Liquid Chromatography-Mass Spectrometry Analysis of Cytosine Modifications

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used technique in the global analysis of epigenetic DNA modifications. The high-resolution chromatographic separation along with sensitive MS detection permits the identification and quantification of deoxyribonucleosides with precision and reliability. Although there have been tremendous advances in LC and MS instrumentation in recent years, sample preparation has not experienced a similar rate of development and is often a bottleneck to chemical analysis. Here we present a protocol for identification and quantification of cytosine modifications that combines a robust and efficient method to generate single nucleosides from genomic DNA samples followed by direct LC-MS/MS analysis.