1 electrospray ionization mass spectrometry of the interaction of oligonucleotides with metals,...

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-



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


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


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


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


Re

lati

ve

Inte

ns

ity

[ZnBLMd(5'GCATGC)2 - 6H]4-

[ZnBLMd(5'GCATGC) - 4H]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


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-


[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

1 electrospray ionization mass spectrometry of the interaction of oligonucleotides with metals,...

Documents

gasphase experiments

gasphase stability

dna interactions

relative gas phase stabilities

metal ion ligand

dissociation pathway

conclusions esicid

dissociation mechanisms