1 electrospray ionization mass spectrometry of the interaction of oligonucleotides with metals,...
TRANSCRIPT
1
Electrospray Ionization Mass Electrospray Ionization Mass Spectrometry of the Interaction of Spectrometry of the Interaction of
Oligonucleotides with Metals, Oligonucleotides with Metals, Small Molecules and DrugsSmall Molecules and Drugs
Janna Anichina Diethard K. Bohme
York University Department of Chemistry
Centre for Research in Mass Spectrometry Toronto, CANADA
ASMS 2007
2
Why Metal Ion – Ligand – Oligonucleotides?
In vivo processes with DNA are mediated by interactions with metal ions, small molecules and proteins.
The mechanisms of action of many anti-tumor drugs include the formation of their adducts with strands of
DNA only in the presence of metal ions.
ESI-MS has been successfully utilized in the study of interactions between DNA and Pt complexes important in chemotherapy (Beck et al. Mass Spec. Rev., 2001, 20, 61).
Systematic ESI-MS studies of metal ion - ligand - DNA interactions remain insufficient.
3
AA, CC, GG, TT, CCC, ATAT,
GCAT, GCGC, CATAC, ACTCG, AGTCTG,
TTAGGG, GCATGC
N
S
SN
O
NH
S+CH3
H3C
NH
OH
HO CH3H
HN
O
H CH3HO
H
CH3H
NH
OHHN
OOH
O
O
NH2O
OH
HO
O
HO
HO OHO
H
N
NH
ON N
CH3
H2N
H
NHH NH2
O
NH2
O NH2
NN
H2N
HN
NH
NH2
Na+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Fe3+, Co3+
Binding of oligonucleotides with metal complexes
Bleomycin A2
1,10-phenanthroline
Triethylenetetramine
4
Experimental
Instruments: MDS SCIEX API 2000 and Q Trap 2000.
Duplexes of the hexamers were prepared by heating 70 M ss in 70 mM aqueous NH4CH3COO to 900C for 10 min, neutral pH, and then cooled down slowly over a 3-hour period.
20 M solution prepared in 20:80 (vol/vol) methanol/water was injected into the ESI sources of the mass spectrometers.
Ratios metal cation to ligand to ss were 5:varied:1, [ss]0 = 20 M.
Flow rate: 5 L/min; N2 as the collision gas; collision voltages : -1 to -100 V in the negative mode.
y = 0.0415x + 1.2798y =0; x = -30.8 V
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
-55-50-45-40-35-30-25-20-15-10-50
Laboratory Collision Voltage /V
Re
lati
ve
Inte
ns
ity
TV
[d(5'GCATGC)2 - 3H]3-
5
Formation of duplexes ?!
0
10
20
30
40
50
60
70
80
90
100
400 600 800 1000 1200 1400 1600 1800
m/z
Re
lati
ve
Ab
un
da
nc
e (
%)
461
614.7
922.5
[d(5'TTAGGG) - 3H]3-
[d(5'TTAGGG) - 4H]4-
[d(5'TTAGGG) - 2H]2-
0
10
20
30
40
50
60
70
80
90
100
400 600 800 1000 1200 1400 1600 1800
Re
lati
ve
Ab
un
da
nc
e (
%)
596.3
447894.5
447 - [d(5'GCATGC) - 4H]4-
596.3 - [d(5'GCATGC) - 3H]3-
894.5 - [d(5'GCATGC) - 2H]2-
0
10
20
30
40
50
60
70
80
90
100
400 600 800 1000 1200 1400 1600 1800
m/z
Re
lati
ve
Ab
un
da
nc
e (
%)
614.7
922.5
1229.3
[d(5'TTAGGG)2 - 3H]3-
0
10
20
30
40
50
60
70
80
90
100
400 600 800 1000 1200 1400 1600 1800
Re
lati
ve
Ab
un
da
nc
e (
%)
894.5
1193.6
596.3
1343.11432.6 1791
1193.6 - [d(5'GCATGC)2 - 3H]3-
1343 - [d(5'GCATGC)3 - 4H]4-
SS DS
6
[d(5’TTAGGG)2 -3H]3- --> [d(5’TTAGGG) -H] - + [d(5’TTAGGG) -2H]2- 1229 1846 922.5
MS/MS of the duplexes of the hexamers
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
-50-46-42-38-34-30-26-22-18-14-10-6-2
Laboratory Collision Voltage /V
Re
lati
ve
Inte
ns
ity
[d(5'GCATGC)2 -3H]3-
[d(5'GCATGC) -2H]2-
[d(5'GCATGC) -H]-
w1-
[d(5’GCATGC)2 -3H]3- --> [d(5’GCATGC) -H] - + [d(5’GCATGC) -2H]2-
Underlined species were not observed due to the limited mass range
7
Metallation of ds hexamers
-Q1: 60 MCA scans from Sample 3 (q1ms_Cu(II)_BLM_TTAGGGan) of Janna_Feb5_07.wiff Max. 2.0e6 cps.
1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350 1360 1370m/z, amu
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
In
te
ns
ity, c
ps
1312.85
1292.95
1271.15 1293.75
1272.05
1315.15
1299.85 1321.551230.45 1251.05
1237.351231.25 1311.551273.35 1343.551251.851333.751213.15 1301.45
1285.05 1369.451241.05 1345.451274.951244.751264.95 1303.85 1306.95 1341.051210.15 1221.45
1288.851254.751226.15 1339.55 1355.551267.051228.15 1296.75
-Q1: 60 MCA scans from Sample 4 (q1ms_Zn(II)_TTAGGGan_neg) of Janna_Feb15_06.wiff Max. 8.1e6 cps.
1220 1230 1240 1250 1260 1270 1280 1290 1300 1310m/z, amu
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
Inte
ns
ity, c
ps
1230.05
1237.15
1251.151235.95
1271.551231.85 1241.45 1292.551263.551242.15 1258.05 1273.851234.35 1313.651274.55 1285.25 1306.351300.051245.851217.25 1233.65 1260.751223.35
[d(5’T2AG3)2 - 3H]3-
[d(5’T2AG3)2 - 3H]3-
[Znd(5’T2AG3)2-5H)]3-
[Zn2d(5’T2AG3)2-7H)]3-
[Zn3d(5’T2AG3)2-9H)]3-
[Nad(5’T2AG3)2-4H)]3-
[Cud(5’T2AG3)2-5H)]3-
[Cu2d(5’T2AG3)2-7H)]3-
[Cu3d(5’T2AG3)2-9H)]3-[Cu4d(5’T2AG3)2-11H)]3-
15 : 1
5 : 1
8
Dissociation of metallated duplexes
0
10
20
30
40
50
60
70
80
90
100
1200 1250 1300 1350 1400
m/z
Re
lati
ve
Ab
un
da
nc
e (
%)
1212.6
1231.6
1250.6
1269.6
1343
1212.6 - [Cod(5'GCATGC)2 - 5H]3-
1231.6 - [Co2d(5'GCATGC)2 - 7H]3-
1250.6 - [Co3d(5'GCATGC)2 - 9H]3-
1269.6 - [Co4d(5'GCATGC)2- 11H]3-
1343 - [d(5'GCATGC)3 - 4H]4-
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
-50-46-42-38-34-30-26-22-18-14-10-6-2
Laboratory Collision Voltage /V
Re
lati
ve
Inte
ns
ity
[Znd(5'GCATGC)2 - 5H]3-
[Znd(5'GCATGC) - 4H]2-
[d(5'GCATGC) - 2H]2-
[d(5'GCATGC) -H]-
Metallated duplexes dissociate into two
strands!
9
Dissociation pathways of metallated duplexesM = Mn, Fe, Co, Ni - pathway (1) dominates
M = Cu, Zn - pathways (1) and (2) are nearly equal
[Md(5’GCATGC)2 - 5H]3-
[Mss - 3H]- + [ss- 2H]2- (1)
[Mss - 4H]2-+ [ss- H]- (2)Pathway (3) dominates for all metals
[M2d(5’GCATGC)2 - 7H]3-[Mss - 3H]- + [Mss- 4H]2- (3)
[M2ss - 5H]- + [ss- 2H]2- (4)
[M3d(5’GCATGC)2 - 9H]3- [M2ss - 5H]- + [Mss- 4H]2- (5)
Underlined species were not observed due to the limited mass range
10
-48
-43
-38
-33
-280 5
Tan
gen
t V
olt
age
/V
Mn
CoNi
CuZn
Fe
Tangent voltages for the dissociation of singly, doubly and triply metallated double-stranded 5’GCATGC3’ trianions.
TV ([d(5’GCATGC)2 - 3H]3-) = -(30.7 0.2) V
[Md(5’GCATGC)2 - 5H]3-
[M2d(5’GCATGC)2 - 7H]3-
[M3d(5’GCATGC)2 - 9H]3-
No metal present
11
[Fe(II)BLM]2+ [O2Fe(II)BLM]2+
H2O2O2
[HOOFe(III)BLM]2+ [Fe(III)BLM]3+
1e, 1H+
"activated BLM"
"peroxide shunt"
substrate DNA
oxidation products(DNA strand scission)
Kryatov et al. Chem. Rev., 2005, 105, 2175-2226
2+
Metallated Bleomycin A2 adducts with ds hexamers
N
S
SN
O
NH
S+CH3
H3C
NH
OH
HO CH3H
HN
O
H CH3HO
H
CH3H
NH
OHHN
OOH
O
O
NH2O
OH
HO
O
HO
HO OHO
H
N
NH
ON N
CH3
H2N
H
NHH NH2
O
NH2
O NH2
Metal binding domain
Linker region
alfa -D-Mannose
alfa -L-Gulose
Pyrimidinylpropionamide
beta-Aminoalanine
12Arrows indicate potential hydrogen-bond donors or acceptors. Note the
crescent shape of the fragment
Chen, J. and Stubbe J. Cur. Op. Chem. Biol., 2004, 8, 175 - 181
N
S S
N
O
NS+
H3C
H3C
N
H
O
H
13
[MBLMd(5’GCATGC)2 - 6H]4- [d(5’GCATGC) - 2H]2- +[MBLMd(5’GCATGC) - 4H]2-
CID profiles of [MBLMd(5’GCATGC)2 - 6H]4-
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
-50-46-42-38-34-30-26-22-18-14-10-6-2
Laboratory Collision Voltage /V
Re
lati
ve
Inte
ns
ity
[MnBLMd(5'GCATGC)2 - 6H]4-
[MnBLMd(5'GCATGC) - 4H]2-
[d(5'GCATGC) - 2H]2--45
-43
-41
-39
-37
-35
-33
-31
-29
-27
-250 6
Ta
ng
en
t V
olt
ag
e /V
TTAGGG
GCATGC
Mn
Ni
Co
Cu
Zn
BLM = (Bleomycin A2 - H+); M = Mn, Co, Ni, Cu
14
[d(5’GCATGC) – 2H]2- + [ZnBLMd(5’GCATGC) – 4H]2-
[ZnBLMd(5’GCATGC)2 – 6H]4-
[Znd(5’GCATGC) – 4H]2- + [BLMd(5’GCATGC) – 2H]2-
Special case of Zn(II) containing complex
0
0.1
0.2
0.3
0.4
0.5
0.6
-50-46-42-38-34-30-26-22-18-14-10-6
Laboratory Collision Voltage /V
Re
lati
ve
Inte
ns
ity
[ZnBLMd(5'GCATGC)2 - 6H]4-
[ZnBLMd(5'GCATGC) - 4H]2-
[d(5'GCATGC) - 2H]2-
[Znd(5'GCATGC) - 4H]2-
[BLMd(5'GCATGC) - 2H]2-
Zn2+ has higher affinity for the phosphate groups of DNA compared to BLM!
15[MLnds - 5H]3- [MLn-1ds - 5H]3- + L with n = 1-3
0.00.1
0.20.30.40.5
0.60.70.8
0.91.0
-50-46-42-38-34-30-26-22-18-14-10-6-2
Laboratory Collision Voltage /V
Re
lati
ve
Inte
ns
ity [NiL 3d(5'GCATGC3')2 -5H]3-
[d(5'GCATGC3') -2H]2-
[NiL 2d(5'GCATGC3')2 -5H]3-
[NiL d(5'GCATGC3')2 -5H]3-
[Ni(5'GCATGC3')2 -5H]3-
ESI/CID of 1,10 - phenanthroline-containing complexes
0.E+00
1.E+05
2.E+05
3.E+05
4.E+05
5.E+05
6.E+05
7.E+05
1100 1200 1300 1400 1500 1600 1700 1800
m/z
Inte
ns
ity
, cp
s
ds3-
LCods3-
Cods 3-
L2Cods3-
Co 2ds 3- LCo2ds3- L2Co2ds3-
L3Cods3-Co 3ds 3-
L3Co2ds3-
L4Co2ds3-
LCo3ds3-
L2Co3ds3-
L3Co3ds3-
L4Co3ds3-
[LnComd(5'GCATGC)2 - (2xm+3)H]3-
with n in the range 1 - 3m, m being 1, 2 and 3
NN
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
-30-28-26-24-22-20-18-16-14-12-10-8-6-4-2
Re
lati
ve
Inte
ns
ity
[CoL 2 d(5'GCATGC3')2 -5H]3-
[CoL d(5'GCATGC3')2 -5H]3-
[Cod(5'GCATGC3')2 -5H]3-
[d(5'GCATGC3') -2H]2-
Co2+ : Phen : ss 5:5:1
16
Species ssML2- ssML22- ssML3
2- dsML3- dsML23- dsML3
3- logK1 logK2 logK3
Co -(22.20.4) -(23.10.4) -(22.40.5) -(26.00.3) -(25.60.3) -(36.20.4) 7.02 6.7 6.28
Ni -(30.10.3) -(24.70.3) -(30.40.4) -(32.20.4) -(34.60.4) -(34.10.3) 8.0 8.0 7.9
Cu -(25.00.4) -(23.50.4) -(25.70.7) -(29.50.2) -(27.00.3) -(36.00.4) 8.82 6.67 5.02
Species ssML2- ssML22- ssML3
2- dsML3- dsML23- dsML3
3- logK1 logK2 logK3
Co -(22.20.4) -(23.10.4) -(22.40.5) -(26.00.3) -(25.60.3) -(36.20.4) 7.02 6.7 6.28
Ni -(30.10.3) -(24.70.3) -(30.40.4) -(32.20.4) -(34.60.4) -(34.10.3) 8.0 8.0 7.9
Cu -(25.00.4) -(23.50.4) -(25.70.7) -(29.50.2) -(27.00.3) -(36.00.4) 8.82 6.67 5.02
Onset energies (in volts) for the dissociation of thesingly metallated double-stranded d(5’GCATGC)
containing 1, 2 and 3 Phens
The last 3 columns contain common logarithms for the binding constants of stepwise coordination ofCo(II), Ni(II) and Cu(II) with1,10-phenanthroline at 298 K and ionic strength =0.1
OEs (in volts) of the dissociation of 1:1, 1:2and 1:3 d(5’GCATGC)2 - [M(II)L3]
2+
complexes
Co Ni Cu
dsML33- -(36.20.4) -(34.10.3) -(36.00.4)
dsM2L63- -(39.0 0.4) -(38.10.6) -(39.90.5)
dsM3L93- -(79.8 0.5) -(81.30.7) not determined
Relative stabilities of Phen-containing species
Coggan et al. Inorg. Chem., 1999, 38, 20, 4496
-82
-77
-72
-67
-62
-57
-52
-47
-42
-37
0 .5
Ta
ng
en
t V
olt
ag
e /V
-82
-77
-72
-67
-62
-57
-52
-47
-42
-37
0 .5
Ta
ng
en
t V
olt
ag
e /V
[dsML3]3-
[ds(ML3)2]3-Co Cu
Ni
-82
-77
-72
-67
-62
-57
-52
-47
-42
-37
0 .5
Ta
ng
en
t V
olt
ag
e /V
Co Ni
[ds(ML3)3]3-
-
N
N
N
N
N
N
M
N
N
N
N
N
N
M
17
Special case of Trien as the ligand
Trien forms mixed complexes with oligonucleotides only in the presence of copper (II) !
CID spectrum of [CuTriend(5'GCATGC)2 - 5H]3-
(1262.6) at Lab Collision Voltage - 40 V
0.0E+00
2.0E+02
4.0E+02
6.0E+02
8.0E+02
1.0E+03
1.2E+03
0 500 1000 1500
m/z
Inte
ns
ity
, cp
s
1262.61214
895
[Cud(5'GCATGC)2 - 5H]3-
[d(5'GCATGC) - 2H]2-
[CudsTrien]3- -(35.70.3) V [CudsPhen]3- -(29.50.2) V
NHNH
NH2NH2
Cu
2+
18
Conclusions
ESI/CID provides insight into Metal ion - Ligand - DNA interactions: the stoichiometry and mode of binding, the dissociation pathway and relative gas phase stabilities.
Future Plans
Systematic ESI/CID studies of Metal ion - Drug - DNA interactions are needed to establish general trends in the gas-phase stability, dissociation mechanisms.
Investigation of the intrinsic reactivity of metallated biological ions toward gaseous carcinogens and other harmful compounds using the Q-trap 2000 and ESI SIFT QqQ instruments.
Solution and gas-phase experiments with double-stranded sequences containing a mismatching base pair and various intercalating species.
19
Prof. D. K. Bohme
Greg Koyanagi
Michael JarvisAndrea DasicSara HashemiTuba Gozet Stefan Feil
Mike DuhigVoislav Blagojevic
$$ NSERC MDS SCIEX
Acknowledgements