Global DNA 5mC Quantification by LC-MS/MS


Global DNA methylation levels in the genome are tissue-specific and decrease in the context of specific developmental and pathological states. Therefore, an accurate assay for quantifying global DNA methylation is required. In addition, global DNA methylation detection can better understand the prevalence of DNA methylation in samples prior to further research using more advanced methods. Measurement of global DNA methylation is challenging because high levels of inter-individual variability and background noise require highly accurate and sensitive analysis. Among the many assays used to assess global DNA methylation levels, liquid chromatography tandem mass spectrometry/mass spectrometry (LC-MS/MS) is regarded as the “gold standard”. Creative BioMart provides customers with a global DNA 5mC quantification service with solid expertise and state-of-the-art LC-MS/MS equipment.

What Is LC-MS/MS for Global Methylation Analysis?

The proportion of 5mC as a percentage of all cytosines in genomic DNA can be accurately measured using stable isotope-dilution LC-MS/MS. The LC-MS/MS assay produces three modes of selectivity. Firstly, HPLC separates the different deoxyribonucleotides in a digested sample of DNA, yielding different retention times on the chromatographic column. Secondly, in tandem mass spectrometry, the deoxyribonucleotides are ionized by electrospray ionization producing protonated ions with unique mass/charge ratios (m/z). The first mass analyzer sequentially selects these molecules, and they are accelerated into a collision cell with sufficient kinetic energy to cause fragmentation upon collision with an inert target gas that is present in the cell. Thirdly, a unique pattern of fragment ions is produced from the selected ion when the second mass analyzer is scanned. Quantification is performed with utmost sensitivity when both mass analyzers are set to pass (i) the protonated molecules of interest and (ii) an abundant fragment ion respectively in what is called the selected reaction monitoring mode.

Chromatogram of a typical injection of digested DNA for global methylation using LC-MS/MS

Figure 1. Chromatogram of a typical injection of digested DNA for global methylation using LC-MS/MS. RT: retention time. (Liu J.; et al. 2013)

Features of Global DNA 5mC Quantification by LC-MS/MS

  1. Extremely high sensitivity and repeatability
  2. Independent of DNA quality
  3. Able to analyze DNA from any species since it is not sequence dependent
  4. The flexibility of the assay allows the reliable comparison of the obtained 5mC levels using variations of the LC-MS/ MS method
  5. Measuring absolute 5mC content in DNA samples and is not appropriate to determine the precise genomic location of methylation

Our Advantages

  1. Tolerance of multiple sample types: we currently accept a wide range of biological and clinical samples including genomic DNA, cells, tissues, formalin-fixed and paraffin-embedded (FFPE) samples. Sample sources include human, animals, plants, and microorganisms.
  2. Flexible and one-stop service: customers only need to provide samples, and we will complete the full LC-MS/MS experiment and data analysis. The complete report is provided to you with both raw and analyzed data, graphs, detailed protocols, and a summary of findings.
  3. Extensive experience & First-class technology platform: we have advanced LC-MS/MS equipment, and our technical team has accumulated years of expertise and experience in complicated mass spectrometry specifications.

Workflow of Global DNA 5mC Quantification by LC-MS/MS at Creative BioMart

Workflow of Global DNA 5mC Quantification by LC-MS/MS at Creative BioMart

Figure 2. Workflow of Global DNA 5mC Quantification by LC-MS/MS at Creative BioMart

With years of professional and abundant experience in this field, Creative BioMart can provide you an advanced global DNA 5mC quantification service with our mature LC-MS/MS platform, quick turnaround time, and uncompromising quality of service. contact us for more information.

References
1. Quinlivan E P.; Gregory J F. DNA methylation determination by liquid chromatography-tandem mass spectrometry using novel biosynthetic [U-15N] deoxycytidine and [U-15N] methyldeoxycytidine internal standards. Nucleic Acids Research. 2008, 36(18): e119.
2. Liu J.; et al. Liquid chromatography tandem mass spectrometry for the measurement of global DNA methylation and hydroxymethylation. Journal of Proteomics & Bioinformatics. 2013, S2: 005. doi: 10.4172/jpb. S2-005

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