Antibodies have also been used to elucidate the biological functions of DNA
epigenetic marks by studying their tissue-specific levels and distribution. Global
levels of epigenetic DNA marks can be evaluated by staining cells or tissues with a
specific monoclonal antibody [53]. Globisch et al. utilized immunostaining experiments with a commercially available 5hmC-specific antibody to map the distribution
of 5hmC in cells and tissues. They found that 5hmC was localized in the cell nuclei
and that virtually all cells contained 5hmC. Furthermore, the use of immunostaining
allowed these authors to confirm that the highest levels of 5hmC are found in the
brain, followed by kidney and nasal epithelia [23].
Antibodies specific for certain epigenetic marks can also be used for affinity
enrichment of DNA regions. Antibodies can be used to immunoprecipitate
modification-containing DNA fragments, allowing for their enrichment. This technique can then be coupled to methylation-specific oligonucleotide (MSO)
microarrays, locus-specific PCR, or whole-genome approaches such as nextgeneration sequencing or microarrays, to enable for low-resolution mapping of
epigenetic marks within the genome (see Sect. 3.2).
2.3 High Performance Liquid Chromatography: Mass
Spectrometry
Global levels of 5mC, 5hmC, 5fC, 5caC, N6MedA, and 4mC in genomic DNA can
be accurately quantified by isotope dilution liquid chromatography-mass spectrometry. In this approach, DNA is enzymatically digested using nucleases and phosphatases, and the resulting nucleosides are analyzed by HPLC-ESI-MS/MS. HPLC
with UV detection can be used for measuring 5mC in biological samples due to its
high content (4–5% of all cytosine bases) [54], while other epigenetic modifications
(5hmC, 5fC, 5caC, N6MedA, and 4mC) are significantly less common and thus
require more sensitive methods such as HPLC-ESI-MS/MS [54].
HPLC-ESI-MS/MS is widely accepted as the most accurate technique for quantification of global levels of 5mC and its oxidized forms [54]. In this approach, DNA
is digested to individual nucleosides. Epigenetically modified DNA nucleosides are
detected based on their characteristic molecular weights, HPLC retention times, and
MS/MS fragmentation (Fig. 6a). 5mC, 5hmC, 5fC, 5caC, and N6MedA can be
readily identified by mass spectrometry based on their distinct mass to charge ratios
and MS/MS fragmentation patterns (Table 1) [54]. Absolute quantitation is
performed using stable isotope-labeled analogues of each nucleoside that are spiked
into samples (Fig. 6b) or by using external calibration curves. This methodology
makes it possible to accurately quantify global amounts of each epigenetic mark of
DNA, although it does not provide their distribution along the genome [55].
Methods that employ HPLC-MS/MS techniques to quantify epigenetic modifications of DNA have been developed for a variety of applications and instruments
[56–58]. Due to the large number of methods that have been published for analyzing
496
C. L. Seiler et al.
epigenetic marks by studying their tissue-specific levels and distribution. Global
levels of epigenetic DNA marks can be evaluated by staining cells or tissues with a
specific monoclonal antibody [53]. Globisch et al. utilized immunostaining experiments with a commercially available 5hmC-specific antibody to map the distribution
of 5hmC in cells and tissues. They found that 5hmC was localized in the cell nuclei
and that virtually all cells contained 5hmC. Furthermore, the use of immunostaining
allowed these authors to confirm that the highest levels of 5hmC are found in the
brain, followed by kidney and nasal epithelia [23].
Antibodies specific for certain epigenetic marks can also be used for affinity
enrichment of DNA regions. Antibodies can be used to immunoprecipitate
modification-containing DNA fragments, allowing for their enrichment. This technique can then be coupled to methylation-specific oligonucleotide (MSO)
microarrays, locus-specific PCR, or whole-genome approaches such as nextgeneration sequencing or microarrays, to enable for low-resolution mapping of
epigenetic marks within the genome (see Sect. 3.2).
2.3 High Performance Liquid Chromatography: Mass
Spectrometry
Global levels of 5mC, 5hmC, 5fC, 5caC, N6MedA, and 4mC in genomic DNA can
be accurately quantified by isotope dilution liquid chromatography-mass spectrometry. In this approach, DNA is enzymatically digested using nucleases and phosphatases, and the resulting nucleosides are analyzed by HPLC-ESI-MS/MS. HPLC
with UV detection can be used for measuring 5mC in biological samples due to its
high content (4–5% of all cytosine bases) [54], while other epigenetic modifications
(5hmC, 5fC, 5caC, N6MedA, and 4mC) are significantly less common and thus
require more sensitive methods such as HPLC-ESI-MS/MS [54].
HPLC-ESI-MS/MS is widely accepted as the most accurate technique for quantification of global levels of 5mC and its oxidized forms [54]. In this approach, DNA
is digested to individual nucleosides. Epigenetically modified DNA nucleosides are
detected based on their characteristic molecular weights, HPLC retention times, and
MS/MS fragmentation (Fig. 6a). 5mC, 5hmC, 5fC, 5caC, and N6MedA can be
readily identified by mass spectrometry based on their distinct mass to charge ratios
and MS/MS fragmentation patterns (Table 1) [54]. Absolute quantitation is
performed using stable isotope-labeled analogues of each nucleoside that are spiked
into samples (Fig. 6b) or by using external calibration curves. This methodology
makes it possible to accurately quantify global amounts of each epigenetic mark of
DNA, although it does not provide their distribution along the genome [55].
Methods that employ HPLC-MS/MS techniques to quantify epigenetic modifications of DNA have been developed for a variety of applications and instruments
[56–58]. Due to the large number of methods that have been published for analyzing
496
C. L. Seiler et al.