Highly Efficient Gel Electrophoresis for Accurate Quantification of Nucleic Acid Modifications via in-Gel Digestion with UHPLC-MS/MS

Gel electrophoresis is a powerful technique for the characterization of sequences, sizes and conformations of nucleic acids due to its remarkable separation efficiency. In parallel, liquid chromatography–mass spectrometry (LC-MS) has established itself as a staple tool for the meticulous characterization and accurate quantification of a multitude of DNA modifications. In this study, we devised an in-gel digestion method for coupling gel electrophoresis with LC-MS/MS. This process involves the enzymatic digestion of DNA within the gel by nucleases and release single nucleosides, which subsequently serve as a preprocessing step for (LC-MS/MS) analysis. We demonstrated that ethylenediaminetetraacetic acid (EDTA) in the routine gel electrophoresis buffer reduced the enzymatic digestion efficiency, while Mg2+ could mitigate this inhibition. We further showed EDTA-free gel electrophoresis and the process of digestion of genomic DNA and plasmid DNA within a gel was fluorescently imaged, proving the efficient digestion of DNA. By this improvement, the efficiency of an in-gel digestion could reach 60% or more of the control, compared with direct in-solution digestion. The measured abundances of DNA modifications (5-methylcytosine and N6-methyladenine) via in-gel digestion are consistent with that measured by in-solution digestion. Collectively, we showed an in-gel digestion method, which is a very useful pretreatment technique for the precise quantification of epigenetic modifications in diverse DNA molecules.