Abstract
5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are important epigenetic biomarkers and are demonstrated to be promising in early cancer diagnosis. The investigation on the global levels and distribution of these two cytosine modifications can reveal their biology functions and help develop diagnostic methods based on these biomarkers. However, compared with a variety of sequencing methods which aim at investigating the distribution of cytosine modifications, HPLC-MS/MS is a powerful technique to quantify the global levels of cytosine modifications. The newly developed sheathless CESI-MS/MS was introduced in this chapter. The method is a sensitive and simple technique to evaluate the global levels of 5mC and 5hmC in different kinds of DNA samples. The protocols for the sample preparation, instrumentations, and data analysis were proposed. With CESI-MS/MS, the limits of detection (LODs) of cytosine modifications can reach attomole level. Meanwhile, CESI-MS/MS can quantify the 5mC/5hmC levels in practical DNA sample less than 10 ng. Considering the ultrahigh sensitivity and the low DNA consumption, CESI-MS/MS can either be used in the preliminary study before sequencing in a research setting or as a final detection technique for the clinical diagnosis.
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Acknowledgements
This work was supported by the National Natural Science 443Foundation of China (Grant Nos. 21675004, 21575005, and 44421775006).
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Yuan, F., Ma, YF., Zhou, YL., Zhang, XX. (2022). Analysis of 5-Methylcytosine and 5-Hydroxymethylcytosine in Genomic DNA by Capillary Electrophoresis-Mass Spectrometry. In: Yuan, BF. (eds) DNA Modification Detection Methods . Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1229-3_3
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DOI: https://doi.org/10.1007/978-1-0716-1229-3_3
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