Charge state effect on the zwitterion influence on stability of non-covalent interaction of single-stranded DNA with peptides

JOURNAL OF MASS SPECTROMETRYJ. Mass Spectrom. 2007; 42: 1613–1622Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jms.1359

Review

Charge state effect on the zwitterion influence onstability of non-covalent interaction of single-strandedDNA with peptides

Sandra Alves,1 Amina Woods2 and Jean Claude Tabet1∗

1 Laboratoire de Chimie Structurale Organique et Biologique, UMR 7613/BP45, Universite Pierre et Marie Curie, 4 Place Jussieu, Paris 75252, France2 NIDA IRP, NIH, 333Cassell Drive, Baltimore, MD 21224, USA

Received 25 May 2007; Accepted 27 October 2007

Negative ion ESI mass spectrometry was used to study the gas-phase stability and dissociation pathwaysof peptide–DNA complexes. We show that bradykinin and three modified peptides containing the basicresidue arginine or lysine form stable interactions with single-stranded oligonucleotides. ESI-MS/MSof complexes of T8 with PPGFSPFRR resulted in a major dissociation pathway through cleavage ofthe peptide covalent bond. The stability of the complex is due to electrostatic interaction between thenegatively charged phosphate group and the basic side chain of the arginine and lysine residues asdemonstrated by Vertes et al. and Woods et al. In fact, the present work establishes the role played byzwitterions on complex stabilisation. The presence of protons in nucleobase and/or amino acid contributesin reinforcing the strength of the salt bridge (SB) interaction. The zwitterionic form of the most basicof amino acid residues, arginine, is assumed to form a strong SB interaction to the negatively chargedphosphate groups of DNA. This non-covalent complex is stable enough to withstand disruption of thenon-covalent interaction and to first break the covalent bond. Moreover, the dependence of fragmentationpatterns upon the complex charge state is explained by the fact that the net number of negative chargesmodulates the number of zwitterionic sites, which stabilise the complexes. Finally, the weak influence ofthe nucleobase is assumed by the existence of competition for proton addition between the nucleobaseand the R/K side chain leading to a decrease in the stabilisation of the SB interaction. Copyright 2007John Wiley & Sons, Ltd.

KEYWORDS: noncovalent complexes; salt bridge interaction; ESI mass spectrometry

INTRODUCTION

The search for better understanding of protein struc-ture–function belongs to the forefront of biochemicalresearch, owing to the crucial role of proteins in biologicalprocesses. Nucleic acid/protein non-covalent interactionsare involved in DNA processing and packing as wellas in gene regulation and expression. Non-covalent com-plexes have been extensively studied in the gas phaseusing mass spectrometric techniques1 – 3 most often usingelectrospray ionisation (ESI).4 Indeed, the soft ionisation fea-ture of electrospray allows the weakly bound non-covalentcomplexes formed in solution to be transferred into thegas phase and detected by mass spectrometry. Molecu-lar weight and consequently complex stoechiometry1 were

ŁCorrespondence to: Jean Claude Tabet, Laboratoire de ChimieStructurale Organique et Biologique, UMR 7613/BP45, UniversitePierre et Marie Curie, 4 Place Jussieu, Paris 75252, France.E-mail: [email protected]

determined, and recently analytical approaches were devel-oped to determine the binding constants.5 However, in mostexamples non-covalent complexes are preferentially formedand folded in the presence of salts, leading to ESI signaldecrease or suppression.6 Efforts have been made to useMALDI ionisation,7 although it is a more energetic ionisa-tion method.8 A few examples demonstrate that MALDI isindeed an alternative technique within optimised experimen-tal conditions,9 – 11 as shown by the use of a less acidic matrix.The sample preparation using 6-aza-2-thiothymine (ATT) asmatrix in the presence of ammonium citrate allowed thedetection of intact double-stranded oligonucleotides.12 Sim-ilarly ATT as matrix was used for the detection of specificDNA–peptide complexes.13 – 15

In the present work, we use ESI mass spectrometry toinvestigate the gas-phase stability and dissociation pathwaysof non-covalent DNA–peptide complexes. Different types ofinteractions are able to stabilise DNA complexes in solution,16

including hydrogen bonds, van der Waals, hydrophobicand electrostatic interactions. There is no consensus on the

Copyright 2007 John Wiley & Sons, Ltd.

1614 S. Alves, A. Woods and J. C. Tabet

quantitative contribution of these interactions. But gas-phasecomplex structures17 are expected to interact mainly throughsalt bridge (SB) formation and/or hydrogen-bonding. Somegas-phase studies of the peptide–DNA interactions havebeen undertaken.13 – 15,18,19 Vertes et al.18 first demonstratedionic interactions in DNA–peptide complexes. Woods et al.highlighted the role of electrostatic interactions between pep-tides containing several basic residues (R or K) and peptidescontaining acidic20,21 or phosphorylated residues21 – 25 andalso with the phosphate groups in DNA strands.13,14

In our case, we observed that a single DNA strandand different modified bradykinins that contain basic Rand/or K residues (one containing the motif RR, denoted-RR) interact non-covalently and show different dissociationpatterns depending upon the peptide sequence and alsoupon the charge state of the complex.26 ESI MS/MSof �-RR C DNA � 3H�3� and �−RR C DNA � 2H�2� anionsrevealed a major dissociation pathway in which the complexis dissociated along a covalent peptidic bond (loss of PPmotif). Covalent bond fragmentation from non-covalentspecies has been already reported for DNA duplexes27,28 andDNA complexes with ligand.29 The purpose of this study isto identify the structural elements that drive the interaction.Our group previously reported the detection of non-covalentcomplexes between highly acidity oligonucleotides andhighly basic PNA molecules using ESI mass spectrometryand assumed the existence of the zwitterion (ZW).30 We aimat establishing the role played by the ZW on DNA–peptidecomplex stabilisation.

Amino acids, the building blocks of peptides and pro-teins, are known to exist in aqueous solution as ZWs. But theexistence of zwitterionic form in the gas phase is less com-mon and is still a topic of on-going discussion. A stabilisationof charge separation is dependent on the gas-phase basic-ity of the proton-donating group and the gas-phase acidityof the proton acceptor groups involved.31,32 The most basicamino acid, arginine, has an extremely basic guanidiniumgroup, and calculations of the zwitterionic and neutral formsof arginine suggest that they are comparable in energy,although the neutral form is still more stable.33,34 Neverthe-less, recent studies showed that small molecules can existas charge-separated; for exemple, methylation of the argi-nine side chain is sufficient to favour the zwitterionic state.35

Most of the studies have investigated the increased stabilityof ZWs by an external factor. Zwitterionic states can be sta-bilised by the addition of solvent molecules,36 and/or nearbycharges.32,37 – 41 Counterions stabilise charge-separated struc-tures through the formation of SB interactions.32,37 – 39,42 – 46 It isinteresting to note that only a few water molecules are neededto stabilise ZWs, and hydrated gas-phase biomoleculescan be formed by electrospray ionisation.47 ZW states arealso favoured by cluster formation.40,48 Protonated dimerof arginine33,49 forms SB structures (where a monomer isin the ZW state and interacts with neutral arginine), andlarger neutral arginine clusters are stable in ZW states.50

The existence of ZWs for peptides is less well documented,but is probably promoted by self-solvation. SB interac-tions have been shown for peptide ions. Charge-separatedstructures have been assumed in different experimental

studies (H/D exchange,49,51 – 53 fragmentation pathways ofcationised species54). The most stable structure of proto-nated bradykinin (denoted as R-R) displays zwitterionicposition stabilised by self-solvation49,51,52,55,56 (intramolec-ular H-bonding between protonated guanidinium groupswith the deprotonated carboxylic of C-terminal). However,in most short peptides, the zwitterionic form implies a fold-ing to bring the NH3

C to the COO� group, which constrainsthe peptide backbone (unfavourable structures).57,58

The zwitterionic form at the bradykinin C-terminal sideis assumed to interact through an SB with the deprotonatedsugar phosphate groups of DNA strands, which stronglystabilise the complexes. The aim of the present work is toestablish the role played by (1) ZWs on complex stabilisationand (2) the charge state influence on the cleavage orientationof multiply charged species and on the presence of ZWs.

EXPERIMENTAL

Peptide and DNA sample preparationPeptides (PPGFSPFRR, RPPGFSPFR, PPGFSPFKK andKPPGFSPFK denoted as – RR, R-R, -KK and K-K, respec-tively) were purchased from Bachem and dissolved inmilli-Q-water to a concentration of 1 mM (stock solution at1 mg/ml). The oligonucleotides were purchased from Pro-ligo France SAS, at the concentration of about 400 µM. Thepeptide–DNA mixture with a ratio (1 : 1, v : v) was dissolvedin methanol/water at a final concentration of 5 µM.

Mass spectrometric experimentsExperiments were performed using an electrospray sourcein negative ion mode combined with an LTQ-Orbitraphybrid instrument (Thermoelectron Corporation). The proof-of-principle of the Orbitrap was first described by Hardmanand Makarov59 and is not discussed here. Recently, anLTQ-Orbitrap hybrid instrument was developped by Ther-moelectron Corporation:60 briefly, the front part is a LinearTrap61 LTQ capable of detecting MS or MS2 spectra (butwith low resolution). Ions can be further released into acurved C-trap (an r.f. only quadrupole), which accumulatesand stores the ions and they are consequently transferred toand analysed in the Orbitrap, an electrostatic ion trap, whichdemonstrated a high resolving power and mass accuracy.62

The general ionisation conditions were as follows: accelerat-ing voltage 3.4 kV; sheath gas flow 25 (ua); no auxiliary gas;ion transfer tube temperature 250 °C; and tube lens voltage180 V. Full scan MS spectra were acquired in the Orbitrapwith a resolution of R D 60 000 (at m/z 400), after accumula-tion to a target value of 105 and a mass range from m/z 300to 2000. For the CID spectra, the target complex ions wereisolated for fragmentation in the linear ion trap and frag-mented using collisionnally induced dissociation under thefollowing conditions: variable normalised collision energy63

(in %); default activation q D 0.250 and fixed activation time1.5 ms. The resulting fragments ions were recorded in thelinear Ion Trap (after radial ejection) or eventually in theOrbitrap. In the latter case, the fragment ions were recordedwith high mass accuracy and resolution, so that the isotopicprofile made it easy to identify the charge state. All data

Copyright 2007 John Wiley & Sons, Ltd. J. Mass Spectrom. 2007; 42: 1613–1622DOI: 10.1002/jms

Zwitterion in DNA–peptide complexes 1615

were acquired using external calibration with a mixture ofcaffeine, MRFA peptide and Ultramark 1600 dissolved in50/50 (v : v) water/acetonitrile solution.

RESULTS AND DISCUSSION

Peptide sequence dependenceTo explore the possibility of observing DNA–peptide non-covalent interactions, mixtures of equal amounts of �dpT�8

(denoted as T8) oligonucleotide and bradykinin solutions(with a sequence RPPGFSPFR and denoted as R-R) wereanalysed by ESI mass spectrometry in negative ion mode.Note that the positive ion mode shows mainly the peptideions (data not shown) and is not discussed here. The negativeion mass spectrum displayed in Fig. 1 (detected in theOrbitrap analyser) shows the formation of the peptide �R-R �H�� and the olgonucleotide �T8 � 4H�4�, �T8 � 3H�3� and�T8 � 2H�2� multiply deprotonated molecules. Formation ofbinary complexes13 – 15 �R-R C T8 � 4H�4�, �R-R C T8 � 3H�3�

and �R-R C T8 � 2H�2� and tertiary �2 ð R-R C T8 � 3H�3�

species (Fig. 1) was observed. We have undertaken anextensive study of the complex fragmentation behaviourbetween a set of modified bradykinin (containing twoR or K residues at C- and or N-terminus side) withT8 (or A8) oligonucleotide strands. The ESI mass spectradisplay monomer anions and similar DNA–peptide multi-deprotonated complexes (data not shown). Note that withpeptides containing lysine residues (K-K and -KK), lowercharge state complexes are produced.

The behaviour of the most abundant multiply depro-tonated binary complexes is first studied. CID spectra ofdifferent triply deprotonated DNA–peptide complexes arerecorded23 (Fig. 2). In Fig. 2(a), �R-R C T8 � 3H�3� complexesdissociate into monomeric �R-R � H�� and �T8 � 2H�2�

anions after disruption of the non-covalent interactions. Thetwo monomers do not have the same tendency to keepthe charges.27 Doubly deprotonated oligonucleotide are pro-duced in higher abundance than the singly deprotonatedpeptide, which should be directed by the respective gas-phase acidities of the oligonucleotide64 and peptide65 chains.At higher excitation conditions (data not shown), covalentbond fragmentations occur from the oligonucleotide strand(by loss of thymine base, by thymidine nucleoside and/ornucleotide unit release). Furthermore, peptide product ionsat m/z 1028.5 are also detected, which correspond to theloss of CH2O from the deprotonated serine of the peptidesequence. Note also the presence of triply charged prod-uct ions at m/z 1100.5 due to direct loss of the thyminebase from the complexes3,28,29 and competitive/consecutiveoligonucleotide chain dissociations by increasing the excita-tion conditions.

Non-covalent complexes with peptides containing twolysine residues (K-K and – KK) are also studied (Fig. 2(c) and(d)). CID spectra of anions display similar fragmentationpatterns to bradykinin (R-R) complexes but with higherdissociation rate constants. Indeed, we observed a highercomplex fragmentation extent whereas lower excitationamplitude is used (Fig. 2). The non-covalent dissociation ofthe binary complexes into monomer strands takes place andis the major pathway, while extensive covalent bond cleavagefrom the complexes is not observed. The lower stability oflysine-containing complexes is related to its thermo-chemicalgas-phase data as compared to those of arginine.66

Surprisingly, dissociation of �-RR C T8 � 3H�3� ions(Fig. 2(b)) showed two different pathways: a minor pathwayis the disruption of non-covalent interaction and a majortriply deprotonated product ion is observed at m/z 1077.9.This fragment came from the loss of the PP motif (194 u)

Figure 1. Negative ion mode of an equimolar mixture of bradykinin (R-R) peptide and T8 oligonucleotide solutions analysed in theOrbitrap. (Note that the isotopic spectral mass makes the charge state determination easy).



Fig

ure

2.C

IDsp

ectr

aof

com

ple

xes

frag

men

ted

and

anal

ysed

inth

eLT

Q:(

a)�R

-RC

T 8�

3H�3�

(45%

ofno

rmal

ised

colli

sion

ener

gy),

(b)�

-RR

CT 8

�3H

�3�(4

5%of

norm

alis

edco

llisi

onen

ergy

),(c

)�K

-KC

T 8�

3H�3�

(35%

ofno

rmal

ised

colli

sion

ener

gy)a

nd(d

)�-K

KC

T 8�

3H�3�

(35%

ofno

rmal

ised

colli

sion

ener

gy).

(The

pea

ksla

bel

led

‘*’c

orre

spon

dto

olig

onuc

leot

ide

frag

men

tio

ns,w

here

asth

ose

lab

elle

d‘°

’are

pro

duc

edfr

omco

mp

lexe

sb

yco

vale

ntb

ond

clea

vage

).



by peptide backbone cleavage from the triply deprotonatedcomplexes (production of yn product ions). Direct prod-uct ions from DNA bond cleavage without non-covalentinteraction dissociation of the complexes are producedat higher excitation conditions (as example �-RR C wi�2�

and/or �-RR C �ai-B��2� labelled ‘°’ in the following figures).The observation of DNA covalent bond fragmentation

has been already reported for DNA duplexes27,28 andcomplexes with ligand29 and DNA–PNA–DNA triplexes.53

DNA bond cleavage from duplexes was assumed to occurby a partial unzipping and backbone cleavage (multi-step process28). Such observation has been explained bythe existence of hydrogen bonding and maybe weakbase stacking between complementary bases,3,27,29 whichstabilises non-covalent interaction and could overcome thecoulombic repulsion between stands.

In our study, experimental observations made frombinary DNA–peptide complexes with modified bradykininimply that DNA interactions with the basic R or K residuesplay a major role in the complex fragmentation patterns. Wethen expect that the DNA strand interacts with the peptideby SB formation and some hydrogen bonding between basicside chains and acidic phosphate groups.13 – 15,21 – 25 Hydrogenbonding can be also assumed between nucleobase andcharged or neutral amino acids. Non-covalent interactionstability should be related to the chemical properties ofbasic side chains and their location in the peptide sequence.Arginine and lysine residues possess side chains that canact as hydrogen donor group whereas phosphate groups ofnucleotide units can receive hydrogen. Woods and Ferre24

proposed that it is likely that protons are sought after by thedelocalised lone pair of electrons on the phosphate group.

If SB formations are assumed in DNA–peptide complexesto explain their strong stability in the gas phase, then tobalance the positively charged AA side chains an increaseof negative charges is required. Such hypothesis can berationalised by considering zwitterionic forms in binarycomplexes. Intrinsic stability of the zwitterionic forms ofthe peptide’s amino acid side chains depends upon theirgas-phase basicity.31,32 The R side chain contains a stronglybasic guanidinium group where proton addition is stabilisedby a delocalised positive charge. So the zwitterionic formof peptides containing a C-terminal R (hence a guanidiniumgroup) is assumed to form an SB with the negatively chargedphosphate group of DNA, stable enough to withstanddisruption of the non-covalent interaction and to breakfirst the covalent bond (Fig. 3). However, the existenceof hydrogen bonding cannot be eliminated. Nevertheless,ion–dipole interactions between amino acid guanidiniumor amino groups and phosphate should be characterisedby a weak stability67 (large difference of their respectivegas-phase acidities64,66). Similarly, a weak influence of H-bonding between nucleobase and amino acid is expected incomparison with the ionic interaction.

In the case of RR motif-containing complexes, zwiterionicsites are assumed in both R residues in the C-terminus,so two delocalised negative charges located in the DNAstrand interact strongly with the adjacent guanidiniumgroups, which facilitates the covalent bond cleavage on

Figure 3. Cartoon showing the salt bridge interactionsbetween the guanidinium group of arginine and the phosphategroup of the oligonucleotide strand.

the N-terminal side, where proline’s rigidity68 favoursthe loss of the PP motif and the production of yn ions.With bradykinin peptide, SB interactions could occur atboth termini, so the covalent bond cleavages are highlyunfavourable. Nevertheless, oligonucleotide chain cleavagescan be produced in both R-containing complexes despite apossible complex structure change. In comparison, lysine-containing complexes display a lower stability under CIDconditions and dissociate only by disruption of the non-covalent complex, because of the lower gas-phase basicity ofthe K residue,69 making the production of ZWs unfavourable.

Charge state dependenceThe most sticking observation is that fragmentation patternsof multi-deprotonated DNA–peptide complexes are depen-dent upon the complex charge state (Fig. 4). The comparisonof CID spectra of different complex anions between – RRpeptide and T8 DNA strand (Figs 2(b), 4(a) and (b)) showedthat �-RR C T8 � 2H�2� anions dissociate by loss of PP motiffrom the peptide to a larger extent to �-RR C T8 � 3�3� anions,whereas �-RR C T8 � 4H�4� anions display a weak production by peptide covalent bond cleavage (at m/z 808.1) anda major pathway by non-covalent dissociation of the com-plexes. Similarly, �-KK C T8 � 2H�2� complex ions dissociateinto product ions at m/z 1528.9 by loss of PP motif from thepeptide which is never observed for �-KK C T8 � 4H�4� and�-KK C T8 � 3H�3� ions, which display only the disruptionof the non-covalent interaction. Note that peptide covalentbond cleavages are never observed from complexes withR–R and K–K peptides.

In summary, covalent bond dissociations are promotedin the lower charge state and only for peptides containingadjacent basic residues (-RR and – KK peptides). Suchobservations were explained for DNA duplexes by alowering of coulombic repulsion for the low duplex chargestate. Indeed, the increase of coulombic energy with thecomplex charge state leads to a lowering of the barrierfor the duplex unzipping mechanism. The lowering ofcoulombic repulsion with the lower charge state values canbe also assumed for DNA–peptide complexes.29 Unzippingmechanism should be easier at higher charge, if weconsider the multiple hydrogen bonds in DNA–peptide



Figure 4. CID spectra of complexes fragmented and analysed in the LTQ: (a) �-RR C T8 � 4H�4� (35% of normalised collisionenergy), (b) �-RR C T8 � 2H�2� (45% of normalised collision energy), (c) �-KK C T8 � 4H�4� (35% of normalised collision energy) and(d) �-KK C T8 � 2H�2� (35% of normalised collision energy). (The peaks labelled ‘*’ correspond to oligonucleotide fragment ions,whereas those labelled ‘°’ are produced from complexes by covalent bond cleavage).

complex. Our explanation assumes the existence of strongSB interaction thanks to the zwitterionic forms of peptides.So, the net number of negative charges modulates thenumber of zwitterionic sites, which stabilise the non-covalentcomplexes. Multi-deprotonated species are characterised bylower gas-phase acidity.65 Then, by increasing the complexcharge state, a smaller number of ZW sites should contributeto the strength of the complex interaction. Non-covalentinteraction cleavage of the complex is promoted rather thancovalent bond cleavage.

Nucleobase natureIn order to determine whether the base of the DNAplays a role in the stability of DNA–peptide complex, A8

strand was employed to test its ability to bind modifiedbradykinins. The ESI mass spectra indicated the formationof analogous binary and tertiary complexes in each case(data not shown). However, the CID spectra of binarycomplexes show differences. First, the binary complexesare characterised by a lower stability in comparison withT8 strand. Moreover, CID of �R-R C A8 � 3H�3� anions inFig. 5(a) displays a large diversity of A8 strand product ions(limited with the T8 strand). We observed also a larger extentof direct fragment ions from complexes by adenine baserelease (giving product ions at m/z 1121.6) and adenosineunit(s) cleavages (labelled as ‘°’ in the Fig. 5). Non-covalentcomplexes with lysine residue (K-K and – KK) were alsostudied. The CID spectra of anions display the non-covalent



Figure 5. CID spectra of complexes fragmented and analysed in the LTQ: (a) �R-R C A8 � 3H�3� (45% of normalised collisionenergy), (b) �-RR C A8 � 3H�3� (45% of normalised collision energy), (c) �K-K C A8 � 3H�3� (35% of normalised collision energy) and(d) �-KK C A8 � 3H�3� (35% of normalised collision energy). (The peaks labelled ‘*’ correspond to oligonucleotide fragment ions,whereas those labelled ‘°’are produced from complexes by covalent bond cleavage).

dissociation of the complexes and consecutive product ionsfrom DNA strand (with lower stability in comparison with R-containing complexes). But, we do not detect covalent bondfragmentation of DNA strand and/or peptide backbonefrom the complexes. Moreover, CID of �-RR C A8 � 3H�3�

complex anions (Fig. 5(b)) showed the additional presenceof fragment ions at m/z 1102.8, corresponding to theloss of PP motif, followed by consecutive DNA backbonecleavages, which are not observed from other modifiedbradykinins.

Such behaviour is in contrast with complexes containingthe T8 strand, in which oligonucleotide chain fragmentationsare very limited. The extensive fragmentation of the A8

strand should be related to nucleobase thermo-chemicaldata. Previous work has shown that adenosine nucleotidesare less acidic than thymidine64 ones but adenosine ischaracterised by a higher gas-phase basicity,70,71 makingadenine a good leaving group. The influence of DNA

sequence on the covalent bond fragmentation from duplexeshas been reported and explained by the contribution ofweak base stacking.27 However, such a hypothesis cannot beassumed here. Nevertheless, the relative values of gas-phasebasicity of nucleobase70,71 relative to that of the amino acid66

should influence the zwitterionic forms of deprotonatedcomplexes. With the assumption of zwitterionic form, wehypothesise a competition for proton addition and thena competition for ZW location between the basic adeninebase and R or K side chain. Hydrogen bonding can alsocontribute to the stabilisation of non-covalent complex andshould be stronger for the adenine base in comparison withthe thymine base. Nevertheless, the observation of covalentbond fragmentation and the lower stability of complexes andthe promption of DNA strand cleavages are consistent withthe likely protonation of adenine base rather than amino acidside chain, implying a lowering of the interaction strength(unfavourable SB formation).



CONCLUSION

Mixtures of oligonucleotide and bradykinins solutions wereanalysed by ESI mass spectrometry in negative ion mode.Their mass spectra showed the presence of major binaryDNA–peptide complexes as well as tertiary species. CIDspectra of DNA–peptide triply deprotonated complexeswere recorded, which showed the dissociation of complexesinto monomers as the major pathways except for the RRmotif-containing peptides which displayed non-covalentproduct ions by covalent bond cleavage. Furthermore,fragmentation patterns of DNA–peptide binary complex aredependent upon the complex charge state. Peptide covalentbond fragmentations were promoted in the lower chargestate and only for peptides containing adjacent arginineresidues. Instead of T8, the A8 strand was employed to testits ability to bind modified bradykinins. CID of complexanions displays similar fragmentation patterns but with alarge diversity of DNA product ions and direct product ionsfrom complexes by DNA chain cleavages.

All experimental observations can be rationalised byconsidering zwitterionic forms in binary complexes (locatedat basic amino acid side chains). The presence of protons innucleobase and or amino acid contributes to reinforcing thestrength of interaction. We then expect that DNA interactswith peptide by SB formation between protonated aminoacid side chain and deprotonated phosphate groups of DNAstrand. Above all, the R residue possesses a guanidiniumgroup which forms an electrostatic interaction stable enoughto withstand disruption of the non-covalent interaction andto break first the covalent bonds. In the case of RR motif-containing complexes, two delocalised negative chargesinteract strongly with the adjacent protonated guanidiniumgroups located at the C-terminus, which facilitates covalentbond cleavage at the N-terminal side. The complex withbradykinin peptide (as R-R) displays different fragmentationpathways because of the small structural change (related toarginine residue location). In the same way, the decreaseof complex charge state induces a smaller number of ZWssites, which contribute to strength of complex interaction.Then, non-covalent bond fragmentation of the complex ispromoted rather than covalent bond cleavage. The weakinfluence of the basic adenine base should be related toits higher gas-phase basicity. With a more basic base inthe DNA strand, it can be assumed that a competition forproton addition between the adenine nucleobase and theR/K side chain takes place, leading to a change in ZW sitelocation (DNA strand) and/or reinforcing the contributionof hydrogen bonding for the stabilisation of non-covalentcomplexes. Both hypotheses would result in a loweringstabilisation of SB formation between DNA peptide chains.

REFERENCES1. Heck AJR, van den Heuvel RHH. Investigation of intact protein

compexes by mass spectrometry. Mass Spectrometry Reviews 2004;23: 368.

2. Di Tullio A, Reale S, De Angelis F. Molecular recognition massspectrometry. Journal of Mass Spectrometry 2005; 40: 845.

3. Pan S, Sun X, Lee JK. DNA stability in the gas versus solutionphases: a systematic study of thirty-one duplexes with varying

length, sequence and charge level. Journal of the American Societyfor Mass Spectrometry 2006; 17: 1383.

4. Whitehouse CM, Dreyer RN, Yamashita M, Fenn JB. Electro-spray interface for liquid chromatogaphs and mass spectrome-ters. Analytical Chemistry 1985; 57: 675.

5. Wortmann A, Rossi F, Lelais G, Zenobi R. Determination of zincto beta-peptide binding constants with electrospray ionizationmass spectrometry. Journal of Mass Spectrometry 2005; 40: 777.

6. Beaudry F, Vachon P. Electrospray ionization suppression, aphysical or chemical phenomenon? Biomedical Chromatography2006; 20: 200.

7. Karas M, Hillenkamp F. Laser desorption ionization of prteinswith molecular masses exceeding 10 000 daltons. AnalyticalChemistry 1988; 60: 2301.

8. Luo G, Marginean I, Vertes A. Internal energy of ions generatedby matrix assisted laser desorption/ionization. AnalyticalChemistry 2002; 74: 6185.

9. Woods AS, Buchabaum JC, Worr TA II, Berg JM, Cotter RJ.Matrix-assisted laser desorption/ionization of noncovalentlybound compounds. Analytical Chemistry 1995; 67: 4462.

10. Distler AM, Allison J. Additives for the stabilization of double-stranded DNA in UV-MALDI MS. Journal of the American Societyfor Mass Spectrometry 2002; 13: 1129.

11. Zehl M, Allmaier G. Instrumental parameters in the MALDI-TOF mass spectrometric analysis of quaternary proteinstructures. Analytical Chemistry 2005; 77: 103.

12. Lecchi P, Pannell LK. The detection of intact double-strandedDNA by MALDI. Journal of the American Society for MassSpectrometry 1995; 6: 972.

13. Lin S, Cotter RJ, Woods AS. Characterization of the ‘‘helixclamp’’ motif of HIV-1 reverse transcriptase using MALDI-TOFMS and surface plasmon resonance. Proteins Structure, Functionand Genetics 1998; 2: 12.

14. Lin S, Long S, Ramirez SM, Cotter RJ, Woods AS. Characteriza-tion of the ‘‘helix Clamp’’ motif of HIV-1 reverse transcriptaseusing MALDI-TOF MS and surface plasmon resonance. Analyti-cal Chemistry 2000; 72: 2635.

15. Luo S–Z, Li Y–M, Quiang W, Zhao Y–F, Abe H, Nemoto T,Qin X–R, Nakanishi H. Detection of specific noncovalentinteraction of peptide with DNA by MALDI-TOF. Journal ofthe American Society for Mass Spectrometry 2004; 15: 28.

16. Vasquez ME, Caamano AM, Mascarenas JL. From transcriptionfactors to designed sequence-specific DNA-binding peptides.Chemical Society Reviews 2003; 32: 338.

17. Schmidt A, Karas M. The influence of electrostatic interactionson the detection of heme-globin complexes in ESI-MS. Journal ofthe American Society for Mass Spectrometry 2001; 12: 1092.

18. Tang X, Callahan JH, Zhou P, Vertes A. Noncovalent protein-oligonucleotide interactions monitored by matrix-assisted laserdesorption/ionization mass spectrometry. Analytical Chemistry1995; 67: 4542.

19. Terrier P, Tortajada J, Buchmann W. A study of non covalentcomplexes involving single-stranded DNA and polybasiccompounds using nanospray mass spectrometry. Journal of theAmerican Society for Mass Spectrometry 2007; 18: 346.

20. Woods AS, Huestis MA. A study of peptide-peptide interactionby matrix-assisted laser desorption/ionization. Journal of theAmerican Society for Mass Spectrometry 2001; 12: 88.

21. Ciruela F, Burgueno J, Casado V, Canals M, Marcellino D,Goldberg SR, Bader M, Fuxe K, Agnati LF, Lluis C, Franco R,Ferre S, Woods AS. Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization.Direct epitope-epitote electrostatic nteractions betweenadenosine A2A and dopamine D2 receptors. Analytical Chemistry2004; 76: 5354.

22. Woods AS. The mighty arginine, the stable quaternity amines,the powerful aromatics, and the aggressive phosphate: their rolein the noncovalent minuet. Journal of Proteome Research 2004; 3:478.



23. Jackson SN, Wang H-YJ, Woods AS. Study of the fragmentationpatterns of the phosphate-arginine noncovalent bond. Journal ofProteome Research 2005; 4: 2360.

24. Woods AS, Ferre S. Amazing stability of the arginine-phosphateelectrostatic interaction. Journal of Proteome Research 2005; 4: 1397.

25. Jackson SN, Wang H-YJ, Yergey A, Woods AS. Phosphatestabilization of intermolecular interaction. Journal of ProteomeResearch 2006; 5: 122.

26. Delvolve A, Alves S, Afonso C, Nielsen PE, Burlina F,Fournier F, Tabet J-C, Effect of charge state on the influenceof zwitterions on the stability of non-covalent interaction ofsingle stranded DNA with peptides and PNAs. Proceedings ofthe 54th ASMS Conference on Mass Spectrometry and Allied Topics,2004.

27. Gabelica V, De Pauw E. Collision-induced dissociation of 16-mer DNA duplexes with various sequences: evidence forconservation of the double helix conformation in the gas phase.International Journal of Mass Spectrometry 2002; 219: 151.

28. Gabelica V, De Pauw E. Comparison of the collision-induceddissociation of duplex DNA at different collision regimes:evidnece for a multistep dissociation mechanism. Journal of theAmerican Society for Mass Spectrometry 2002; 13: 91.

29. Keller K, Zhang J, Oehlers L, Brodbelt JS. Influence of initialcharge state on fragmentation patterns for noncovalentdrug/DNA duplex complexes. Journal of Mass Spectrometry 2005;40: 1362.

30. Devolve A, Tabet J-C, Bregant S, Afonso C, Burlina F,Fournier F. Charge dependent behaviour of PNA/DNA/PNAtriplexes in the gas phase. Journal of Mass Spectrometry 2006; 41:1498.

31. Strittmatter EF, Williams ER. The role of proton affinity, acidity,and electrostatics on the stability of neutral versus ion-pair formsof molecular dimmers. International Journal of Mass Spectrometry2001; 212: 287.

32. Lemoff AS, Bush MF, Williams ER. Binding energies of water tosodiated valine and structural isomers in the gas phase: the effectof proton affinity on zwitterion stability. Journal of the AmericanChemical Society 2003; 125: 13576.

33. Price WD, Jockusch RA, Lemoff AS, Williams ER. Is arginine azwitterion in the gas phase? Journal of the American ChemicalSociety 1997; 119: 11988.

34. Chapo CJ, Paul JB, Provencal RA, Roth K, Saykally RJ. Is arginiezwitterionic or neutral in the gas phase ? Results from IR cavityringdown spectroscopy. Journal of the American Chemical Society1998; 120: 12956.

35. Julian RR, Jarrold MFJ. Phys. gas-phase zwitterions in theabsence of a net charge. Journal of Physical Chemistry A 2004;108: 10861.

36. Jensen JH, Gordon MS. On the number of water moleculesnecessary to stabilize the glycine zwitterion. Journal of theAmerican Chemical Society 1995; 117: 8159.

37. Jockusch RA, Lemoff AS, Williams ER. Hydration of valine-cation complexes in the gas phase: on the number of watermolecules necessary to form a zwitterion. Journal of PhysicalChemistry A 2001; 105: 10929.

38. Lemoff AS, Bush MF, Wu C–C, Williams ER. Structures andhydration enthalpies of cationized glutamine and structuralanalogues in the gas phase. Journal of the American ChemicalSociety 2005; 127: 10276.

39. Lemoff AS, Bush MF, Williams ER. Structures of cationizedpraline analogues: evidence for the zwitterionic form. Journalof Physical Chemistry A 2005; 109: 1903.

40. Lemoff AS, Bush MF, O’Brien JT, Williams ER. Structures oflithiated lysine and structural analogues in the gas phase: effectsof water and proton affinity on zwitterionic stability. Journal ofPhysical Chemistry A 2006; 110: 8433.

41. Xu S, Nilles JM, Bowen KH. Zwitterion formation in hydratedamino acid, dipole bound anions: how many water moleculesare required? Journal of Chemical Physics 2003; 119: 10696.

42. Wyttenbach T, Witt M, Bowers MT. On the stability of aminoacid zwitterions in the gas phase: the influence of derivatization,

proton affinity, and alkali addition. Journal of the AmericanChemical Society 2000; 122: 3458.

43. Cerda BA, Wesdemiotis C. Zwitterionnic vs. charge-solvatedstructures in the binding of arginine to alkali metal ions inthe gas phase. Analyst 2000; 125: 657.

44. Kapota C, Lemaire J, Maıtre P, Ohanessian G. Vibrational vs.charge-solvated structures in the binding of arginine to alkalimetal ions in in the gas phase. Journal of the American ChemicalSociety 2004; 126: 1836.

45. Strittmatter EF, Lemoff AS, Williams ER. Journal of PhysicalChemistry A 2000; 104: 9793.

46. Ai H, Bu Y, Li P, Zhang C. The regulatory roles of metal ions(MC/2C D LiC, NaC, KC, Be2C, Mg2C, and Ca2C) and watermolecules in stabilizing the zwitterionic form of glycinederivatives. New Journal of Chemistry 2005; 29: 1540.

47. Rodriguez-Cruz SE, Klassen JS, Williams ER. Hydration of gasphase ions formed by electrospray ionization. Journal of theAmerican Society for Mass Spectrometry 1999; 10: 958.

48. Nemes P, Schlosser G, Vekey K. Amino acid cluster formationstudied by electrospray ionization mass spectrometry. Journal ofMass Spectrometry 2005; 40: 43.

49. Lifshitz C. A review of gas-phase H/D exchange experiments:the protonated arginine dimmer and bradykinin nonapeptidesystems. International Journal of Mass Spectrometry 2004; 234: 63.

50. Julian RR, Beauchamp JL, Goddard WA III. Cooperative saltbridge stabilization of gas-phase zwitterions in neutral arginieclusters. Journal of Physical Chemistry A 2002; 106: 32.

51. Freitas MA, Marshall AG. Rate and extent of gas-phasehydrogen/deuterium exchange of bradykinins: evidence forpeptide zwitterions in the gas phase. International Journal of MassSpectrometry 1999; 182/183: 221.

52. Levy-Seri E, Koster G, Kogan A, Gutman K, Reuben BG,Lifshitz C. An electrospray ionization-flow tube study of H/Dexchange in protonated bradykinin. Journal of Physical ChemistryA 2001; 105: 5552.

53. Boutin M, Bich C, Afonso C, Burlina F, Fournier F, Tabet J-C. Negative charge driven fragmentations for evidencingzwitterionic forms from doubly charged coppered peptides.Journal of Mass Spectrometry 2007; 41: 25.

54. Beauchamp JL, Kim HS, Lee S–W. Salt bridge chemistry appliedto gas-phase peptide sequencing: selective fragmentation ofsodiated gas-phase peptide ions adjacente to aspartic acidresidues. Journal of the American Chemical Society 1998; 120: 3188.

55. Schnier PD, Price WD, Jockusch RA, Lemoff AS, Williams ER.Blackbody infrared radiative dissociation of bradykinin and itsanalogues: energetics, dynamics, and evidence for salt-bridgestructures in the gas phase. Journal of the American ChemicalSociety 1996; 118: 7178.

56. Kjeldesen F, Silivra OA, Zubarev R. Zwitterionic sates in gasphases poly-peptide ions revealed by 157 nm ultra-violet photo-dissociation. J. Am. Chem. Eur. J. 2006; 12: 7920.

57. Wyttenbach T, Bushnell JE, Bowers MT. Salt bridge structures inthe absence of solvent ? The case for the oligoglycines. Journal ofthe American Chemical Society 1998; 120: 3188.

58. Antoine R, Broyer M, Dugourd P, Breaux G, Hagemeister FC,Pippen D, Hudgins RR, Jarrold MF. Direct probing of zwitterionformation in unsolvated peptides. Journal of the American ChemicalSociety 2003; 125: 8996.

59. Hardman M, Makarov AA. Interfacing the orbitrap massanalyzer to an electrospray ion source. Analytical Chemistry 2003;75: 1699.

60. Olsen JV, de Godoy LMF, Li G, Macek B, Mortensen P, Peach R,Makarov A, Lange O, Horning S, Mann M. Parts per millionmass accuracy on an orbitrap mass spectrometer via lock massinjection into a C-trap. Molecular and Cellular Proteomics 2005;4.12: 2010.

61. Douglas DJ, Frank AJ, Mao D. Linear ion traps in massspectrometry. Mass Spectrometry Reviews 2005; 24: 1.

62. Hu Q, Noll RJ, Li H, Makarov A, Hardman M, Cooks RG. Theorbitrap: a new mass spectrometer. Journal of Mass Spectrometry2005; 40: 430.



63. Gabelica V, Karas M, De Pauw E. Calibration of ion effectivetemperatures achieved by resonant activation in a quadrupoleion trap. Analytical Chemistry 2003; 75: 5152.

64. Jones CM, Bernier M, Carson E, Colyer KE, Metz R, Pawlow A,Wischow ED, Webb I, Andriole EJ, Poutsma JC. Gas-phaseacidities of the 20 protein amino acids. International Journal ofMass Spectrometry 2007; 267: 54.

65. Zhang X, Cassady CJ. Apparent gas-phase acidities of multiplyprotonated peptide peptide ions: uniquitin, insulin insulin Band renin substrate. Journal of the American Society for MassSpectrometry 1996; 7: 1211.

66. O’Hair RAJ, Bowie JH, Gronert S. Gas phase acidities of the ˛amino acids. International Journal of Mass Spectrometry and IonProcesses 1992; 117: 23.

67. Mautner M. The ionic hydrogen bond. Chemical Reviews 2005;105: 213.

68. Paizs B, Suhai S. Fragmentation pathways of protonatedpeptides. Mass Spectrometry Reviews 2005; 24: 508.

69. Harrison AG. The gas-phase basicities and proton affinities ofamino acids and peptides. Mass Spectrometry Reviews 1997; 16:201.

70. Armentano D, De Munno G, Di Donna L, Napoli A, Sindona G,Giorgi G, Salvini L. Self-assembling of cytosine nucleoside intotriply-bound dimers in acid media. A comprehensive evaluationof proton-bound pyrimidine nucleosides by electrospray tandemmass spectrometry, x-rays diffractometry, and theoreticalcalculations. Journal of the American Society for Mass Spectrometry2004; 15: 268.

71. Di Donna L, Napoli A, Sindona G, Athanassopoulos C. Acomprehensive evaluation of the kinetic method applied in thedetermination of the proton affinity of the nucleic acid molecules.Journal of the American Society for Mass Spectrometry 2004; 15: 1080.


Documents

Charge state effect on the zwitterion influence on stability of non-covalent interaction of single-stranded DNA with peptides