Basics: Mass spectrometry is an analytical method that determines the mass-to-charge ratio (m/z) of ions. Molecules are ionized in the source and accelerated by an electric field into the analyzer. In time-of-flight (TOF) mass spectrometry (see Figure 1), the analyzer is a chamber under vacuum that contains no electric fields. The ions drift through the analyzer with the kinetic energy obtained from the potential energy of the electric field. The kinetic energy the ions obtain is defined by Equation 1 where eV is the potential energy of the electric field, KE is the kinetic energy, m is mass-to-charge ratio, and v is velocity.
If all ions obtain the same kinetic energy, the ions of lesser m/z will have greater velocity than ions of greater m/z. Therefore, as ions traverse the analyzer, they separate in space. A detector is positioned at the end of the analyzer to measure the arrival time of ions. Ions of lesser m/z arrive first, followed by ions of greater m/z. A plot of intensity or abundance versus time is made to show the arrival time distribution of the ions detected.
So the polymers will hit the detector, the small ones first, then the big ones. They hit completely in order by mass. All the polymer molecules of the same molecular weight will hit the detector together. When they hit the detector, the detector registers a peak. The size of the peak is proportional to the number of molecules that hit at one time. For large samples such as biomolecules, molecular masses can be measured to within an accuracy of 0.01% of the total molecular mass of the sample i.e. within a 4 Daltons (Da) or atomic mass units (amu) error for a sample of 40,000 Da. This is sufficient to allow minor mass changes to be detected, e.g. the substitution of one amino acid for another, or a post-translational modification.
We use an analysis that utilizes base-specific cleavage of a desired target region. Samples are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method permits the high-throughput identification of methylation sites and their semi-quantitative measurement at multiple CpG positions. MALDI-MS is a method that offers a high degree of automation. The idea behind this technology is to generate PCR amplification products on bisulfite-treated DNA from cases and controls. Sodium Bisulfite converts unmethylated, but not methylated, cytosines into uracils, which can then be amplified as thymine in a subsequent PCR, thereby enabling differentiation of methylated and unmethylated sequences. The bisulfite treated samples are then processed for analysis with MALDI-TOF. This way, it is possible to identify genomic regions at which DNA methylation profiles display statistically significant variation due to stochastical, biological or environmental influences.
Fig. 2. Analysis of methylation by base-specific cleavage and MALDI-TOF MS. Bisulfite treated samples are amplified with primers of whom one is tagged with a T7 promoter sequence. The PCR product is transcribed into a RNA transcript and cleaved base-specific. The cleavage products are analyzed with a mass spectrophotometer and characteristic mass signal patterns are obtained. Each spectra can read several CpG dinucleotides so that, for example, a whole promoter can be analyzed in one experiment.
This epigenotyping assay allows for accurate discrimination of methylation levels that differ by as low as 5%. Our technology enables us also to do multiplexing of samples (e.g. pooled disease or control samples), which may help us to detect disease genes without the need to for exact reconstruction of methylation sites, therefore speeding up the process of gene discovery.
Typical output for a genomic region:
This Methylation Analysis provides the following benefits:
* No need for tedious cloning of PCR products
* Rapid, quantitative assessment of the degree of methylation
* Automated discovery of up to 30 methylated CpG positions in regions of 200-600 bp
* Detection of methylation levels as low as 5% in sample mixtures
* Results may be obtained from various sample types, e.g. blood, frozen tissue, mouth swabs or paraffin embedded tissue
* High precision, reproducibility and throughput on an established Autoflex system
