Detection of 5-Carboxyl Cytosine


5-carboxylcytosine (5caC) is an important derivative of cytosine modification in DNA demethylation pathway. However, the accurate quantification of 5caC is a challenge, especially in mammalian tissue. This plight probably results from the trace amount of 5caC in mammalian and the inadequate sensitivity of the existing technologies. Herein, we report a novel LC-MS/MS method to precisely quantify 5caC in mammalian cells. The 5caC standard reagent was synthesized, and the genomic DNA was hydrolyzed with formic acid and the target compound 5caC was detected by HILIC LC-MS/MS. The results showed 5caC generally exist in mice organs and cancer tissues. The content of 5caC in mice brain was higher than in lungs and liver, and was obviously decreased in colorectal cancer tissues compared with the adjacent tissue. This result suggests that 5caC probably associates with tumorigenesis and plays a dominated role in epigenetic control of neuronal function.

Due to the importance of DNA demethylation process in mammalian, the intermediates including 5mC, 5hmC and 5fC have been consecutively quantified in different biological samples to explore their function. However, the quantification of 5caC is extremely difficult. So far, quantification of 5caC is mainly reported in embryonic stem cells (approximately 3/106 cytosines). Pfaffeneder et al attempted to perform an HPLC-MS assay for simultaneous determination of 5fC and 5caC. Unfortunately, none 5caC signal was detected. Considering the fact that 5caC had not been detected in mammals cell, He etc. deduced that 5caC might be actively removed from genomic DNA immediately after its generation in cells.

Qualitative test based on the synthetic 5caC standard was performed with Agilent 6430A LC-MS/MS using nitrogen as the nebulizer gas. The MS was operated at unit mass resolution for both precursor and product ions. Qualification of 5caC was achieved in multiple-reaction monitoring (MRM) mode, which could be seen from full-scan mass spectrum of 5caC

In summary, we have confirmed and quantified the 5caC in mammalian tissues with excellent accuracy, and found that 5caC was normally presented in mammalian organs and the content was higher in tumor-adjacent tissues than colorectal cancer congeners. In accordance with the results, 5caC was probably suggested to participate in regulation of neuronal activities and cancer development

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