High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry

Highlights: • We present an ultra-rapid (• The method overcomes issues of liquid chromatography like carryover and biased retention of nucleosides of different hydrophobicities. • We demonstrate that routinely used targeted approaches overlook the formation of nucleoside byproducts affecting quantification. • We optimized instrument settings to minimize biases in quantification. We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of 2 > 0.98) and 0.13%–1.75% (R² > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.