles of 5-fodC and 5-cadC in response to environmental
stresses