Nucleic Acids Research
Volume 3 no.11 November 1976
Detection and identification of minor nucleotides in intact deoxyribonucleic acids
by mass spectrometry.
J.L.Wiebers
Department of Biological Sciences, Purdue University, West Lafayette,
IN 47907, USA
Received 26 July 1976
ABSTRACT
A mass spectral method is described for the detection and identification
of unusual nucleotide residues present in DNAs. Analysis by this method of
intact, underivatized DNA from salmon sperm, calf thymus, mouse L-cells,
wheat germ, M. lysodeikticus, 15. coli, and the bacteriophages 0X-174, fd, and
lambda, yields diagnostic ions for the four common components of DNA as well
as characteristic ions for 5-methyldeoxycytidine residues. The spectrum from
T2 DNA contains ions indicative of 5-hydroxymethyldeoxycytidine and 5-methyldeoxycytidine components but no ions corresponding to deoxycytidine residues.
The DNAs of phages fd and 0X-174 also display ion products indicative of
N6-methyldeoxyadenosine residues. Additional series of ions in the spectra of
all four bacteriophage DNAs suggest the presence of 5-substituted deoxyuridine
residues. The detection method exhibits considerable sensitivity in that
amounts of DNA as low as 0.01 A 2 6 0 nm u n i t s c a n b e u s e d i n t n e analysis, and
thus, the procedure should prove of some value in the detection and location
of modified components in specific regions of the various genomes by analysis
of the appropriate endonuclease restriction fragments.
INTRODUCTION
It has been demonstrated previously that intact DNA or polydeoxyrlbonucleotides can be subjected to mass spectrometric analysis without prior
derivatization or chemical or enzymatic hydrolysis (1,2).
When a polydeoxy-
ribonucleotide is exposed to the pyrolytic and electron impact conditions of
the mass spectrometer, the primary fragmentation process involves cleavage at
the phosphodiester bonds linking the nucleotide residues.
Subsequent frag-
mentations result in ion products which are diagnostic for the common nucleotide components of the polynucleotide (3,4).
The general scheme proposed for
the mass spectral fragmentation together with the suggested structures of the
ion products are outlined in Figure 1.
Ion a^ is considered to be the first
volatile product that is released from the polynucleotide chain and, thus, is
susceptible to electron impact, which fragments the molecule to the purine or
pyrimidine base and to methylfuran.
Subsequently, ions b_ and c_ are formed
through the attachment of one or two methylfuran moieties to ion <i (3,5).
Ion
d^ is the consequence of the attachment of a PO3 moiety to the exocyclic amino
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© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
Nucleic Acids Research
BASE
BASE
BASE
ION d
lONo
CH
BASE FRAGMENT
.BASE
CH,
• INDICATES ATTACHMENT SITE
NOT RESOLVED. METHYLFURAN
LOSES I H WHEN ATTACHED.
ION b
CH
3
ION c
Figure 1.
Proposed scheme for the electron impact and pyrolytic
fragmentation of polydeoxyribonucleotides.
group of the base (presumably through a phosphoramidate linkage) and this ion
only appears in spectra of nucleotides that contain a free exocylic amino
group (3). The purine or pyrimidine base fragment and the ion types a_, b_, c^,
and d^, are specific for each of the common deoxyribonucleotide residues
found in DNA and they have been used to yield sequence information in a novel
method for the ordering of nucleotides in oligodeoxyribonucleotides (3,4).
This communication demonstrates that the same types of ion products
documented above can be used to detect modified nucleotide residues present
in polydeoxyribonucleotides, and, describes a mass spectral method for the
detection and identification of unusual components in DNAs from various
animal, plant, bacterial, and viral sources.
The method differs from other
methods used to detect modified residues in that
(a) no prior chemical or
enzymatic treatment of the DNA molecule is required before the analysis, and,
(b) exceedingly small amounts of the intact material can be analyzed directly,
an attribute that should prove particularly useful in the detection of
modified nucleotides in limited amounts of endonuclease restriction fragments
of DNA.
MATERIALS AND METHODS
The DNAs from the bacteriophages T2, lambda, fd, 0X-174, and the DNA from
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M. lysodiekticus were obtained from Miles Laboratories, Elkhart, IN. DNAs
from calf thymus and wheat germ as well as the satellite DNA from mouse L-cells
were kindly provided by Dr. John H. Spencer, McGill University.
The wheat
germ DNA had been prepared as described previously (6); the calf thymus DNA
by the method of Kay e_t aL. (7) ; and the mouse L-cell satellite DNA as
described by Harbers et_ aX. (8). DNA from 12. coli and from salmon sperm was
obtained from Calbiochem, La Jolla, CA. Poly(dA-dT) and poly(dl-dC) were
obtained from Miles Laboratories, Elkhart, IN.
5-Methyldeoxycytidine
5'-phosphate, deoxyinosine 5'-phosphate, and 5-hydroxymethyldeoxycytidine
were obtained from Sigma Chemical Co., St. Louis, MO.
Mass Spectral Analysis.
Solutions containing 0.01 to 1.0 A2gQnm
un
*ts
of the compounds were introduced into capillary sample tubes and taken to
T ie s a m
dryness in vacuo in a desiccator containing P2°5'
*
P l e s were intro-
duced into the spectrometer (DuPont 21-490 B) by direct probe, slowly heated
to about 250°, and, the analysis was carried out at 70 eV, 3 X 10
source temperature, 200°.
Torr,
Spectra were recorded at the point at which the
maximum number of ions were generated as indicated by the ion monitor.
Multiple analyses were performed on each compound to confirm the reproducibility of the spectra.
RESULTS AND DISCUSSION
Diagnostic ions for common and minor components of DNA.
The mass spectrum of salmon sperm DNA CFigure 2) indicates the major
100
135
A
Fig.
8
T
126
A
215
c
in
8
.ATIVE INTENSIIIT
2
SALMON SPERM DNA
80-
A
oe
G
231
T
117
A
c
162
20-
C
ll
100
ll J llljll 1
20
160
140
.f
&
-gjT
180
TC
1 ll, 1 l l 2 0 6
200 ' 220
256
A ;,
1 ,f
240
2&I
C
f5
271 Q j j
280
'
1
300
G
311
A
I^S
. , .
320
• :
340
1
365
i.T
3to '
,r
3 80
G
391
1.
400
Figure 2. Mass spectrum of salmon sperm DNA. A, G, C, and T, indicate
the ions that arise from deoxyadenosine, deoxyguanosine,
deoxycytidine and thymidine residues, respectively. In
Figures 2-14, peaks indicated by broken lines are drawn at
10 times their actual relative intensity.
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ions that can be used to detect the four common nucleotide components of DNA.
The mass values for these diagnostic ions are listed in Table I.
The corre-
sponding ion types appear in spectra of unusual nucleotides such as deoxyinosine 5'-phosphate or 5-methyldeoxycytidine 5'-phosphate (Table I ) .
Table I.
For
Mass Values (m/e) of Diagnostic Ions Derived from Residues in DNA
and Polydeoxyribonucleotides.
Nucleoside Residue
Ion Type
dA
dC
dG
dT
d-m5C
d-hm5C
d-m6A
dl
135
111
151
126
125
141
149
136
a
215
191
231
206
205
221
229
216
b
295
271
311
286
285
301
309
296
c
375
186
391
202
366
*
365
d
351
162
381
192
389
*
376
*
Base + H
176
*
Ions belonging to the d_ class are derived from PO3 attachment to exocyclic amino groups of the bases and do not appear when such groups are
either absent or alkylated.
example, the spectrum of the latter compound (Figure 3) shows ion a_ at m/e
205; ion b_ at m/e 285; ion c_ at m/e 365; and ion c[ at m/e 176.
It may be
observed (Table I) that the ion mass values deriving from a 5-methyldeoxycytidine residue are distinct from those originating from a cytidine moiety,
and, although the values of the modified residue differ by only one mass unit
from those ions generated from a thymidine residue, the presence of the ion
type d^ serves to further distinguish the two residues since this ion cannot
be formed from thymidine.
By analogy with these results, the expected values
for the ions from N6-methyldeoxyadenosine and 5-hydroxymethyldeoxycytidine
residues can be calculated (Table I).
For all of the nucleoside residues,
the ion types ji, b_, and c_ have particularly useful diagnostic value because
their peaks fall in the higher mass range of the spectrum and consequently
are not masked by the numerous ions that appear in the lower mass region of
the spectrum.
Furthermore, these ion species have intensity values that
permit them to be distinguished from the spectral background even when the
ions are derived from minor components of the DNA.
Detection of 5-methyldeoxycytidine residues in DNA.
5-Methylcytosine is known to be present in the DNAs of numerous members
of the animal and plant kingdoms (9), and the mass spectral analyses of DNAs
of some representative species clearly indicate the presence of this minor
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00
E5
Fig. 3
S-METHYLDEOXYCYTIDINE-S-PHOSPHATE
80-
60
HO-?-O-CH, " ^ N ^ °
f\, J]
40-
Oh H
20"
176
IO9
1,
100
120
205
162
140
160
180
200
,256
.285
i
260
2 8 0 3 6 0 3 2 0 3 4 0 ' 3 6 0 ' 3 8 0 ' 4 b 0
; 365
1.
.1.
220
240
WHEAT GERM DNA
>
<i 40
262
|
J28S
I
308
•LJ
100
120
140
160
180
200
oo-
220
a«0
260
280
300
320
340
360
380
400
35
Fig. 5
135
CALF THYMUS DNA
80-
60
126
3«
40-
20"
ii
100
117
,62
72
l|l 1 ||
120
111,, 1 ,,,
140
160
282
,1845
.
176
180
1, Iff Hi
200
220
T
J
^
240
295
348
311
_ iT 1
2ei 6 27i
260
] j?86.!
280
300
,39,
,382 jj
320
340
360
380
400
MOUSE L-CELL SATELLITE DNA
_,
11,11 yu
(40
160
" " —uii-ju •" • " , '""
• "• ['!•
" '•
•" "
^"-1-—|—LU—,
pi 1
,
, '•,• ,—u—,—j^-,
^
r_j
I 8 O 2 0 0 2 2 0 2 4 O 2 6 O 2 8 0 3 O O 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0
m/e
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component.
For example, the spectra of equal quantities (1.0 A o , n
unit) of
the DNA from salmon sperm (Figure 2), wheat germ (Figure 4 ) , calf thymus
(Figure 5 ) , and mouse L-cell satellite DNA (Figure 6) exhibit the ions at m/e
176, 205, 285, and 365 that are diagnostic for this particular residue as well
as the corresponding ions for the common components.
The spectra of bacterial
DNA from E. coli (Figure 7) and M. lysodeikticus (Figure 8) also display the
ions that are indicative of this minor residue.
As a control, analyses on the
same quantities of the synthetic polynucleotides poly(dA-dT) (Figure 9) and
poly(dl-dC) (Figure 10) yield only the expected peaks for dA, dT, dC, and dl
(Table I ) .
Although the lower limit of detectability of 5-mechyldeoxycytidine in
DNA by this method has not yet been determined, the content of this component
in some DNAs has been derived by chemical and chromatographic methods, and the
mass spectral results can be compared with the published values.
For example,
the value for wheat germ DNA has been reported as 5.6 moles per 100 gram atoms
of DNA phosphorous (6), and the corresponding values for calf thymus DNA (10)
and mouse L-cell satellite DNA (8, 11, 12) are 1.3 to 1.9 moles and 3.5 to
4.6 moles respectively.
In this regard, it is of interest that mass spectra
units) than the
recorded on sample sizes 100-fold less (i.e., 0.01 A-,260nm
amounts indicated above still permit the detection of the characteristic ions
for the 5-methyldeoxycytidine residue.
Detection of 5-hydroxymethyldeoxycytidine residues in T2 DNA.
The mass spectrum of bacteriophage T2 DNA (Figure 11) is of particular
interest because this DNA is known to have all of its deoxycytidine residues
hydroxymethylated (13). The spectrum of T2 DNA exhibits none of the diagnostic
ions that originate from deoxycytidine residues (Table I ) , but rather, the
expected values for ions deriving from 5-hydroxymethylcytidine residues
(Table I) . The results from this particular DNA emphasize the validity
of the mass spectral detection method since they qualitatively confirm the
findings of the chemical composition studies on the DNA.
Mass spectra of DNAs from bacteriophages fd, lambda, 0X-174, and T2.
The spectra derived from the four bacteriophage DNAs are strikingly
different from those discussed above in that they are much more complex and
indicate the presence of several different minor purine and pyrimidine
components.
The spectra of fd DNA (Figure 12), lambda DNA (Figure 13) and
0X-174 DNA (Figure 14) show the diagnostic ions for the four common components
as well as a number of additional ions.
the latter class are listed in Table II.
2964
Some of the more prominant members of
It can be observed that the ions for
as
on
S3
RELATIVE INTENSITY
RELATIVE INTENSITY
•33
CD
tn
CO
o'
Nucleic Acids Research
T2 DNA
u f mii I.I
100
120
• ••••^•-
•"!
,!250^ |i
j i , i 1256 !;268
t
i ill
|l
— 11111
140
.1 i, I ill,
160
ISO
^ •
*•
• j
• mi
' • I
••
^
•
200
•
!••
220
.
*
j i
240
Ti
L
• •
260
I i 7
i
•
280
II
1
|
["
300
'
'
•
320
I
343
,|, .348
'
i
340
•
:37=
365 .:. 381
360
OS
Fi«.l2
80-
fd VIRAL DNA
60280
106
2
5
.295
229
170
,iii|i,ii,,iin III
00
J
III
348
183
Hi! II i, i ii
1 ,,, 1
I2?5,: .i: 2 ? 9
ii,?"
330
375
389
i
35
Fig. 13
LAItfBDA PHAGE DNA
80
60
40ce in
20
11
100
231
r'O
126
1 120
„„ 1 140
1
'•7
160
B3B6,a . a 2 ?
1. 1 1. 1. ]3 »
180
200
T U
^°
"d220 ji!2 4 0
2 3
f 271 282
260
320
300
368
343
311
^
280
340
360
380
400
~26~
Fkj.M
<J>X-174 ONA
as
91
KX)
120
140
160
180
206&I
22?236
l i _ IJTRlli
kl I L
200
220
240
• , Saoasa
if;
iiii! l i i i
ill U
"f?:
Lii !• : ' : !
•'. ~ i
I si
m/e
2966
260
280
••
300
SU
320
340
369
™,!
36»f.
382^:;
360
380
400
Nucleic Acids Research
Table II.
Diagnostic Ions Derived from Modified Residues in Bacteriophage
DNAs.
Residue and Diagnostic Ions
T2
X
fd
0X-174
5-methyldeoxycytidine
m/e 176,205,285,365
+
+
+
+
5-hydroxymethyldeoxycytidine
m/e 141,221,301,381
+
-
-
-
N6-methyldeoxyadenosine
m/e 149,229,309,389
-
-
+
+
5-carboxymethyldeoxyuridine
m/e 152,170,250,330
+
+
+
+
5-(4',5'-dihydroxypentyl)deoxyuridine
m/e 128,153,183,214,263,343
+
+
+
+
5-methyldeoxycytidine residues appear In all four viral DNAs, whereas ions
representative of 5-hydroxymethyldeoxycytidine residues appear only in the T2
DNA.
Ion products that have mass values corresponding to those which could
originate from N6-methyldeoxyadenosine residues (Table I) can be observed in
the spectra of the DNA from fd and 0X-174 (Table II). This modified residue
has already been reported to be present in some viral DNAs (9,14).
All four DNAs show two series of Ions (Table II) that could be characteristic of substituted deoxyuridine residues.
While there is at present no firm
evidence that these ion products actually derive from such types of modified
nucleosides in these DNAs, a possible explanation for their presence is that
they arise in the one case from 5-carboxymethyldeoxyuridine residues and, in
the other, from 5-(4',5'-dihydroxypentyl)deoxyuridine residues.
For example,
the ion at m/e 170 corresponds to the parent ion for 5-carboxymethyluracil
(15), and the ions found at m/e 250 and 330 are the values expected for ion
types a_ and b_ derived from the corresponding nucleoside.
With regard to
5-(4',5'-dihydroxypentyl)deoxyuridine residues, Brandon et al. (16) have reported that, in the bacteriophage SP-15 of J3. subtilis, about 43% of the
thymidine residues are replaced by this modified component.
In determining
the structure of the pyrimidine base derived from this modified nucleoside,
these investigators showed that its mass spectrum contained a parent ion at
m/e 214 (in low abundance) with major fragment ions at m/e 128, 153, and 183.
The same series of ions appear in the spectra of the four bacteriophage DNAs
in this study (Table II) together with ions at m/e 263 and 343.
It is not
unlikely that this latter pair of mass values correspond to ions &_ and b_
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Nucleic Acids Research
that, in this case, are formed by the attachment of one or two methylfuran
moieties to the c o H ii N 2°3
structure
(m/e
183
:
M+
~
3 1
) • which, in turn, de-
rives from the unstable parent ion at m/e 214.
In the spectra of the four phage DNAs there are a number of other series
of ion products that appear to correspond to the a_, b_, and c^ pattern, and
these are currently under investigation.
CONCLUSION
The results of this investigation demonstrate that minor residues such
as 5-methyldeoxycytidine, 5-hydroxymethyldeoxycytidine, and N6-methyldeoxyadenosine in DNAs can be detected and identified by mass spectral analysis of
the intact, untreated DNA.
It will be necessary to carry out further studies
on a number of DNAs as well as model nucleotides to provide reliable identification of other modified components.
Such studies will be carried out on the
DNA of the bacteriophage SP-15 of 15. subtilis, mentioned above; the DNA of
Pseudomonas acidovorans in which about 50% of the thymidine residues are
replaced by 5-(4-amlnobutylaminoethyl)deoxyuridine residues (17); bacteriophage PBS2 DNA in which all of the thymidine residues are replaced by deoxyuridine residues (18), and bacteriophage 0c DNA in which 5-hydroxymethyldeoxyuridine replaces the thymidine residues (19). In view of the sensitivity
of the method, it will also be necessary to consider the possibility of contamination of such bacteriophage DNA by host DNA and thus, analyses of both
host and viral DNA should be performed.
Furthermore, it will be advantageous
to define the limits of sensitivity of the method for the detection of minor
components in DNA.
With regard to this aspect, further study on the bacterio-
phage 0X-174 should prove of interest since it has been reported (20) that DNA
from a lysis-defective mutant of phage 0X-174, am3, contains a single 5-methyldeoxycytidine residue per DNA molecule and that this residue is located in a
specific region of the 0X-174 genome, very likely in gene H.
It should be
possible to confirm this conclusion by a mass spectral study of this DNA and
its endonuclease restriction fragments.
Mass spectrometry has been used previously to detect minor nucleosides in
DNA C21); however, the method differs markedly from the one described here in
that it involves hydrolysis of large quantities of DNA, a series of manipulations for the preparation of the hydrolysate prior to derivatization, subsequent trifluoroacetylation, and, in some cases, the introduction of the
sample into the mass spectrometer via gas chromatography.
An attribute of
the method is that exact mass values for the derivatized minor nucleosides
can be obtained from the high resolution spectra and, thus, the method is of
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Nucleic Acids Research
particular value for confirmatory purposes.
ACKNOWLEDGEMENT
This investigation was supported by National Science Foundation Grant
No. BMS-74-22213.
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Gray, M. W. and Lane, B. G. (1968) Biochemistry 7, 3441-3453.
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Takahashi, I., and Marmur, J. (1963) Nature (London), 197, 794-795.
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Roscoe, D. H., and Tucker, R. G. (1966) Virology 29, 157-166.
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Nucleic Acids Research
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Lee, A. S. and Sinsheimer, R. L. (1974) J. Virol. 14, 872-877.
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Koenig, W. A., Smith, L. C , Crain, P. F., and McCloskey, J. A. (1971)
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