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Contents lists available at SciVerse ScienceDirect

International Journal of Mass Spectrometry

j ourna l ho me page: www.elsev ier .com/ locate / i jms

he binding of Ca2+, Co2+, Ni2+, Cu2+, and Zn2+ cations to angiotensin Ietermined by mass spectrometry based techniques

atthew S. Glover, Jonathan M. Dilger, Feifei Zhu, David E. Clemmer ∗

epartment of Chemistry, Indiana University, Bloomington, IN 47405, United States

a r t i c l e i n f o

rticle history:eceived 6 May 2013eceived in revised form 18 June 2013ccepted 18 June 2013

a b s t r a c t

The interaction of a series of doubly charged metal cations (M2+ = Ca, Co, Ni, Cu, and Zn) with angiotensinI (AngI, Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10) is examined by collision-induced disso-ciation (CID) and ion mobility spectrometry–mass spectrometry (IMS–MS). The series of CID patternscombined with IMS–MS data for [AngI+M+H]3+ ions provides information about the metal–peptide bind-

vailable online xxx

eywords:on mobility spectrometryollision-induced dissociationetal–peptide interactions

eptide ion structure

ing sites. Overall, Ca2+ favors association with oxygen atoms spanning the peptide backbone; whereas,the transition metals favor binding at a site that involves association at the His6 and His9 sites. From theseexperiments, it is possible to derive insight into the populations of different metal coordination sites thatare sampled in solution prior to introduction of species into the gas phase.

© 2013 Published by Elsevier B.V.

. Introduction

Metal ion–biomolecule interactions are important for many bio-ogical processes including those involving structural stabilization,edox reactions, and signal transduction [1–4]. The interactions ofetal ions with favored binding sites (i.e., with specific amino acid

esidues or sequence motifs) also are known to induce structuralhanges, such that enzymatic activity is regulated by metal binding3]. Thus, it is important to understand fundamental interactionsf metals with polypeptide chains. One means of understandingetal binding interactions involves analysis by mass spectrom-

try (MS) or tandem mass spectrometry (MS–MS) techniques.S analyses provide direct information about the stoichiome-

ry of the metal–biopolymer complex [5]. MS–MS analyses, inhich collision-induced dissociation (CID) is used to break apartetal-containing biopolymers, sometimes show specific sites of

ragmentation – providing insight into metal binding sites [6–19].everal studies have explored unique fragmentation mechanismsnd it has been suggested that such specificity may be useful forirect sequencing or fingerprinting biopolymers by MS [16–18].trictly, MS–MS techniques provide insight about the metal-

Please cite this article in press as: M.S. Glover, et al., Int. J. Mass Spectrom.

inding sites at the transition states associated with dissociation.ittle is known about the distribution of metal ion–peptide confor-ations prior to dissociation [20–28] and even less is understood

∗ Corresponding author.E-mail address: clemmer@indiana.edu (D.E. Clemmer).

387-3806/$ – see front matter © 2013 Published by Elsevier B.V.ttp://dx.doi.org/10.1016/j.ijms.2013.06.014

about transitions between states as ions are activated and ulti-mately dissociate.

In this paper, we use ion mobility spectrometry (IMS)–MStechniques and MS–MS dissociation studies to investigate theinteraction of a model peptide angiotensin I (AngI, Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10) with Ca2+, Co2+, Ni2+,Cu2+, and Zn2+. IMS–MS methods are an effective means of detec-ting multiple structures of biological molecules in the gas phase[29–31]. We note that the slow heating [32] associated with CIDcan induce structural transitions in the precursor ion along thepathway to dissociation [33]; thus, we supplement our studieswith multidimensional IMS techniques (e.g., IMS–IMS–MS) thatmonitor structural transitions that occur upon collisional activa-tion [34,35]. The AngI model peptide contains several residuesthat are known to interact with metal cations – the side chains ofAsp1, Arg2, Tyr4, His6, His9; additionally, ten-residue AngI is suf-ficiently long that backbone carbonyl oxygen atoms and amidenitrogen atoms may also coordinate the metal cation. The workpresented here provides insight into the low-lying conformationsthat are present prior to dissociation and allows for a comparisonbetween metal-mediated conformations prior to and after colli-sional activation and the fragments produced by CID. Finally, infavorable cases, the similarity of IMS peaks observed before andafter activation allows us to speculate about what conformations

(2013), http://dx.doi.org/10.1016/j.ijms.2013.06.014

are favored in solution for these metals. Overall, our data are inagreement with known binding sites for these metals that wouldbe predicted from structures taken from the protein data bank(PDB).

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. Experimental method

.1. Instrumentation

CID experiments were performed on a LTQ Velos mass spec-rometer (Thermo Scientific, San Jose, CA). Fragment ions wereroduced in the linear ion trap by applying a resonant rf excitationaveform for 10 ms, an activation q of 0.25, and normalized colli-

ion energy of 16%. A list of candidate fragment ions was generatednline with the MS-product tool [36]. Metal-containing fragmenton m/z values were calculated from expected fragment productslus the mass of the metal cation.

Ion mobility experiments were conducted on a home-builtnstrument described in detail previously [34,35]. Briefly, electro-prayed ions are stored in an hourglass-shaped ion funnel [37].ackets of ions are periodically released into the drift tube by low-ring an electrostatic gate on the source funnel for 150 �s. The driftube is operated with 3.00 ± 0.01 Torr He buffer gas and a uniformlectric field (∼10 V cm−1). Ions are radially focused in funnels athe middle and end of the drift tube. Ions exit the drift tube through

differentially pumped region and are transferred to the time-of-ight (TOF) mass spectrometer. Ion drift times and mass-to-chargealues are recorded in a nested fashion [38].

We have described the experimental design and analytical util-ty of IMS–IMS–MS in detail previously [34,35]. Briefly, we operatehe linear drift tube as two independent drift tubes separated byn ion funnel. An electrostatic gate in the middle funnel is imple-ented to select ions of a single mobility or range of mobilities

o be transmitted to the second drift tube. Mobility-selected ionsan be collisionally activated before entering the second region ofhe drift tube; at low activation voltages structural transitions arenduced, and at higher activation voltages fragmentation occurs.tructural transitions can be monitored by measuring the mobili-ies of annealed ions following separation of the ions through theecond region of the drift tube.

.2. Generation of [AngI+M+H]3+ ions

Peptide ions were produced by nanoelectrospray ionizationsing a Triversa Nanomate autosampler (Advion Biosciences,

nc, Ithaca, NY). Concentrations of 4–20 �M angiotensin ISigma–Aldrich, St. Louis, MO) and 24–100 �M metal werelectrosprayed from 50:50 water:acetonitrile solutions. Metal-ted peptides were formed by addition of metal acetate saltsSigma–Aldrich, St. Louis, MO) to the solution containing peptides.he concentrations of each metal were varied to obtain abundantignals of the [AngI+M+H]3+ ions.

.3. Measuring collision cross sections

Although strictly speaking we record drift time distributions, its often useful to plot data on a cross section scale. As long as theharge state is known this is a simple conversion. Collision crossections are determined according to [39]

= (18�)1/2

16ze

(kbT)1/2

[1

MI+ 1

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760P

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273.21N

(1)

here tD is the time required for the ion to traverse the drift tube,e is the charge of the ion, kb is Boltzmann’s constant, MI and MB arehe masses of the ion and buffer gas, E is the electric field, L is therift tube length, T and P are the temperature and pressure, and N is

Please cite this article in press as: M.S. Glover, et al., Int. J. Mass Spectrom.

he neutral number density at STP. The drift tube contains two ionunnels which are operated at higher electric fields (∼12 V cm−1)han the rest of the drift tube with an applied RF voltage. In fact, wese two methods for obtaining collision cross sections measured on

Scheme 1. Abundant fragment ions produced from CID of [AngI+M+H]3+ ions.

this instrument. The time it takes a single conformation to traversethe section of the drift tube between the source and middle funnelgate is measured. This region of the drift tube does not containan ion funnel and has a linear electric field, so the collision crosssection can be evaluated directly from the drift time with Eq. (1); inthis method, tD is the time between the source pulse and the timerequired to select the ion of interest. Additionally, we can measurethe time it takes for ions to traverse the entire drift tube. In orderto account for the nonlinear field in the ion funnels, collision crosssections are calibrated to values determined by the aforementionedmethod.

3. Results and discussion

3.1. Discussion of related prior results

Previous MS–MS studies [8,9] of the [AngI+M]2+ ion by Looand coworkers showed that Co2+, Ni2+, Cu2+, and Zn2+ are coor-dinated by the histidine residues at position 6 and 9 (His6 andHis9). Additionally, they concluded that Cu2+ also interacts withthe C-terminus and Tyr4. In subsequent work, Vachet and cowork-ers confirmed the binding reported by Loo’s group for Co2+, Ni2+,and Cu2+ by using a metal-catalyzed oxidation MS technique [14].These findings are in agreement with earlier nuclear magnetic res-onance (NMR) studies which revealed that Zn2+ interacts with theHis6 and His9 residues in dimethyl sulfoxide (DMSO) and watersolutions [40]. Below, we use IMS–MS techniques to investigatethese systems in more detail. In addition, we extend the stud-ies of metals bound to AngI by including the Ca2+ alkaline earthcation. One difference between the results published here and thosepresented previously is that the previous work [9,14] focused onthe [AngI+M]2+ ion; here, we focus on the [AngI+M+H]3+ ion. The[AngI+M+H]3+ ion is chosen because it is possible to resolve moreconformations for this charge state.

3.2. MS–MS data reported in this study

Fragmentation spectra of the [AngI+M+H]3+ ions are displayedin Fig. 1. Metalated fragment ion labels are based on singly chargedprotonated fragment ions. For example, the [b9+Ca]3+ ion is a[b9]+ ion with the addition of Ca2+. Overall, these spectra appearmuch simpler than fragmentation spectra obtained for protonatedpeptides. Consistent with Loo’s and Vachet’s observations for the[AngI+M]2+ system, dissociation of [AngI+M+H]3+ is limited to spe-cific sites along the backbone that depend on the associated metal[8,9,14].

Fig. 1 shows that Co2+, Ni2+, Cu2+, and Zn2+ binding lead tofragmentation near the His6 residue as the [b6+M−H]2+ and [y4]+

fragments are observed in high abundance. For each of these cationswe determine that the sum of intensities for the [b6+M−H]2+,[a6+M−H]2+, and [y4]+ peaks comprises ∼24–40%. An illustration ofthe positions in which the peptide is cleaved to produce these frag-ments is provided in Scheme 1. Fragmentation near the His6 residueis observed in low abundance when M = Ca2+ (Fig. 1e). For Ca2+ a

2+ 2+ +

(2013), http://dx.doi.org/10.1016/j.ijms.2013.06.014

sum of ion intensities for the [b6+M−H] , [a6+M−H] , [y4] indi-cates that only 0.3% of ions dissociate near His6. For convenience,a summary of the relative populations of fragment ions that arisefrom dissociation near His6 is provided in Table 1.

Please cite this article in press as: M.S. Glover, et al., Int. J. Mass Spectrom. (2013), http://dx.doi.org/10.1016/j.ijms.2013.06.014

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Fig. 1. MS–MS spectra from the [AngI+Zn+H]3+ (a), [AngI+Cu+H]3+ (b), [AngI+Ni+H]3+ (c), [AngI+Co+H]3+ (d), and [AngI+Ca+H]3+ (e) ions fragmented by CID. Ion abundancesare normalized to unity for each spectrum. The most abundant fragments are labeled according to the notation explained in the text.

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Table 1Fragmentation near His6 and elongated conformations.

Metal Normalized abundance (%)

[b6+M−H]2+,[a6+M−H]2+, [y4]+ a

Elongated conformationsb

Ca2+ 0.3 0.0Co2+ 34.5 56.1Ni2+ 34.4 27.4Cu2+ 39.9 90.2Zn2+ 24.2 88.7

a Fragment ion abundances are normalized to the total ion count for each MS–MSspectrum (Fig. 1).

c

bgsCTir

iHetbp[fTCsipp[w

Faimd

b Elongated conformation (>326 A2) abundances are normalized to the total ionount for the [AngI+M+H]3+ ion from each activated distribution (Fig. 5).

The aforementioned trend is in good agreement with trends ininding interactions that are known from solution and crystallo-raphic studies available from the PDB [41,42]. From the manytructures that are available we find that histidine interacts witho2+, Ni2+, Cu2+, and Zn2+ at a much greater frequency than Ca2+.he MS–MS fragment ions that indicate M-His6 interactions aren agreement with metal ion coordination in solution systemseported in the PDB [41,42].

Despite the large difference in abundance of the [b6+M−H]2+

on between calcium and the transition metals, fragment ions nearis9 (i.e., [b9+M]3+) are observed in relatively high abundance forvery metal cation (Fig. 1). Although this would be expected forransition metals, it is less likely that Ca2+ would interact with His9

ecause Ca2+ is almost always coordinated by oxygen ligands inroteins [41,42]. In addition to fragments near His9, we observe they9+M]3+ and [RVYIHPFH+M]3+ fragment ions in greater abundanceor the [AngI+Ca+H]3+ ion than any other metal species (Fig. 1).herefore, the presence of large fragment ions from both the N- and-terminal ends of the peptide may indicate Ca2+-AngI interactionspan a large portion of the peptide backbone. Previous MS–MS stud-es by Gross and coworkers showed that Ca2+-coordinated peptidesroduce large fragments with the N- and C-terminal regions of the

Please cite this article in press as: M.S. Glover, et al., Int. J. Mass Spectrom.

eptides ‘trimmed’ away [10]. Therefore, the high abundance of theb9+Ca]3+ and [y9+Ca]3+ ion may result from a similar mechanismhere the Ca2+ cation is coordinated by a large portion of AngI.

ig. 2. Two-dimensional IMS–MS plot of ions produced by ESI of AngI upon theddition of zinc acetate in 50:50 water:acetonitrile. The top drift time distributions obtained by integrating each drift time bin across the entire range of m/z. The side

ass spectrum is obtained by integrating each m/z bin across the entire range ofrift times.

Fig. 3. Collision cross section distributions for the [AngI+M+H]3+ ions (M = 2H+, Ca2+,Co2+, Ni2+, Cu2+, and Zn2+) produced by ESI. The distributions have been obtainedfrom drift time (m/z) nested measurements by integrating every drift time bin across

the m/z range for each ion of interest. Distributions have been normalized to the totalion abundance for each trace.

Singly charged fragments that contain the doubly charged metalare observed in greatest abundance for the [AngI+Ni+H]3+ ion(Fig. 1c). We observe [bx+Ni−2H]+ fragments outside of the His6-His9 binding motif (x = 2–6). Such fragments may indicate Ni2+

interacts with amide nitrogen atoms on the peptide backbone. Con-densed phase studies have shown that Ni2+ lowers the pKa of amidenitrogen atoms and interact with deprotonated nitrogen atoms insmall peptides [43]. Interestingly, the two cations with the mostabundant signal for the [b3+M−2H]+ and [y7]2+ complementaryions (Fig. 1) are the two cations that have been shown to mostsignificantly lower the pKa of amide nitrogen atoms, Cu2+ and Ni2+

(2013), http://dx.doi.org/10.1016/j.ijms.2013.06.014

[43]. Also, recent infrared multiple-photon dissociation (IRMPD)studies of metals interacting with small peptides show that Ni2+

interacts with backbone nitrogen atoms [44].

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Fig. 4. Collision cross section distribution for the [AngI+Zn+H]3+ ion. The bottom trace in both panels shows the distribution obtained from ESI without selection or activation.The distributions labeled selection correspond to the two major conformations of the [AngI+Zn+H] ion at 308 A2 (a) and 343 A2 (b) marked by dotted vertical lines. Thed e selee

3

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istributions labeled 40, 90, and 130 V correspond to the collisional activation of thach trace.

.3. Ion mobility distributions

Fig. 2 shows the nested tD (m/z) distributions for the AngI sam-le with the addition of zinc acetate. Although several charge statesre observed, the spectrum is dominated by the [AngI+3H]3+ andAngI+Zn+H]3+ ions. Visual inspection of these data allows one touickly surmise that substitution of a zinc cation for 2 protons influ-nces the structure of the AngI ion. More insight can be obtainedy comparing specific cross section distributions for the differ-nt metals. Fig. 3 shows the cross section distributions for theAngI+M+H]3+ ions. From these data, it is clear that the metal cationas an influence on the range of structures that AngI adopts. Whenound to a metal, the range of conformations is large for a peptidef this size, varying from 291 to 354 A2 (or ∼22%). In comparison,

Please cite this article in press as: M.S. Glover, et al., Int. J. Mass Spectrom.

nly a single abundant conformation at 337 A2 is observed for theriply protonated [AngI+3H]3+ ion (Fig. 3).

It is interesting to compare the cross section distributionsith the general properties of the metal ions such as preferred

cted conformer. Distributions have been normalized to the total ion abundance for

coordination number. One apparent difference is between alkalineearth cation Ca2+ and transition metals Co2+, Ni2+, Cu2+, Zn2+. Themost abundant conformation of the [AngI+Ca+H]3+ ion is 302 A2

(Fig. 3). The Ca2+ coordinated peptide is >10% smaller than the mostabundant conformations for the Co2+, Cu2+, and Zn2+ coordinatedpeptides which have major features at 340, 340, and 343 A2, respec-tively (Fig. 3). One explanation for Ca2+ inducing more compactconformations is that the preferred coordination numbers of Ca2+

is 6–8 [4]. Coordination numbers for Co2+, Ni2+, Cu2+, and Zn2+ aresmaller on average (4–6) [4]. The preference for more elongatedconformations may result from a smaller portion of the peptideinteracting with the metal cation.

The different metal cations influence not only the collision crosssection but also the number of stable structures observed. Our

(2013), http://dx.doi.org/10.1016/j.ijms.2013.06.014

results show that many more structures are stabilized for the tran-sition metals. A good example of this is the [AngI+Ni+H]3+ ion,having at least 7 partially resolved conformers, and the largestrange of conformations (291–354 A2). In solution, Ni2+ is known

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6 al of Mass Spectrometry xxx (2013) xxx– xxx

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Fig. 5. Collision cross section distributions for the [AngI+M+H]3+ (M = 2H+, Ca2+,Co2+, Ni2+, Cu2+, and Zn2+) ions produced after collisional activation (IMS–IMS–MS).See text for details of activation voltages. The distributions have been obtained fromdrift time (m/z) nested measurements by integrating every drift time bin across the

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or its flexibility of coordination numbers and geometries [45].n contrast, we only observe a single major conformation for theAngI+Ca+H]3+ ion.

.4. Activated mobility distributions

To gain insight into the structural transitions that occur uponollisional activation, we have conducted IMS–IMS–MS studies.ollision cross section distributions were measured for the selec-ion and activation of [AngI+M+H]3+ ions. Conformations wereelected and activated from 0 to 170 V in 10 V increments. A repre-entative data set for the selection and activation of the two mostbundant conformations of the [AngI+Zn+H]3+ ion is displayed inig. 4. At low activation voltages we observe structural transi-ions that are dependent on the original conformation selected. Forxample, when the two major conformations of the [AngI+Zn+H]3+

re activated with 40 V, at least 5 stable conformations are formedhen selecting the conformation at 308 A2, but only a single confor-ation is observed upon selecting and activating the conformation

t 343 A2 (Fig. 4).At activation voltages that are slightly below the onset of sig-

ificant fragmentation we observe a nearly identical collision crossection distribution, regardless of the initial conformation selected.

e have previously referred to data obtained under these con-itions as quasi-equilibrium distributions [33]. That is, the ionsre heated over all of the barriers between states and upon cool-ng they establish the preferred gas-phase distribution of states. Aharacteristic of the quasi-equilibrium distribution is that the IMSistribution of annealed ions remains the same regardless of whatolutions are used to produce ions [46]. Once the gas-phase distri-utions are known, we can use differences that are found (under

ower energy, non-equilibrium conditions) to obtain insight abouthat populations of states are favored in solution. Returning to

he case of Zn2+, we observe that the activated quasi-equilibriumistribution for the [AngI+Zn+H]3+ ions is very different than theon-activated initial distribution obtained directly from the source.

n the original source distribution the conformation at 308 A2 isimilar in abundance to the more elongated conformation at 343 A2,ut at higher activation voltages the peak at 343 A2 dominates thepectrum (Fig. 4). We attribute this change in distributions to dif-erences in the potential energy landscapes that are associated withhe peptide in solution and in the gas phase.

Fig. 5 shows representative collision cross section distributionsor activated [AngI+M+H]3+ ions. The distributions are obtainedrom the highest activation voltage that the total fragment ion sig-al is <5% of the precursor signal. The activation voltages for Ca2+,o2+, Ni2+, Cu2+, Zn2+, and 2H+ are 140, 110, 120, 100, 140, and40 V, respectively. With the exception of [AngI+Ca+H]3+, everyollisionally activated species forms a markedly different distri-ution of structures than is observed from the ESI source. Ineneral, a distribution consisting of fewer and more elongatedeatures than the source distribution is observed upon collisionalctivation.

.5. Structure–fragment relationships

It is worthwhile to compare the activated ion distributionsith the fragment ions produced by CID. This is especially intrigu-

ng because the populations of conformations that are found justrior to dissociation should approach the populations of transi-ion states that lead to dissociation products. Table 1 includes theormalized abundances for fragment ions near His6 and elongated

Please cite this article in press as: M.S. Glover, et al., Int. J. Mass Spectrom.

onformations (>326 A2) of the activated distributions for all of theetal ions presented in this study. We note that calcium behaves

ery differently than the transition metals. Unlike the transitionetals, calcium does not favor the elongated state under gas-phase

m/z range for each ion of interest. Distributions have been normalized to the totalion abundance for each trace. The dotted traces are obtained by multiplying by 3 toshow features of low abundance.

quasi-equilibrium conditions. Moreover, calcium does not pro-duce fragments associated with the His6 site. As mentioned above,the fragment ions produced from the [AngI+Ca+H]3+ ion are largewith the major fragments being small neutral losses and the N-and C-terminal regions of the peptide cleaved away. The IMS–IMSdata supports the idea that Ca2+ is able to coordinate a largeportion of the peptide and that it does not “open-up” upon acti-vation, or form the [b6+Ca−H]2+, [a6+Ca−H]2+, and [y4]+ fragmentions.

In contrast, all of the transition metal coordinated ions shift tomore elongated conformations (Fig. 5) upon activation and pro-duce the [b6+M−H]2+, [a6+M−H]2+, and [y4]+ fragments near His6

(Fig. 1). The [AngI+Ni+H]3+ion is also interesting as this is the

(2013), http://dx.doi.org/10.1016/j.ijms.2013.06.014

only transition metal containing species that does not prefer aconformation >326 A2 after activation. As mentioned above, the[AngI+Ni+H]3+ produced the most abundant signal for [bx+Ni−2H]+

fragment ions. Thus, the metal–peptide interactions that produce

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he [bx+Ni−2H]+ fragment ions outside the His6-His9 bindingocket may stabilize compact conformations.

.6. Insight into populations of metal binding sites in solution

One final issue of interest involves what these results implybout metal binding sites in solution. It is fascinating that upon acti-ation ions prefer structures that have mobilities that are identicalo peaks observed directly from the source. That is, the activationrocess converts the distribution from that produced by the sourceo that preferred in the gas phase; however, there are no new peaks.hus, it appears that the differences in these source- and gas-phaseistributions are only changes in the populations of preferred struc-ures, rather than the creation of new gas-phase conformations.his interpretation suggests that multiple metal binding sites areopulated in solution. All of the transition metals appear to shown elongated peak in the non-activated source distributions. Thus,hese metals display one binding configuration near the His6 as wells other binding configurations that lead to more compact ions.inding at His6 is consistent with information about metal bindinghat is obtained from the PDB. It will be interesting to gain insightnto the other binding configurations and structures that are sug-ested from these studies, but not immediately apparent based onxamination of the PDB and CID studies.

. Summary and conclusions

MS-based techniques have been used to study metal–peptidenteractions for the [AngI+M+H]3+ ion. From a combination of CIDnd IMS–MS studies, we find evidence that transition metals bindear the His6 reside, both in the gas-phase prior to dissociation asell as in solution. Calcium does not appear to favor this binding

ite.

cknowledgements

The instrumentation in this work is supported by a grant fromhe NIH (NIH-5R01GM93322-2); other support was provided byhe Indiana University METACyt initiative that is funded by a grantrom the Lilly Endowment.

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11] B.A. Cerda, L. Cornett, C. Wesdemiotis, Probing the interaction of alkali and

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