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Anal Chem ; 92(2): 2301-2309, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31845797


Ribonucleotide analogues and their related phosphorylated metabolites play critical roles in tumor metabolism. However, determination of the endogenous ribonucleotides from the complex biological matrix is still a challenge due to their high structural similarity and high polarity that will lead to the low retention and low detection sensitivities by liquid chromatogram mass spectrometry analysis. In this study, we developed the diazo reagent labeling strategy with mass spectrometry analysis for sensitive determination of ribonucleotides in the living organism. A pair of light and heavy stable isotope labeling reagents, 2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (d5-2-DMBA), were synthesized to label ribonucleotides. 2-DMBA showed high specificity and high efficiency for the labeling of ribonucleotides. Our results demonstrated that the detection sensitivities of 12 ribonucleotides increased by 17-174-fold upon 2-DMBA labeling. The obtained limits of detection (LODs) of ribonucleotides ranged from 0.07 fmol to 0.41 fmol. Using this method, we achieved the sensitive and accurate detection of ribonucleotides from only a few cells (8 cells). To the best of our knowledge, this is the highest detection sensitivity for ribonucleotides ever reported. In addition, we found that the contents of almost all of these ribonucleotides were significantly increased in human breast carcinoma tissues compared to tumor-adjacent normal tissues, suggesting that endogenous ribonucleotides may play certain functional roles in the regulation of cancer development and formation. This method also can be potentially applied in the analysis of phosphorylated compounds.

Nucleic Acids Res ; 47(3): 1268-1277, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30517733


In addition to DNA cytosine methylation (5-methyl-2'-deoxycytidine, m5dC), DNA adenine methylation (N6-methyl-2'-deoxyadenosine, m6dA) is another DNA modification that has been discovered in eukaryotes. Recent studies demonstrated that the content and distribution of m6dA in genomic DNA of vertebrates and mammals exhibit dynamic regulation, indicating m6dA may function as a potential epigenetic mark in DNA of eukaryotes besides m5dC. Whether m6dA undergoes the further oxidation in a similar way to m5dC remains elusive. Here, we reported the existence of a new DNA modification, N6-hydroxymethyl-2'-deoxyadenosine (hm6dA), in genomic DNA of mammalian cells and tissues. We found that hm6dA can be formed from the hydroxylation of m6dA by the Fe2+- and 2-oxoglutarate-dependent ALKBH1 protein in genomic DNA of mammals. In addition, the content of hm6dA exhibited significant increase in lung carcinoma tissues. The increased expression of ALKBH1 in lung carcinoma tissues may contribute to the increase of hm6dA in DNA. Taken together, our study reported the existence and formation of hm6dA in genomic DNA of mammals.

Adenina/metabolismo , Metilação de DNA/genética , DNA/genética , Epigênese Genética , Adenina/análogos & derivados , Adenina/síntese química , Adenina/farmacologia , Animais , DNA/efeitos dos fármacos , DNA/metabolismo , Genoma/efeitos dos fármacos , Células HeLa , Humanos , Hidroxilação/efeitos dos fármacos , Mamíferos
ACS Chem Biol ; 12(6): 1636-1643, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28448110


Toxic heavy metals have been considered to be harmful environmental contaminations. The molecular mechanisms of heavy-metals-induced cytotoxicity and carcinogenicity are still not well elucidated. Previous reports showed exposures to toxic heavy metals can cause a change of DNA cytosine methylation (5-methylcytosine, 5-mC). However, it is still not clear whether heavy metals have effects on the recently identified new epigenetic marks in both DNA and RNA, i.e., 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Here, we established a chemical labeling strategy in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive detection of eight modified cytidines in DNA and RNA. The developed method allowed simultaneous detection of all eight modified cytidines with improved detection sensitivities of 128-443-fold. Using this method, we demonstrated that the levels of 5-hmC, 5-foC, and 5-caC significantly decreased in both the DNA and RNA of mouse embryonic stem (ES) cells while exposed to arsenic (As), cadmium (Cd), chromium (Cr), and antimony (Sb). In addition, we found that treatments by heavy metals induced a decrease of the activities of 10-11 translocation (Tet) proteins. Furthermore, we revealed that a content change of metabolites occurring in the tricarboxylic acid cycle may be responsible for the decline of the derivatives of 5-mC. Our study shed light on the epigenetic effects of heavy metals, especially for the induced decline of the derivatives of 5-mC in both DNA and RNA.

5-Metilcitosina/análise , Metais Pesados/farmacologia , Células-Tronco/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Animais , Células Cultivadas , Citosina/análogos & derivados , Citosina/análise , Citosina/metabolismo , DNA/metabolismo , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Camundongos , RNA/metabolismo , Células-Tronco/efeitos dos fármacos , Espectrometria de Massas em Tandem
Anal Chem ; 89(7): 4153-4160, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28271879


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.

5-Metilcitosina/química , DNA/análise , RNA/análise , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Metilação , Estrutura Molecular
Sci Rep ; 5: 15217, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26458332


2-hydroxyglutarate (2HG) is a potent competitor of α-ketoglutarate (α-KG) and can inhibit multiple α-KG dependent dioxygenases that function on the epigenetic modifications. The accumulation of 2HG contributes to elevated risk of malignant tumors. 2HG carries an asymmetric carbon atom in its carbon backbone and differentiation between D-2-hydroxyglutarate (D-2HG) and L-2-hydroxyglutarate (L-2HG) is crucially important for accurate diagnosis of 2HG related diseases. Here we developed a strategy by chiral derivatization combined with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive determination of D-2HG and L-2HG enantiomers. N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was used to derivatize 2HG. The formed diastereomers by TSPC labeling can efficiently improve the chromatographic separation of D-2HG and L-2HG. And derivatization by TSPC could also markedly increase the detection sensitivities by 291 and 346 folds for D-2HG and L-2HG, respectively. Using the developed method, we measured the contents of D-2HG and L-2HG in clear cell renal cell carcinoma (ccRCC) tissues. We observed 12.9 and 29.8 folds increase of D-2HG and L-2HG, respectively, in human ccRCC tissues compared to adjacent normal tissues. The developed chiral derivatization combined with LC-ESI-MS/MS analysis offers sensitive determination of D-2HG and L-2HG enantiomers, which benefits the precise diagnosis of 2HG related metabolic diseases.

Glutaratos/metabolismo , Metaboloma , Metabolômica , Neoplasias/metabolismo , Carcinoma de Células Renais/metabolismo , Cromatografia Líquida/métodos , Glutaratos/urina , Humanos , Metabolômica/métodos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos