3 research outputs found
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
Metal Oxide-Based Selective Enrichment Combined with Stable Isotope Labeling-Mass Spectrometry Analysis for Profiling of Ribose Conjugates
Some modified ribonucleosides
in biological fluids have been evaluated
as cancer-related metabolites. Detection of endogenous modified ribonucleosides
in biological fluids may serve as a noninvasive cancers diagnostic
method. However, determination of modified ribonucleosides is still
challenging because of their low abundance and serious matrix interferences
in biological fluids. Here, we developed a novel strategy for comprehensive
profiling of ribose conjugates from biological fluids using metal
oxide-based dispersive solid-phase extraction (DSPE) followed with <i>in vitro</i> stable isotope labeling and double neutral loss
scan-mass spectrometry analysis (DSPE-SIL-LC-DNLS-MS). Cerium dioxide
(CeO<sub>2</sub>) was used to selectively recognize and capture ribose
conjugates from complex biological samples under basic environment.
The enriched ribose conjugates were subsequently labeled with a pair
of isotope labeling reagents (acetone and acetone-d<sub>6</sub>).
The glucosidic bond of acetone labeled ribose conjugates is readily
ruptured, and the generated ribose that carries an isotope tag can
be lost as a neutral fragment under collision induced dissociation
(CID). Since the light (acetone) and heavy (acetone-d<sub>6</sub>)
labeled compounds have the same chemical structures and can generate
different neutral loss fragments (NL 172 and 178 Da), it is therefore
highly convenient to profile ribose conjugates by double neutral loss
scan mode in mass spectrometry analysis. In this respect, the light
and heavy labeled compounds were ionized at the same condition but
recorded separately on MS spectra, which can significantly improve
the detection specificity and facilitate the identification of ribose
conjugates. Using the developed DSPE-SIL-LC-DNLS-MS strategy, we profiled
the ribose conjugates in human urine, and 49 ribose conjugates were
readily identified, among which 7 ribose conjugates exhibited significant
contents change between healthy controls and lymphoma patients. The
DSPE-SIL-LC-DNLS-MS strategy combines the selective enrichment, stable
isotope labeling, and double neutral loss scan - MS analysis, which
therefore can efficiently minimize false positive results, facilitate
the relative quantification, and notably increase the numbers of identified
ribose conjugates in biological fluids samples. Taken together, this
study established a promising strategy for the effective profiling
of urinary modified ribonucleosides, and simultaneous evaluation of
the contents change of multiple modified ribonucleosides should provide
more accurate and conclusive results for the use of urinary modified
ribonucleosides as indicators of cancers
Formation and Determination of Endogenous Methylated Nucleotides in Mammals by Chemical Labeling Coupled with Mass Spectrometry Analysis
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 <i>N</i>,<i>N</i>-dimethyl-<i>p</i>-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