30–80 μg of genomic DNA, and the limits of detection for 5hmC, 5fC, and 5caC
were reported to be 2–3 modifications per 10
5 cytidines [59].
More recent applications of HPLC-MS/MS for epigenetic marks of DNA have
employed two-dimensional HPLC (2D-LC) tandem mass spectrometry. A general
method for analysis of 5mC, 5hmC, 5fC, and 5caC was reported by Liu et al. [54]. In
this approach, DNA samples are enzymatically digested to nucleosides, followed by
ultrafiltration to remove proteins. A column switching nano-HPLC methodology
using two HPLC columns was used to allow for online sample cleanup prior to highresolution MS/MS analysis using a Q Exactive hybrid quadrupole-Orbitrap mass
spectrometer [54]. Using this methodology, 5mC, 5hmC, 5fC, and 5caC can be
quantified at low femtomole levels [54]. A similar methodology was reported by
Gackowski et al. [60]. 2D-LC allowed for isolation of peaks of interest (5hmC, 5fC,
and 5caC) as they eluted off the first HPLC column and directing them to a second
column, which was connected to a Waters Xevo TQ-S mass spectrometer. Using this
approach, a high level of sensitivity was achieved for 5hmC (LOD (limit of
detection), 3 fmol; LOQ (limit of quantitation), 10 fmol), 5fC (LOD, 0.3 fmol;
LOQ, 0.8 fmol), and 5caC (LOD, 0.05 fmol; LOQ, 0.13 fmol), and the amounts of
each were quantified in matched cancer and normal human colon tissue samples
[60]. By minimizing sample processing and analyte cleanup steps, 2D-LC methodologies significantly simplify sample analyses and minimize the cost, allowing for
high-throughput quantification of large numbers of samples.
Although the vast majority of mass spectrometry-based techniques do not allow
for mapping epigenetic marks along the genome, a recent report by Lin et al.
employed a mass spectrometry-based approach to determine local concentrations
of 5mC at specific genes [61]. Following endonuclease cleavage with BbvI, DNA
sequences of interest were captured by hybridization to complementary sequences
immobilized on streptavidin beads [61]. The captured DNA was heated in formic
acid to release 5mC as a free base, which was detected using an LTQ mass
spectrometer. Lin et al. used this method to probe methylation status of four
promoter regions: GSTP1, BCL2, ESR1, and HIC1 in DNA from clinical prostate
tissue samples digested with BbvI [61]. The capture methodology was able to
effectively distinguish cancer tissue from normal tissue based on the level of
promoter methylation of GSTP1, BCL2, ESR1, and HIC1 [61].
Biological samples from clinical studies are often precious, requiring method
minimization to reduce DNA input. Le et al. have developed an LC-MS/MS method
for the analyses of 5mC and 5hmC in 50 ng of DNA [62]. This effort employed an
Agilent 6,460 QQQ, an Agilent Eclipse C18 (2.1 Â 50 mm, 1.8 μm particle size)
column eluted at a flow rate of 100 μL/min. External calibration curves were
constructed using known concentrations of 5mC and 5hmC. This method was able
to quantify 0–10% 5mC and 0–2% 5hmC [62].
The recent discovery of N6MedA (Fig. 1) in mammalian embryonic stem cells
[29] was accomplished by offline HPLC separation of enzymatic DNA digests,
followed by HPLC-ESI-MS/MS analysis of N6MedA using a Waters UPLC coupled
to a TSQ Quantum Ultra triple-quadrupole mass spectrometer. Quantitation of the
N6MedA was performed with external calibration curves, and the identity of the
498
C. L. Seiler et al.
were reported to be 2–3 modifications per 10
5 cytidines [59].
More recent applications of HPLC-MS/MS for epigenetic marks of DNA have
employed two-dimensional HPLC (2D-LC) tandem mass spectrometry. A general
method for analysis of 5mC, 5hmC, 5fC, and 5caC was reported by Liu et al. [54]. In
this approach, DNA samples are enzymatically digested to nucleosides, followed by
ultrafiltration to remove proteins. A column switching nano-HPLC methodology
using two HPLC columns was used to allow for online sample cleanup prior to highresolution MS/MS analysis using a Q Exactive hybrid quadrupole-Orbitrap mass
spectrometer [54]. Using this methodology, 5mC, 5hmC, 5fC, and 5caC can be
quantified at low femtomole levels [54]. A similar methodology was reported by
Gackowski et al. [60]. 2D-LC allowed for isolation of peaks of interest (5hmC, 5fC,
and 5caC) as they eluted off the first HPLC column and directing them to a second
column, which was connected to a Waters Xevo TQ-S mass spectrometer. Using this
approach, a high level of sensitivity was achieved for 5hmC (LOD (limit of
detection), 3 fmol; LOQ (limit of quantitation), 10 fmol), 5fC (LOD, 0.3 fmol;
LOQ, 0.8 fmol), and 5caC (LOD, 0.05 fmol; LOQ, 0.13 fmol), and the amounts of
each were quantified in matched cancer and normal human colon tissue samples
[60]. By minimizing sample processing and analyte cleanup steps, 2D-LC methodologies significantly simplify sample analyses and minimize the cost, allowing for
high-throughput quantification of large numbers of samples.
Although the vast majority of mass spectrometry-based techniques do not allow
for mapping epigenetic marks along the genome, a recent report by Lin et al.
employed a mass spectrometry-based approach to determine local concentrations
of 5mC at specific genes [61]. Following endonuclease cleavage with BbvI, DNA
sequences of interest were captured by hybridization to complementary sequences
immobilized on streptavidin beads [61]. The captured DNA was heated in formic
acid to release 5mC as a free base, which was detected using an LTQ mass
spectrometer. Lin et al. used this method to probe methylation status of four
promoter regions: GSTP1, BCL2, ESR1, and HIC1 in DNA from clinical prostate
tissue samples digested with BbvI [61]. The capture methodology was able to
effectively distinguish cancer tissue from normal tissue based on the level of
promoter methylation of GSTP1, BCL2, ESR1, and HIC1 [61].
Biological samples from clinical studies are often precious, requiring method
minimization to reduce DNA input. Le et al. have developed an LC-MS/MS method
for the analyses of 5mC and 5hmC in 50 ng of DNA [62]. This effort employed an
Agilent 6,460 QQQ, an Agilent Eclipse C18 (2.1 Â 50 mm, 1.8 μm particle size)
column eluted at a flow rate of 100 μL/min. External calibration curves were
constructed using known concentrations of 5mC and 5hmC. This method was able
to quantify 0–10% 5mC and 0–2% 5hmC [62].
The recent discovery of N6MedA (Fig. 1) in mammalian embryonic stem cells
[29] was accomplished by offline HPLC separation of enzymatic DNA digests,
followed by HPLC-ESI-MS/MS analysis of N6MedA using a Waters UPLC coupled
to a TSQ Quantum Ultra triple-quadrupole mass spectrometer. Quantitation of the
N6MedA was performed with external calibration curves, and the identity of the
498
C. L. Seiler et al.