A Rigorous Nuclear DNA-Excluding Mass Spectrometry Assay for Accurate Identification of 5-Methylcytosine in Mitochondrial DNA

5-Methylcytosine (5mC) functions as a well-characterized epigenetic DNA mark in nuclear DNA, but its presence in mitochondrial DNA (mtDNA) remains elusive. Here, we report a new and rigorous nuclear DNA (nDNA)-excluding mass spectrometry assay enabling the reliable and accurate identification of 5mC in mtDNA for the first time. First, circular mtDNA is enriched over 809-946-fold by combining alkaline lysis and linear DNA-specific RecBCD cutting; nDNA accounts for ∼12-19% of the DNA remaining after this step. Second, assisted by the restrictive endonucleases BbsI (for human mtDNA) and EcoRV (for mouse mtDNA), circular mtDNA was cut into only one or two linearized mtDNA fragments, while the residual nuclear DNA was efficiently degraded into shorter fragments; thus, the linearized mtDNA fragment(s) could be well isolated from the residual degraded nDNA via gel electrophoresis. Finally, the linearized mtDNA bands are excised and subjected to in-gel digestion followed by precise stable isotope-diluted LC-MS/MS analysis. With this sensitive and accurate method, we demonstrated that mtDNA is hypomethylated in a normal mouse cell line, which is rationally attributed to de novo methylation. Overall, we provide a powerful, gold-standard mass spectrometry assay for screening and identifying mtDNA 5mC in diverse scenarios.