High--throughput Measurement of DNA Mono
High
Mono-- and Oligo
Oligo--nucleotides by RapidFire/TOFRapidFire/TOF-MS
William A. LaMarr1, Deyu Li2, Vipender Singh2, John M. Essigmann2, and Peter T. Rye1
1) Agilent Technologies, Wakefield, MA. 2) MIT, Departments of Chemistry and Biological Engineering, Cambridge, MA.
2 Measuring
2.
M
iResults
Oligo-nucleotides
Oli and Discussion
l id (Example
(E
l 1)
Measuring structural changes to DNA is pivotal to
understanding epigenetics, gene expression, and cancer.
Accordingly, instrumentation that facilitates the analysis of
DNA reactants is valuable to discovering drugs with wide
therapeutic potential. This work contains three demonstrations
in which the Agilent RapidFire High-throughput Mass
Spectrometry System was used to measure mono- and oligonucleotides from in vitro samples, establishing the viability of
the platform for DNA-based applications. The RapidFire system
performed sample preparation,
preparation from 96
96- and 384
384-well
well plates,
plates
with a sustained throughput of ~8 seconds per sample, and
permitted the DNA species to be measured directly by online
time-of-flight (TOF) mass spectrometry (MS). TOF-MS collects
full scan data over time and thereby permitted the abundance
of any given DNA species to be extracted from the data set
post run.
2A
160000
1000
1500
2000
2500
3A
AUC of MS Signal
Figure 2AB. In efforts to evaluate the ability of the RapidFire/TOF-MS System to measure modifications to short oligonucleotides, the repair of an
Ethenoadenine (A) residue by the AlkB enzyme was chosen as a model system. The substrate, a 16-mer DNA strand containing a centrally located A
residue, was injected into the TOF-MS (2A). A dose-response plate containing ten 2-fold dilutions of the DNA was analyzed by RapidFire/TOF-MS. The
MS signal for the substrate was extracted using m/z=1637.2797and plotted vs. concentration using Excel (2B).
2C
1629.2797
A
U
10000
12500
1D
AUC of MS Signal
A
60
50
40
20
y = 31.642x ‐ 42.6
R² = 0.9946
A
5000
U
2000
y = 8.0668x + 44.2
R² = 0.9987
1000
y = 4.2866x + 49.8
R² = 0.9996
0
0
50
100
150
[mononucleotide] (nM)
200
250
60
80
100
The RapidFire/TOF-MS system enables the measurement of mono- and oligo-nucleotides from in vitro
mixtures at a sustained rate of ~8 seconds per sample.
30
20
2.
Experiments with mono-nucleotides demonstrate efficient resolution of the different DNA species, good
sensitivity, low carryover, and a linear signal response with dose.
3.
Experiments with oligo-nucleotides demonstrate the ability to monitor enzymatic transformation of
multiple DNA species concurrently,
concurrently facilitating the identification and quantitation of all reaction
products.
4.
Because high-throughput detection of DNA species by MS is fast, accurate, and versatile, it has the
potential to accelerate a broad range of drug discovery efforts.
10
0
0
y = 19.293x + 111.73
R² = 0.9972
G
3000
40
40
10
0
C
4000
20
1.
glycol
70
30
7000
6000
0
Conclusions
50
epoxide
Relative Abundance
7500
[mononucleotide] (nM)
40
Actual % Product
eA
80
0
y = 0.9503x + 7.8292
R² = 0.9916
60
20
1648.9287
glycol
2F
90
y = 14.906x ‐ 5219.4
R² = 0.9905
y = 5.6381x + 1735.1
R² = 0.987
100
100
100000
C
5000
3D
60
y = 17.444x + 2410
R² = 0.9959
2500
5’-GTCATCAGGATCGATGGCAG-3’
m/z=1544.2589
M-4H
0
Percent Conversion
G
0
1642.6064
epoxide
2E
C
200000
5’ … CG … 3’
80
y = 39.968x ‐ 1348.7
R² 0 9991
R² = 0.9991
A
300000
Extracted ion chromatogram
For m/z=1547.7568
A
+ AlkB
1637.2797
A
Figure 2CD. The repair of A to adenine (A) by AlkB involves epoxide
and glycol intermediates (2C), providing an opportunity to measure
four distinct DNA species in every reaction. Figure 2D shows a TOFMS scan, generated from RapidFire/TOF-MS analysis of a reaction, in
which all four DNA species are present.
1C
G
3B
Extracted ion chromatogram
for m/z=1544.2589
(native 20-mer DNA )
CH3
DNMT1
5’ … CG … 3’
2D
3C
5’-GTCATCAGGAT-5mC-GATGGCAG-3’
m/z=1547.7586
M-4H
500000
Figure 1. To demonstrate the ability of RapidFire/TOF-MS to accurately
quantitate mono-nucleotide concentrations, four representative molecules
were chosen for study: adenosine 55’monophosphate
monophosphate (A),
(A) cytidine 55’monophosphate (C), guanosine 5’-monophosphate (G), and deoxyuridine
5’-monophosphate (U). A mixture containing all four molecules was
injected in to the TOF-MS providing accurate m/z values for each DNA
species (1A). Next, a dose response plate containing ten 2-fold dilutions
of the mononucleotides (in triplicate with ‘buffer only’ between each
sample) was analyzed by RapidFire/TOF-MS. The MS signals for the
mononucleotides were extracted (1B) and plotted vs. concentration using
Excel (1C). Figure 1D is the same data as that in 1C, but adjusted to show
the low concentration range in better detail. The data for each
mononucleotide show good signal linearity and sensitivity.
500
[oligo] (nM)
400000
A
40000
0
U
C
80000
0
AUC of MS Signal
G
100000
20000
1B
A
120000
60000
M‐2H
m/z=2456.4196
Results and
Discussion
1. Measuring
Mono-nucleotides
U
5’‐GAAGACCTAGGCGTCC‐3’
MW ~4915
Figure 3. Over the past couple years, use of the RapidFire system of studying
epigenetic modifications has grown significantly. Primarily, however, the
target substrates have been peptide-based. To help establish the applicability
of the platform for investigating epigenetic related changes to DNA,
preliminary experiments were conducted using DNA standards. Two 20-mer
oligonucleotides were synthesized to represent one strand of the DMNT1
reaction substrate and product (3A). Analysis of these standards by TOF-MS
displays optimal ions for the two DNA species that are easily resolved (3B).
Subsequently, six mixtures containing different proportions of the ‘substrate’
substrate
and ‘product’ DNA stands were analyzed by RapidFire/TOF-MS, and the
corresponding MS signals were extracted (3C) and plotted vs. actual %
product using Excel (3D). The data display good correlation with the actual %
product and good signal linearity.
y = 63.297x + 4239.1
R² = 0.9932
140000
In the first demonstration, a set of four mononucleotides was analyzed by RapidFire/TOF-MS, highlighting the potential for
investigations including detection, quantitation, and localization of modified bases in DNA of known sequence. In the second
demonstration, the AlkB enzyme was used to repair an etheno adduct, located within a 16-mer oligonucleotide, and the
reaction products (including substrate, two repair intermediates, and product DNA strands) to be measured discretely. Lastly,
i the
in
th third
thi d demonstration,
d
t ti
th abundance
the
b d
off a single
i l 5-methylcytosine
5 th l t i residue,
id located
l t d within
ithi a 20-mer
20
oligonucleotide,
li
l tid was
measured in the presence of native 20-mer DNA. Taken together, this research provides a framework for high-throughput
detection of DNA species by MS, in general, and can be used to accelerate experimentation in multiple fields.
1A
2B
M‐3H
m/z=1637.2797
M‐4H
m/z=1227.7098
3 Measuring
3.
M
iResults
Oligo-nucleotides
Oli and Discussion
l id (Example
(E
l 2)
Measured % Product
I
Introduction
d i
SLAS 2012
Poster TP67
15
30
45
Time (minutes)
60
75
90
0
20
40
60
80
100
Time (minutes)
Figure 2EF. An eight point time course between AlkB and the eA containing 16-mer substrate was conducted. Chromatograms were extracted for the
substrate (eA, 1637.2797) the epoxide intermediate (epoxide, 1642.6064), the glycol intermediate (glycol, 1648.9287), and the product (A,
1629.2797). The resulting peaks were integrated using RapidFire Integrator and analyzed using Excel. In addition to the decrease of substrate over
time, the appearance of both intermediates as well as the reaction product were observed (2E). Data were also plotted to reflect Percent Conversion of
substrate over time (2F).