Rapid and Definitive Analysis of In Vitro DNA Methylation by Nano-electrospray Ionization Mass Spectrometry
Open Access
- 16 September 2019
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Society for Mass Spectrometry
- Vol. 30 (11), 2335-2346
- https://doi.org/10.1007/s13361-019-02304-5
Abstract
CpG methylation of DNA is an epigenetic marker that is highly related to the regulation of transcription initiation. For analysis of CpG methylation in genomic DNA sequences, bisulfite-induced modification in combination with polymerase chain reaction (PCR) is usually utilized, but it cannot be straightforwardly applied to methylated short- and middle-sized DNAs, such as < 500 base pairs (bp), which are often utilized in structural biology studies. In the present study, we applied nano-electrospray ionization mass spectrometry (nano-ESI-MS) for the characterization of methylated DNA with < 400 bp prepared in vitro. First, double-stranded DNA oligomers were methylated with recombinant M.SssI DNA methylase, which has been reported to modify completely and exclusively CpG sites in the sequence. The fragments generated by the digestion with methylation-insensitive restriction nuclease were then analyzed to identify the methylation levels by nano-ESI-MS, without liquid chromatography (LC) separation. By methylation-insensitive nuclease digestion, we divided the DNA strands into several fragments, and nano-ESI-MS enabled the accurate analysis of methylation levels in the DNA fragments with a relatively small amount of DNA sample prepared under optimized conditions. Furthermore, it was revealed that M.SssI methylase hardly modifies the CpG sites closely positioned at the ends of linear DNA. The present method is similar to the strategy for post-translational modification analysis of proteins and is promising for the rapid and definitive characterization of methylated DNA that may be used in structural biology studies.Keywords
Funding Information
- Japan Agency for Medical Research and Development (JP17am0101076)
- Japan Society for the Promotion of Science (JP17K07313)
- Ministry of Education, Culture, Sports, Science and Technology (JP18H04561)
- Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering
This publication has 37 references indexed in Scilit:
- Functional DNA methylation differences between tissues, cell types, and across individuals discovered using the M&M algorithmGenome Research, 2013
- OPERating ON Chromatin, a Colorful Language where Context MattersJournal of Molecular Biology, 2011
- CpG islands and the regulation of transcriptionGenes & Development, 2011
- The complex language of chromatin regulation during transcriptionNature, 2007
- Epigenetics in human disease and prospects for epigenetic therapyNature, 2004
- Controlling the double helixNature, 2003
- Translating the Histone CodeScience, 2001
- New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioningJournal of Molecular Biology, 1998
- CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation.Genes & Development, 1989
- Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltonsAnalytical Chemistry, 1988