- Email: [email protected]
The binding of Ca2+, Co2+, Ni2+, Cu2+, and Zn2+ cations to angiotensin I determined by mass spectrometry based techniques
International Journal of Mass Spectrometry 354–355 (2013) 318–325
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
The binding of Ca2+ , Co2+ , Ni2+ , Cu2+ , and Zn2+ cations to angiotensin I determined by mass spectrometry based techniques Matthew S. Glover, Jonathan M. Dilger, Feifei Zhu, David E. Clemmer ∗ Department of Chemistry, Indiana University, Bloomington, IN 47405, United States
a r t i c l e
i n f o
Article history: Received 6 May 2013 Received in revised form 18 June 2013 Accepted 18 June 2013 Available online 28 June 2013 Keywords: Ion mobility spectrometry Collision-induced dissociation Metal–peptide interactions Peptide ion structure
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 angiotensin I (AngI, Asp1 -Arg2 -Val3 -Tyr4 -Ile5 -His6 -Pro7 -Phe8 -His9 -Leu10 ) is examined by collision-induced dissociation (CID) and ion mobility spectrometry–mass spectrometry (IMS–MS). The series of CID patterns combined with IMS–MS data for [AngI+M+H]3+ ions provides information about the metal–peptide binding 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 these experiments, it is possible to derive insight into the populations of different metal coordination sites that are sampled in solution prior to introduction of species into the gas phase. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Metal ion–biomolecule interactions are important for many biological processes including those involving structural stabilization, redox reactions, and signal transduction [1–4]. The interactions of metal ions with favored binding sites (i.e., with specific amino acid residues or sequence motifs) also are known to induce structural changes, such that enzymatic activity is regulated by metal binding [3]. Thus, it is important to understand fundamental interactions of metals with polypeptide chains. One means of understanding metal binding interactions involves analysis by mass spectrometry (MS) or tandem mass spectrometry (MS–MS) techniques. MS analyses provide direct information about the stoichiometry of the metal–biopolymer complex [5]. MS–MS analyses, in which collision-induced dissociation (CID) is used to break apart metal-containing biopolymers, sometimes show specific sites of fragmentation – providing insight into metal binding sites [6–19]. Several studies have explored unique fragmentation mechanisms and it has been suggested that such specificity may be useful for direct sequencing or fingerprinting biopolymers by MS [16–18]. Strictly, MS–MS techniques provide insight about the metalbinding sites at the transition states associated with dissociation. Little is known about the distribution of metal ion–peptide conformations prior to dissociation [20–28] and even less is understood
∗ Corresponding author. E-mail address: [email protected] (D.E. Clemmer). 1387-3806/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijms.2013.06.014
about transitions between states as ions are activated and ultimately dissociate. In this paper, we use ion mobility spectrometry (IMS)–MS techniques and MS–MS dissociation studies to investigate the interaction 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 detecting multiple structures of biological molecules in the gas phase [29–31]. We note that the slow heating [32] associated with CID can induce structural transitions in the precursor ion along the pathway to dissociation [33]; thus, we supplement our studies with multidimensional IMS techniques (e.g., IMS–IMS–MS) that monitor structural transitions that occur upon collisional activation [34,35]. The AngI model peptide contains several residues that are known to interact with metal cations – the side chains of Asp1 , Arg2 , Tyr4 , His6 , His9 ; additionally, ten-residue AngI is sufficiently long that backbone carbonyl oxygen atoms and amide nitrogen atoms may also coordinate the metal cation. The work presented here provides insight into the low-lying conformations that are present prior to dissociation and allows for a comparison between metal-mediated conformations prior to and after collisional activation and the fragments produced by CID. Finally, in favorable cases, the similarity of IMS peaks observed before and after activation allows us to speculate about what conformations are favored in solution for these metals. Overall, our data are in agreement with known binding sites for these metals that would be predicted from structures taken from the protein data bank (PDB).
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
319
2. Experimental method 2.1. Instrumentation CID experiments were performed on a LTQ Velos mass spectrometer (Thermo Scientific, San Jose, CA). Fragment ions were produced in the linear ion trap by applying a resonant rf excitation waveform for 10 ms, an activation q of 0.25, and normalized collision energy of 16%. A list of candidate fragment ions was generated online with the MS-product tool [36]. Metal-containing fragment ion m/z values were calculated from expected fragment products plus the mass of the metal cation. Ion mobility experiments were conducted on a home-built instrument described in detail previously [34,35]. Briefly, electrosprayed ions are stored in an hourglass-shaped ion funnel [37]. Packets of ions are periodically released into the drift tube by lowering an electrostatic gate on the source funnel for 150 s. The drift tube is operated with 3.00 ± 0.01 Torr He buffer gas and a uniform electric field (∼10 V cm−1 ). Ions are radially focused in funnels at the middle and end of the drift tube. Ions exit the drift tube through a differentially pumped region and are transferred to the time-offlight (TOF) mass spectrometer. Ion drift times and mass-to-charge values are recorded in a nested fashion [38]. We have described the experimental design and analytical utility of IMS–IMS–MS in detail previously [34,35]. Briefly, we operate the linear drift tube as two independent drift tubes separated by an ion funnel. An electrostatic gate in the middle funnel is implemented to select ions of a single mobility or range of mobilities to be transmitted to the second drift tube. Mobility-selected ions can be collisionally activated before entering the second region of the drift tube; at low activation voltages structural transitions are induced, and at higher activation voltages fragmentation occurs. Structural transitions can be monitored by measuring the mobilities of annealed ions following separation of the ions through the second region of the drift tube. 2.2. Generation of [AngI+M+H]3+ ions Peptide ions were produced by nanoelectrospray ionization using a Triversa Nanomate autosampler (Advion Biosciences, Inc, Ithaca, NY). Concentrations of 4–20 M angiotensin I (Sigma–Aldrich, St. Louis, MO) and 24–100 M metal were electrosprayed from 50:50 water:acetonitrile solutions. Metalated peptides were formed by addition of metal acetate salts (Sigma–Aldrich, St. Louis, MO) to the solution containing peptides. The concentrations of each metal were varied to obtain abundant signals of the [AngI+M+H]3+ ions. 2.3. Measuring collision cross sections Although strictly speaking we record drift time distributions, it is often useful to plot data on a cross section scale. As long as the charge state is known this is a simple conversion. Collision cross sections ˝ are determined according to [39] =
(18)1/2 1 1 ze + 1/2 MI M 16 B (kb T )
1/2 t E 760 D L
T 1 P 273.2 N
(1)
where tD is the time required for the ion to traverse the drift tube, ze is the charge of the ion, kb is Boltzmann’s constant, MI and MB are the masses of the ion and buffer gas, E is the electric field, L is the drift tube length, T and P are the temperature and pressure, and N is the neutral number density at STP. The drift tube contains two ion funnels which are operated at higher electric fields (∼12 V cm−1 ) than the rest of the drift tube with an applied RF voltage. In fact, we use 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 traverse the section of the drift tube between the source and middle funnel gate is measured. This region of the drift tube does not contain an ion funnel and has a linear electric field, so the collision cross section can be evaluated directly from the drift time with Eq. (1); in this method, tD is the time between the source pulse and the time required to select the ion of interest. Additionally, we can measure the time it takes for ions to traverse the entire drift tube. In order to account for the nonlinear field in the ion funnels, collision cross sections are calibrated to values determined by the aforementioned method. 3. Results and discussion 3.1. Discussion of related prior results Previous MS–MS studies [8,9] of the [AngI+M]2+ ion by Loo and coworkers showed that Co2+ , Ni2+ , Cu2+ , and Zn2+ are coordinated by the histidine residues at position 6 and 9 (His6 and His9 ). Additionally, they concluded that Cu2+ also interacts with the C-terminus and Tyr4 . In subsequent work, Vachet and coworkers 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 resonance (NMR) studies which revealed that Zn2+ interacts with the His6 and His9 residues in dimethyl sulfoxide (DMSO) and water solutions [40]. Below, we use IMS–MS techniques to investigate these systems in more detail. In addition, we extend the studies of metals bound to AngI by including the Ca2+ alkaline earth cation. One difference between the results published here and those presented previously is that the previous work [9,14] focused on the [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 more conformations for this charge state. 3.2. MS–MS data reported in this study Fragmentation spectra of the [AngI+M+H]3+ ions are displayed in Fig. 1. Metalated fragment ion labels are based on singly charged protonated fragment ions. For example, the [b9 +Ca]3+ ion is a [b9 ]+ ion with the addition of Ca2+ . Overall, these spectra appear much simpler than fragmentation spectra obtained for protonated peptides. Consistent with Loo’s and Vachet’s observations for the [AngI+M]2+ system, dissociation of [AngI+M+H]3+ is limited to specific sites along the backbone that depend on the associated metal [8,9,14]. Fig. 1 shows that Co2+ , Ni2+ , Cu2+ , and Zn2+ binding lead to fragmentation near the His6 residue as the [b6 +M−H]2+ and [y4 ]+ fragments are observed in high abundance. For each of these cations we determine that the sum of intensities for the [b6 +M−H]2+ , [a6 +M−H]2+ , and [y4 ]+ peaks comprises ∼24–40%. An illustration of the positions in which the peptide is cleaved to produce these fragments is provided in Scheme 1. Fragmentation near the His6 residue is observed in low abundance when M = Ca2+ (Fig. 1e). For Ca2+ a sum of ion intensities for the [b6 +M−H]2+ , [a6 +M−H]2+ , [y4 ]+ indicates that only 0.3% of ions dissociate near His6 . For convenience, a summary of the relative populations of fragment ions that arise from dissociation near His6 is provided in Table 1.
320
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
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 abundances are normalized to unity for each spectrum. The most abundant fragments are labeled according to the notation explained in the text.
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
321
Table 1 Fragmentation near His6 and elongated conformations. Metal
Ca2+ Co2+ Ni2+ Cu2+ Zn2+
Normalized abundance (%) [b6 +M−H]2+ , [a6 +M−H]2+ , [y4 ]+ a
Elongated conformationsb
0.3 34.5 34.4 39.9 24.2
0.0 56.1 27.4 90.2 88.7
a Fragment ion abundances are normalized to the total ion count for each MS–MS spectrum (Fig. 1). b Elongated conformation (>326 A˚ 2 ) abundances are normalized to the total ion count for the [AngI+M+H]3+ ion from each activated distribution (Fig. 5).
The aforementioned trend is in good agreement with trends in binding interactions that are known from solution and crystallographic studies available from the PDB [41,42]. From the many structures that are available we find that histidine interacts with Co2+ , Ni2+ , Cu2+ , and Zn2+ at a much greater frequency than Ca2+ . The MS–MS fragment ions that indicate M-His6 interactions are in agreement with metal ion coordination in solution systems reported in the PDB [41,42]. Despite the large difference in abundance of the [b6 +M−H]2+ ion between calcium and the transition metals, fragment ions near His9 (i.e., [b9 +M]3+ ) are observed in relatively high abundance for every metal cation (Fig. 1). Although this would be expected for transition metals, it is less likely that Ca2+ would interact with His9 because Ca2+ is almost always coordinated by oxygen ligands in proteins [41,42]. In addition to fragments near His9 , we observe the [y9 +M]3+ and [RVYIHPFH+M]3+ fragment ions in greater abundance for the [AngI+Ca+H]3+ ion than any other metal species (Fig. 1). Therefore, the presence of large fragment ions from both the N- and C-terminal ends of the peptide may indicate Ca2+ -AngI interactions span a large portion of the peptide backbone. Previous MS–MS studies by Gross and coworkers showed that Ca2+ -coordinated peptides produce large fragments with the N- and C-terminal regions of the peptides ‘trimmed’ away [10]. Therefore, the high abundance of the [b9 +Ca]3+ and [y9 +Ca]3+ ion may result from a similar mechanism where the Ca2+ cation is coordinated by a large portion of AngI.
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 obtained from 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 total ion abundance for each trace.
Fig. 2. Two-dimensional IMS–MS plot of ions produced by ESI of AngI upon the addition of zinc acetate in 50:50 water:acetonitrile. The top drift time distribution is obtained by integrating each drift time bin across the entire range of m/z. The side mass spectrum is obtained by integrating each m/z bin across the entire range of drift times.
Singly charged fragments that contain the doubly charged metal are 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. Condensed phase studies have shown that Ni2+ lowers the pKa of amide nitrogen atoms and interact with deprotonated nitrogen atoms in small peptides [43]. Interestingly, the two cations with the most abundant signal for the [b3 +M−2H]+ and [y7 ]2+ complementary ions (Fig. 1) are the two cations that have been shown to most significantly lower the pKa of amide nitrogen atoms, Cu2+ and Ni2+ [43]. Also, recent infrared multiple-photon dissociation (IRMPD) studies of metals interacting with small peptides show that Ni2+ interacts with backbone nitrogen atoms [44].
322
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
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 A˚ 2 (a) and 343 A˚ 2 (b) marked by dotted vertical lines. The distributions labeled 40, 90, and 130 V correspond to the collisional activation of the selected conformer. Distributions have been normalized to the total ion abundance for each trace.
3.3. Ion mobility distributions Fig. 2 shows the nested tD (m/z) distributions for the AngI sample with the addition of zinc acetate. Although several charge states are observed, the spectrum is dominated by the [AngI+3H]3+ and [AngI+Zn+H]3+ ions. Visual inspection of these data allows one to quickly surmise that substitution of a zinc cation for 2 protons influences the structure of the AngI ion. More insight can be obtained by comparing specific cross section distributions for the different metals. Fig. 3 shows the cross section distributions for the [AngI+M+H]3+ ions. From these data, it is clear that the metal cation has an influence on the range of structures that AngI adopts. When bound to a metal, the range of conformations is large for a peptide of this size, varying from 291 to 354 A˚ 2 (or ∼22%). In comparison, only a single abundant conformation at 337 A˚ 2 is observed for the triply protonated [AngI+3H]3+ ion (Fig. 3). It is interesting to compare the cross section distributions with the general properties of the metal ions such as preferred
coordination number. One apparent difference is between alkaline earth cation Ca2+ and transition metals Co2+ , Ni2+ , Cu2+ , Zn2+ . The most abundant conformation of the [AngI+Ca+H]3+ ion is 302 A˚ 2 (Fig. 3). The Ca2+ coordinated peptide is >10% smaller than the most abundant conformations for the Co2+ , Cu2+ , and Zn2+ coordinated peptides which have major features at 340, 340, and 343 A˚ 2 , respectively (Fig. 3). One explanation for Ca2+ inducing more compact conformations is that the preferred coordination numbers of Ca2+ is 6–8 [4]. Coordination numbers for Co2+ , Ni2+ , Cu2+ , and Zn2+ are smaller on average (4–6) [4]. The preference for more elongated conformations may result from a smaller portion of the peptide interacting with the metal cation. The different metal cations influence not only the collision cross section but also the number of stable structures observed. Our results show that many more structures are stabilized for the transition metals. A good example of this is the [AngI+Ni+H]3+ ion, having at least 7 partially resolved conformers, and the largest range of conformations (291–354 A˚ 2 ). In solution, Ni2+ is known
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
323
for its flexibility of coordination numbers and geometries [45]. In contrast, we only observe a single major conformation for the [AngI+Ca+H]3+ ion. 3.4. Activated mobility distributions To gain insight into the structural transitions that occur upon collisional activation, we have conducted IMS–IMS–MS studies. Collision cross section distributions were measured for the selection and activation of [AngI+M+H]3+ ions. Conformations were selected and activated from 0 to 170 V in 10 V increments. A representative data set for the selection and activation of the two most abundant conformations of the [AngI+Zn+H]3+ ion is displayed in Fig. 4. At low activation voltages we observe structural transitions that are dependent on the original conformation selected. For example, when the two major conformations of the [AngI+Zn+H]3+ are activated with 40 V, at least 5 stable conformations are formed when selecting the conformation at 308 A˚ 2 , but only a single conformation is observed upon selecting and activating the conformation at 343 A˚ 2 (Fig. 4). At activation voltages that are slightly below the onset of significant fragmentation we observe a nearly identical collision cross section distribution, regardless of the initial conformation selected. We have previously referred to data obtained under these conditions as quasi-equilibrium distributions [33]. That is, the ions are heated over all of the barriers between states and upon cooling they establish the preferred gas-phase distribution of states. A characteristic of the quasi-equilibrium distribution is that the IMS distribution of annealed ions remains the same regardless of what solutions are used to produce ions [46]. Once the gas-phase distributions are known, we can use differences that are found (under lower energy, non-equilibrium conditions) to obtain insight about what populations of states are favored in solution. Returning to the case of Zn2+ , we observe that the activated quasi-equilibrium distribution for the [AngI+Zn+H]3+ ions is very different than the non-activated initial distribution obtained directly from the source. In the original source distribution the conformation at 308 A˚ 2 is similar in abundance to the more elongated conformation at 343 A˚ 2 , but at higher activation voltages the peak at 343 A˚ 2 dominates the spectrum (Fig. 4). We attribute this change in distributions to differences in the potential energy landscapes that are associated with the peptide in solution and in the gas phase. Fig. 5 shows representative collision cross section distributions for activated [AngI+M+H]3+ ions. The distributions are obtained from the highest activation voltage that the total fragment ion signal is <5% of the precursor signal. The activation voltages for Ca2+ , Co2+ , Ni2+ , Cu2+ , Zn2+ , and 2H+ are 140, 110, 120, 100, 140, and 140 V, respectively. With the exception of [AngI+Ca+H]3+ , every collisionally activated species forms a markedly different distribution of structures than is observed from the ESI source. In general, a distribution consisting of fewer and more elongated features than the source distribution is observed upon collisional activation. 3.5. Structure–fragment relationships It is worthwhile to compare the activated ion distributions with the fragment ions produced by CID. This is especially intriguing because the populations of conformations that are found just prior to dissociation should approach the populations of transition states that lead to dissociation products. Table 1 includes the normalized abundances for fragment ions near His6 and elongated conformations (>326 A˚ 2 ) of the activated distributions for all of the metal ions presented in this study. We note that calcium behaves very differently than the transition metals. Unlike the transition metals, calcium does not favor the elongated state under gas-phase
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 from 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 total ion abundance for each trace. The dotted traces are obtained by multiplying by 3 to show features of low abundance.
quasi-equilibrium conditions. Moreover, calcium does not produce fragments associated with the His6 site. As mentioned above, the fragment ions produced from the [AngI+Ca+H]3+ ion are large with the major fragments being small neutral losses and the Nand C-terminal regions of the peptide cleaved away. The IMS–IMS data supports the idea that Ca2+ is able to coordinate a large portion of the peptide and that it does not “open-up” upon activation, or form the [b6 +Ca−H]2+ , [a6 +Ca−H]2+ , and [y4 ]+ fragment ions. In contrast, all of the transition metal coordinated ions shift to more elongated conformations (Fig. 5) upon activation and produce 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 only transition metal containing species that does not prefer a conformation >326 A˚ 2 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
324
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
the [bx +Ni−2H]+ fragment ions outside the His6 -His9 binding pocket may stabilize compact conformations.
[13]
3.6. Insight into populations of metal binding sites in solution [14]
One final issue of interest involves what these results imply about metal binding sites in solution. It is fascinating that upon activation ions prefer structures that have mobilities that are identical to peaks observed directly from the source. That is, the activation process converts the distribution from that produced by the source to that preferred in the gas phase; however, there are no new peaks. Thus, it appears that the differences in these source- and gas-phase distributions are only changes in the populations of preferred structures, rather than the creation of new gas-phase conformations. This interpretation suggests that multiple metal binding sites are populated in solution. All of the transition metals appear to show an elongated peak in the non-activated source distributions. Thus, these metals display one binding configuration near the His6 as well as other binding configurations that lead to more compact ions. Binding at His6 is consistent with information about metal binding that is obtained from the PDB. It will be interesting to gain insight into the other binding configurations and structures that are suggested from these studies, but not immediately apparent based on examination of the PDB and CID studies. 4. Summary and conclusions MS-based techniques have been used to study metal–peptide interactions for the [AngI+M+H]3+ ion. From a combination of CID and IMS–MS studies, we find evidence that transition metals bind near the His6 reside, both in the gas-phase prior to dissociation as well as in solution. Calcium does not appear to favor this binding site. Acknowledgements The instrumentation in this work is supported by a grant from the NIH (NIH-5R01GM93322-2); other support was provided by the Indiana University METACyt initiative that is funded by a grant from the Lilly Endowment.
[15]
[16]
[17]
[18]
[19]
[20] [21]
[22] [23]
[24]
[25]
[26]
[27]
[28]
References
[29]
[1] S.J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry, University Science Books, CA, 1994. [2] R.H. Holm, P. Kennepohl, E.I. Solomon, Structural and functional aspects of metal sites in biology, Chemical Reviews 96 (1996) 2239–2314. [3] T. Dudev, C. Lim, Principles governing Mg, Ca, and Zn binding and selectivity in proteins, Chemical Reviews 103 (2003) 773–787. [4] E.A. Permyakov, Metalloproteomics, Wiley, NJ, 2009. [5] J.A. Loo, Studying noncovalent protein complexes by electrospray ionization mass spectrometry, Mass Spectrometry Reviews 16 (1997) 1–23. [6] P. Hu, M.L. Gross, Strong interactions of anionic peptides and alkaline earth metal ions: metal-ion-bound peptides in the gas phase, Journal of the American Chemical Society 114 (1992) 9153–9160. [7] A. Reiter, J. Adams, H. Zhao, Intrinsic (gas-phase) binding of Co2+ and Ni2+ by peptides: a direct reflection of aqueous-phase chemistry, Journal of the American Chemical Society 116 (1994) 7827–7838. [8] J.A. Loo, P. Hu, R.D. Smith, Interaction of angiotensin peptides and zinc metal ions probed by electrospray ionization mass spectrometry, Journal of the American Society for Mass Spectrometry 5 (1994) 959–965. [9] P. Hu, J.A. Loo, Gas-phase coordination properties of Zn2+ , Cu2+ , Ni2+ , and Co2+ with histidine-containing peptides, Journal of the American Chemical Society 117 (1995) 11314–11319. [10] O.V. Nemirovskiy, M.L. Gross, Determination of calcium binding sites in gasphase small peptides by tandem mass spectrometry, Journal of the American Society for Mass Spectrometry 9 (1998) 1020–1028. [11] B.A. Cerda, L. Cornett, C. Wesdemiotis, Probing the interaction of alkali and transition metal ions with bradykinin and its des-arginine derivatives via matrix-assisted laser desorption/ionization and postsource decay mass spectrometry, International Journal of Mass Spectrometry 193 (1999) 205–226. [12] S.P. Gaucher, J.A. Leary, Influence of metal ion and coordination geometry on the gas phase dissociation and stereochemical differentiation
[30]
[31]
[32] [33]
[34]
[35]
[36] [37]
[38]
[39] [40]
of N-glycosides, International Journal of Mass Spectrometry 197 (2000) 139–148. M.D. Leavell, J.A. Leary, Stabilization, linkage analysis of metal-ligated sialic acid containing oligosaccharides, Journal of the American Society for Mass Spectrometry 12 (2001) 528–536. J.D. Bridgewater, J. Lim, R.W. Vachet, Transition metal–peptide binding studied by metal-catalyzed oxidation reactions and mass spectrometry, Analytical Chemistry 78 (2006) 2432–2438. S.J. Ye, P.B. Armentrout, Absolute thermodynamic measurements of alkali metal cation interactions with a simple dipeptide and tripeptide, Journal of Physical Chemistry A 112 (2008) 3587–3596. M. Roˇzman, S.J. Gaskell, Non-covalent interactions of alkali metal cations with singly charged tryptic peptides, Journal of Mass Spectrometry 45 (2010) 1409–1415. K.K. Morishetti, S.C. Russell, X. Zhao, D.B. Robinson, J. Ren, Tandem mass spectrometry studies of protonated and alkali metalated peptoids: enhanced sequence coverage by metal cation addition, International Journal of Mass Spectrometry 308 (2011) 98–108. H.M. Watson, J.B. Vincent, C.J. Cassady, Effects of transition metal ion coordination on the collision-induced dissociation of polyalanines, Journal of Mass Spectrometry 46 (2011) 1099–1107. J. Lee, L.P. Jayathilaka, S. Gupta, J.-S. Huang, B.-S. Lee, Gold ion-angiotensin peptide interaction by mass spectrometry, Journal of the American Society for Mass Spectrometry 23 (2012) 942–951. J.A. Taraszka, J. Li, D.E. Clemmer, Metal-mediated peptide ion conformations in the gas phase, Journal of Physical Chemistry B 104 (2000) 4545–4551. B.T. Ruotolo, C.C. Tate, D.H. Russell, Ion mobility-mass spectrometry applied to cyclic peptide analysis: conformational preferences of gramicidin S and linear analogs in the gas phase, Journal of the American Society for Mass Spectrometry 15 (2004) 870–878. M. Kohtani, M.F. Jarrold, S. Wee, R.A.J. O’Hair, Metal ion interactions with polyalanine peptides, Journal of Physical Chemistry B 108 (2004) 6093–6097. T. Wyttenbach, D. Liu, M.T. Bowers, Interactions of the hormone oxytocin with divalent metal ions, Journal of the American Chemical Society 130 (2008) 5593–6000. Y. Berezovskaya, C.T. Armstrong, A.L. Boyle, M. Porrini, D.N. Woolfson, P.E. Barran, Metal binding to a zinc-finger peptide: a comparison between solution and the gas phase, Chemical Communications 47 (2011) 412–414. L. Chen, Y.Q. Gao, D.H. Russell, How alkali metal ion binding alters the conformation preferences of gramacidin A: a molecular dynamics and ion mobility study, Journal of Physical Chemistry A 116 (2012) 689–696. J.M. Dilger, S.J. Valentine, M.S. Glover, M.A. Ewing, D.E. Clemmer, A database of alkali metal-containing peptide cross sections: influences of metals on size parameters for specific amino acids, International Journal of Mass Spectrometry 330-332 (2012) 35–45. S.-J. Hyung, A.S. DeToma, J.R. Brender, S. Lee, S. Vivekanandan, A. Kochi, J.-S. Choi, A. Ramamoorthy, B.T. Ruotolo, M.H. Lim, Insights into antiamyloidogenic properties of the green tea extract (−)-epigallocathechin-3-gallate toward metal-associated amyloid- species, Proceedings of the National Academy of Sciences of the United States of America 110 (2013) 3743–3748. J.M. Dilger, S.J. Valentine, M.S. Glover, D.E. Clemmer, A database of alkalineearth-coordinated peptide cross sections: insight into general aspects of structure, Journal of the American Society for Mass Spectrometry 24 (2013) 768–779. D.E. Clemmer, M.F. Jarrold, Ion mobility measurements and their applications to clusters and biomolecules, Journal of Mass Spectrometry 32 (1997) 577–592. T. Wyttenbach, P.R. Kemper, M.T. Bowers, Design of a new electrospray ion mobility mass spectrometer, International Journal of Mass Spectrometry 212 (2001) 13–23. B.C. Bohrer, S.I. Merenbloom, S.L. Koeniger, A.E. Hilderbrand, D.E. Clemmer, Biomolecule analysis by ion mobility spectrometry, Annual Review of Analytical Chemistry 1 (2008) 293–327. S.A. McLuckey, D.E. Goeringer, Slow heating methods in tandem mass spectrometry, Journal of Mass Spectrometry 32 (1997) 461–474. N.A. Pierson, S.J. Valentine, D.E. Clemmer, Evidence for a quasi-equilibrium distribution of states for bradykinin [M+3H]3+ ions in the gas phase, Journal of Physical Chemistry B 114 (2010) 7777–7783. S.L. Koeniger, S.I. Merenbloom, S.J. Valentine, M.F. Jarrold, H.R. Udseth, R.D. Smith, D.E. Clemmer, An IMS–IMS analogue of MS–MS, Analytical Chemistry 78 (2006) 4161–4174. S.I. Merenbloom, S.L. Koeniger, S.J. Valentine, M.D. Plasencia, D.E. Clemmer, I.M.S–I.M.S, IMS–IMS–IMS/MS for separating peptide and protein fragment ions, Analytical Chemistry 78 (2006) 2802–2809. Protein Prospector. http://www.prospector.ucsf.edu/prospector/ (last accessed May 2013). K. Tang, A.A. Shvartsburg, H.-N. Lee, D.C. Prior, M.A. Buschbach, F. Li, A.V. Tolmachev, G.A. Anderson, R.D. Smith, High-sensitivity ion mobility spectrometry/mass spectrometry using electrodynamic ion funnel interfaces, Analytical Chemistry 77 (2005) 3330–3339. C.S. Hoaglund, S.J. Valentine, C.R. Sporleder, J.P. Reilly, D.E. Clemmer, Threedimensional ion mobility/TOFMS analysis of electrosprayed biomolecules, Analytical Chemistry 70 (1998) 2236–2242. E.A. Mason, E.W. McDaniel, Transport Properties of Ions in Gases, Wiley, New York, 1988. A.P. Arnold, D.M. Stanley, J.G. Collins, Zn2+ -induced deprotonation of a peptide nitrogen in angiotensin I, FEBS Letters 289 (1991) 96–98.
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325 [41] M. Babor, H.M. Greenblatt, M. Edelman, V. Sobolev, Flexibility of metal binding sites in proteins on a database scale, Proteins 59 (2005) 221–230. [42] H. Zheng, M. Chruszcz, P. Lasota, L. Lebioda, W. Minor, Data mining of metal ion environments present in protein structures, Journal of Inorganic Biochemistry 102 (2008) 1765–1776. [43] H. Sigel, R.B. Martin, Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands, Chemical Reviews 82 (1982) 385–426.
325
[44] R.C. Dunbar, N.C. Polfer, G. Berden, J. Oomens, Metal ion binding to peptides: oxygen or nitrogen sites? International Journal of Mass Spectrometry 330–332 (330) (2012) 71–80. [45] S.W. Ragsdale, Nickel-based enzyme systems, Journal of Biological Chemistry 284 (2009) 18571–18575. [46] N.A. Pierson, L. Chen, S.J. Valentine, D.H. Russell, D.E. Clemmer, Number of solution states of bradykinin from ion mobility and mass spectrometry measurements, Journal of the American Chemical Society 133 (2011) 13810–13813.
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
The binding of Ca2+ , Co2+ , Ni2+ , Cu2+ , and Zn2+ cations to angiotensin I determined by mass spectrometry based techniques Matthew S. Glover, Jonathan M. Dilger, Feifei Zhu, David E. Clemmer ∗ Department of Chemistry, Indiana University, Bloomington, IN 47405, United States
a r t i c l e
i n f o
Article history: Received 6 May 2013 Received in revised form 18 June 2013 Accepted 18 June 2013 Available online 28 June 2013 Keywords: Ion mobility spectrometry Collision-induced dissociation Metal–peptide interactions Peptide ion structure
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 angiotensin I (AngI, Asp1 -Arg2 -Val3 -Tyr4 -Ile5 -His6 -Pro7 -Phe8 -His9 -Leu10 ) is examined by collision-induced dissociation (CID) and ion mobility spectrometry–mass spectrometry (IMS–MS). The series of CID patterns combined with IMS–MS data for [AngI+M+H]3+ ions provides information about the metal–peptide binding 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 these experiments, it is possible to derive insight into the populations of different metal coordination sites that are sampled in solution prior to introduction of species into the gas phase. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Metal ion–biomolecule interactions are important for many biological processes including those involving structural stabilization, redox reactions, and signal transduction [1–4]. The interactions of metal ions with favored binding sites (i.e., with specific amino acid residues or sequence motifs) also are known to induce structural changes, such that enzymatic activity is regulated by metal binding [3]. Thus, it is important to understand fundamental interactions of metals with polypeptide chains. One means of understanding metal binding interactions involves analysis by mass spectrometry (MS) or tandem mass spectrometry (MS–MS) techniques. MS analyses provide direct information about the stoichiometry of the metal–biopolymer complex [5]. MS–MS analyses, in which collision-induced dissociation (CID) is used to break apart metal-containing biopolymers, sometimes show specific sites of fragmentation – providing insight into metal binding sites [6–19]. Several studies have explored unique fragmentation mechanisms and it has been suggested that such specificity may be useful for direct sequencing or fingerprinting biopolymers by MS [16–18]. Strictly, MS–MS techniques provide insight about the metalbinding sites at the transition states associated with dissociation. Little is known about the distribution of metal ion–peptide conformations prior to dissociation [20–28] and even less is understood
∗ Corresponding author. E-mail address: [email protected] (D.E. Clemmer). 1387-3806/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijms.2013.06.014
about transitions between states as ions are activated and ultimately dissociate. In this paper, we use ion mobility spectrometry (IMS)–MS techniques and MS–MS dissociation studies to investigate the interaction 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 detecting multiple structures of biological molecules in the gas phase [29–31]. We note that the slow heating [32] associated with CID can induce structural transitions in the precursor ion along the pathway to dissociation [33]; thus, we supplement our studies with multidimensional IMS techniques (e.g., IMS–IMS–MS) that monitor structural transitions that occur upon collisional activation [34,35]. The AngI model peptide contains several residues that are known to interact with metal cations – the side chains of Asp1 , Arg2 , Tyr4 , His6 , His9 ; additionally, ten-residue AngI is sufficiently long that backbone carbonyl oxygen atoms and amide nitrogen atoms may also coordinate the metal cation. The work presented here provides insight into the low-lying conformations that are present prior to dissociation and allows for a comparison between metal-mediated conformations prior to and after collisional activation and the fragments produced by CID. Finally, in favorable cases, the similarity of IMS peaks observed before and after activation allows us to speculate about what conformations are favored in solution for these metals. Overall, our data are in agreement with known binding sites for these metals that would be predicted from structures taken from the protein data bank (PDB).
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
319
2. Experimental method 2.1. Instrumentation CID experiments were performed on a LTQ Velos mass spectrometer (Thermo Scientific, San Jose, CA). Fragment ions were produced in the linear ion trap by applying a resonant rf excitation waveform for 10 ms, an activation q of 0.25, and normalized collision energy of 16%. A list of candidate fragment ions was generated online with the MS-product tool [36]. Metal-containing fragment ion m/z values were calculated from expected fragment products plus the mass of the metal cation. Ion mobility experiments were conducted on a home-built instrument described in detail previously [34,35]. Briefly, electrosprayed ions are stored in an hourglass-shaped ion funnel [37]. Packets of ions are periodically released into the drift tube by lowering an electrostatic gate on the source funnel for 150 s. The drift tube is operated with 3.00 ± 0.01 Torr He buffer gas and a uniform electric field (∼10 V cm−1 ). Ions are radially focused in funnels at the middle and end of the drift tube. Ions exit the drift tube through a differentially pumped region and are transferred to the time-offlight (TOF) mass spectrometer. Ion drift times and mass-to-charge values are recorded in a nested fashion [38]. We have described the experimental design and analytical utility of IMS–IMS–MS in detail previously [34,35]. Briefly, we operate the linear drift tube as two independent drift tubes separated by an ion funnel. An electrostatic gate in the middle funnel is implemented to select ions of a single mobility or range of mobilities to be transmitted to the second drift tube. Mobility-selected ions can be collisionally activated before entering the second region of the drift tube; at low activation voltages structural transitions are induced, and at higher activation voltages fragmentation occurs. Structural transitions can be monitored by measuring the mobilities of annealed ions following separation of the ions through the second region of the drift tube. 2.2. Generation of [AngI+M+H]3+ ions Peptide ions were produced by nanoelectrospray ionization using a Triversa Nanomate autosampler (Advion Biosciences, Inc, Ithaca, NY). Concentrations of 4–20 M angiotensin I (Sigma–Aldrich, St. Louis, MO) and 24–100 M metal were electrosprayed from 50:50 water:acetonitrile solutions. Metalated peptides were formed by addition of metal acetate salts (Sigma–Aldrich, St. Louis, MO) to the solution containing peptides. The concentrations of each metal were varied to obtain abundant signals of the [AngI+M+H]3+ ions. 2.3. Measuring collision cross sections Although strictly speaking we record drift time distributions, it is often useful to plot data on a cross section scale. As long as the charge state is known this is a simple conversion. Collision cross sections ˝ are determined according to [39] =
(18)1/2 1 1 ze + 1/2 MI M 16 B (kb T )
1/2 t E 760 D L
T 1 P 273.2 N
(1)
where tD is the time required for the ion to traverse the drift tube, ze is the charge of the ion, kb is Boltzmann’s constant, MI and MB are the masses of the ion and buffer gas, E is the electric field, L is the drift tube length, T and P are the temperature and pressure, and N is the neutral number density at STP. The drift tube contains two ion funnels which are operated at higher electric fields (∼12 V cm−1 ) than the rest of the drift tube with an applied RF voltage. In fact, we use 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 traverse the section of the drift tube between the source and middle funnel gate is measured. This region of the drift tube does not contain an ion funnel and has a linear electric field, so the collision cross section can be evaluated directly from the drift time with Eq. (1); in this method, tD is the time between the source pulse and the time required to select the ion of interest. Additionally, we can measure the time it takes for ions to traverse the entire drift tube. In order to account for the nonlinear field in the ion funnels, collision cross sections are calibrated to values determined by the aforementioned method. 3. Results and discussion 3.1. Discussion of related prior results Previous MS–MS studies [8,9] of the [AngI+M]2+ ion by Loo and coworkers showed that Co2+ , Ni2+ , Cu2+ , and Zn2+ are coordinated by the histidine residues at position 6 and 9 (His6 and His9 ). Additionally, they concluded that Cu2+ also interacts with the C-terminus and Tyr4 . In subsequent work, Vachet and coworkers 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 resonance (NMR) studies which revealed that Zn2+ interacts with the His6 and His9 residues in dimethyl sulfoxide (DMSO) and water solutions [40]. Below, we use IMS–MS techniques to investigate these systems in more detail. In addition, we extend the studies of metals bound to AngI by including the Ca2+ alkaline earth cation. One difference between the results published here and those presented previously is that the previous work [9,14] focused on the [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 more conformations for this charge state. 3.2. MS–MS data reported in this study Fragmentation spectra of the [AngI+M+H]3+ ions are displayed in Fig. 1. Metalated fragment ion labels are based on singly charged protonated fragment ions. For example, the [b9 +Ca]3+ ion is a [b9 ]+ ion with the addition of Ca2+ . Overall, these spectra appear much simpler than fragmentation spectra obtained for protonated peptides. Consistent with Loo’s and Vachet’s observations for the [AngI+M]2+ system, dissociation of [AngI+M+H]3+ is limited to specific sites along the backbone that depend on the associated metal [8,9,14]. Fig. 1 shows that Co2+ , Ni2+ , Cu2+ , and Zn2+ binding lead to fragmentation near the His6 residue as the [b6 +M−H]2+ and [y4 ]+ fragments are observed in high abundance. For each of these cations we determine that the sum of intensities for the [b6 +M−H]2+ , [a6 +M−H]2+ , and [y4 ]+ peaks comprises ∼24–40%. An illustration of the positions in which the peptide is cleaved to produce these fragments is provided in Scheme 1. Fragmentation near the His6 residue is observed in low abundance when M = Ca2+ (Fig. 1e). For Ca2+ a sum of ion intensities for the [b6 +M−H]2+ , [a6 +M−H]2+ , [y4 ]+ indicates that only 0.3% of ions dissociate near His6 . For convenience, a summary of the relative populations of fragment ions that arise from dissociation near His6 is provided in Table 1.
320
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
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 abundances are normalized to unity for each spectrum. The most abundant fragments are labeled according to the notation explained in the text.
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
321
Table 1 Fragmentation near His6 and elongated conformations. Metal
Ca2+ Co2+ Ni2+ Cu2+ Zn2+
Normalized abundance (%) [b6 +M−H]2+ , [a6 +M−H]2+ , [y4 ]+ a
Elongated conformationsb
0.3 34.5 34.4 39.9 24.2
0.0 56.1 27.4 90.2 88.7
a Fragment ion abundances are normalized to the total ion count for each MS–MS spectrum (Fig. 1). b Elongated conformation (>326 A˚ 2 ) abundances are normalized to the total ion count for the [AngI+M+H]3+ ion from each activated distribution (Fig. 5).
The aforementioned trend is in good agreement with trends in binding interactions that are known from solution and crystallographic studies available from the PDB [41,42]. From the many structures that are available we find that histidine interacts with Co2+ , Ni2+ , Cu2+ , and Zn2+ at a much greater frequency than Ca2+ . The MS–MS fragment ions that indicate M-His6 interactions are in agreement with metal ion coordination in solution systems reported in the PDB [41,42]. Despite the large difference in abundance of the [b6 +M−H]2+ ion between calcium and the transition metals, fragment ions near His9 (i.e., [b9 +M]3+ ) are observed in relatively high abundance for every metal cation (Fig. 1). Although this would be expected for transition metals, it is less likely that Ca2+ would interact with His9 because Ca2+ is almost always coordinated by oxygen ligands in proteins [41,42]. In addition to fragments near His9 , we observe the [y9 +M]3+ and [RVYIHPFH+M]3+ fragment ions in greater abundance for the [AngI+Ca+H]3+ ion than any other metal species (Fig. 1). Therefore, the presence of large fragment ions from both the N- and C-terminal ends of the peptide may indicate Ca2+ -AngI interactions span a large portion of the peptide backbone. Previous MS–MS studies by Gross and coworkers showed that Ca2+ -coordinated peptides produce large fragments with the N- and C-terminal regions of the peptides ‘trimmed’ away [10]. Therefore, the high abundance of the [b9 +Ca]3+ and [y9 +Ca]3+ ion may result from a similar mechanism where the Ca2+ cation is coordinated by a large portion of AngI.
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 obtained from 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 total ion abundance for each trace.
Fig. 2. Two-dimensional IMS–MS plot of ions produced by ESI of AngI upon the addition of zinc acetate in 50:50 water:acetonitrile. The top drift time distribution is obtained by integrating each drift time bin across the entire range of m/z. The side mass spectrum is obtained by integrating each m/z bin across the entire range of drift times.
Singly charged fragments that contain the doubly charged metal are 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. Condensed phase studies have shown that Ni2+ lowers the pKa of amide nitrogen atoms and interact with deprotonated nitrogen atoms in small peptides [43]. Interestingly, the two cations with the most abundant signal for the [b3 +M−2H]+ and [y7 ]2+ complementary ions (Fig. 1) are the two cations that have been shown to most significantly lower the pKa of amide nitrogen atoms, Cu2+ and Ni2+ [43]. Also, recent infrared multiple-photon dissociation (IRMPD) studies of metals interacting with small peptides show that Ni2+ interacts with backbone nitrogen atoms [44].
322
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
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 A˚ 2 (a) and 343 A˚ 2 (b) marked by dotted vertical lines. The distributions labeled 40, 90, and 130 V correspond to the collisional activation of the selected conformer. Distributions have been normalized to the total ion abundance for each trace.
3.3. Ion mobility distributions Fig. 2 shows the nested tD (m/z) distributions for the AngI sample with the addition of zinc acetate. Although several charge states are observed, the spectrum is dominated by the [AngI+3H]3+ and [AngI+Zn+H]3+ ions. Visual inspection of these data allows one to quickly surmise that substitution of a zinc cation for 2 protons influences the structure of the AngI ion. More insight can be obtained by comparing specific cross section distributions for the different metals. Fig. 3 shows the cross section distributions for the [AngI+M+H]3+ ions. From these data, it is clear that the metal cation has an influence on the range of structures that AngI adopts. When bound to a metal, the range of conformations is large for a peptide of this size, varying from 291 to 354 A˚ 2 (or ∼22%). In comparison, only a single abundant conformation at 337 A˚ 2 is observed for the triply protonated [AngI+3H]3+ ion (Fig. 3). It is interesting to compare the cross section distributions with the general properties of the metal ions such as preferred
coordination number. One apparent difference is between alkaline earth cation Ca2+ and transition metals Co2+ , Ni2+ , Cu2+ , Zn2+ . The most abundant conformation of the [AngI+Ca+H]3+ ion is 302 A˚ 2 (Fig. 3). The Ca2+ coordinated peptide is >10% smaller than the most abundant conformations for the Co2+ , Cu2+ , and Zn2+ coordinated peptides which have major features at 340, 340, and 343 A˚ 2 , respectively (Fig. 3). One explanation for Ca2+ inducing more compact conformations is that the preferred coordination numbers of Ca2+ is 6–8 [4]. Coordination numbers for Co2+ , Ni2+ , Cu2+ , and Zn2+ are smaller on average (4–6) [4]. The preference for more elongated conformations may result from a smaller portion of the peptide interacting with the metal cation. The different metal cations influence not only the collision cross section but also the number of stable structures observed. Our results show that many more structures are stabilized for the transition metals. A good example of this is the [AngI+Ni+H]3+ ion, having at least 7 partially resolved conformers, and the largest range of conformations (291–354 A˚ 2 ). In solution, Ni2+ is known
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
323
for its flexibility of coordination numbers and geometries [45]. In contrast, we only observe a single major conformation for the [AngI+Ca+H]3+ ion. 3.4. Activated mobility distributions To gain insight into the structural transitions that occur upon collisional activation, we have conducted IMS–IMS–MS studies. Collision cross section distributions were measured for the selection and activation of [AngI+M+H]3+ ions. Conformations were selected and activated from 0 to 170 V in 10 V increments. A representative data set for the selection and activation of the two most abundant conformations of the [AngI+Zn+H]3+ ion is displayed in Fig. 4. At low activation voltages we observe structural transitions that are dependent on the original conformation selected. For example, when the two major conformations of the [AngI+Zn+H]3+ are activated with 40 V, at least 5 stable conformations are formed when selecting the conformation at 308 A˚ 2 , but only a single conformation is observed upon selecting and activating the conformation at 343 A˚ 2 (Fig. 4). At activation voltages that are slightly below the onset of significant fragmentation we observe a nearly identical collision cross section distribution, regardless of the initial conformation selected. We have previously referred to data obtained under these conditions as quasi-equilibrium distributions [33]. That is, the ions are heated over all of the barriers between states and upon cooling they establish the preferred gas-phase distribution of states. A characteristic of the quasi-equilibrium distribution is that the IMS distribution of annealed ions remains the same regardless of what solutions are used to produce ions [46]. Once the gas-phase distributions are known, we can use differences that are found (under lower energy, non-equilibrium conditions) to obtain insight about what populations of states are favored in solution. Returning to the case of Zn2+ , we observe that the activated quasi-equilibrium distribution for the [AngI+Zn+H]3+ ions is very different than the non-activated initial distribution obtained directly from the source. In the original source distribution the conformation at 308 A˚ 2 is similar in abundance to the more elongated conformation at 343 A˚ 2 , but at higher activation voltages the peak at 343 A˚ 2 dominates the spectrum (Fig. 4). We attribute this change in distributions to differences in the potential energy landscapes that are associated with the peptide in solution and in the gas phase. Fig. 5 shows representative collision cross section distributions for activated [AngI+M+H]3+ ions. The distributions are obtained from the highest activation voltage that the total fragment ion signal is <5% of the precursor signal. The activation voltages for Ca2+ , Co2+ , Ni2+ , Cu2+ , Zn2+ , and 2H+ are 140, 110, 120, 100, 140, and 140 V, respectively. With the exception of [AngI+Ca+H]3+ , every collisionally activated species forms a markedly different distribution of structures than is observed from the ESI source. In general, a distribution consisting of fewer and more elongated features than the source distribution is observed upon collisional activation. 3.5. Structure–fragment relationships It is worthwhile to compare the activated ion distributions with the fragment ions produced by CID. This is especially intriguing because the populations of conformations that are found just prior to dissociation should approach the populations of transition states that lead to dissociation products. Table 1 includes the normalized abundances for fragment ions near His6 and elongated conformations (>326 A˚ 2 ) of the activated distributions for all of the metal ions presented in this study. We note that calcium behaves very differently than the transition metals. Unlike the transition metals, calcium does not favor the elongated state under gas-phase
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 from 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 total ion abundance for each trace. The dotted traces are obtained by multiplying by 3 to show features of low abundance.
quasi-equilibrium conditions. Moreover, calcium does not produce fragments associated with the His6 site. As mentioned above, the fragment ions produced from the [AngI+Ca+H]3+ ion are large with the major fragments being small neutral losses and the Nand C-terminal regions of the peptide cleaved away. The IMS–IMS data supports the idea that Ca2+ is able to coordinate a large portion of the peptide and that it does not “open-up” upon activation, or form the [b6 +Ca−H]2+ , [a6 +Ca−H]2+ , and [y4 ]+ fragment ions. In contrast, all of the transition metal coordinated ions shift to more elongated conformations (Fig. 5) upon activation and produce 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 only transition metal containing species that does not prefer a conformation >326 A˚ 2 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
324
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325
the [bx +Ni−2H]+ fragment ions outside the His6 -His9 binding pocket may stabilize compact conformations.
[13]
3.6. Insight into populations of metal binding sites in solution [14]
One final issue of interest involves what these results imply about metal binding sites in solution. It is fascinating that upon activation ions prefer structures that have mobilities that are identical to peaks observed directly from the source. That is, the activation process converts the distribution from that produced by the source to that preferred in the gas phase; however, there are no new peaks. Thus, it appears that the differences in these source- and gas-phase distributions are only changes in the populations of preferred structures, rather than the creation of new gas-phase conformations. This interpretation suggests that multiple metal binding sites are populated in solution. All of the transition metals appear to show an elongated peak in the non-activated source distributions. Thus, these metals display one binding configuration near the His6 as well as other binding configurations that lead to more compact ions. Binding at His6 is consistent with information about metal binding that is obtained from the PDB. It will be interesting to gain insight into the other binding configurations and structures that are suggested from these studies, but not immediately apparent based on examination of the PDB and CID studies. 4. Summary and conclusions MS-based techniques have been used to study metal–peptide interactions for the [AngI+M+H]3+ ion. From a combination of CID and IMS–MS studies, we find evidence that transition metals bind near the His6 reside, both in the gas-phase prior to dissociation as well as in solution. Calcium does not appear to favor this binding site. Acknowledgements The instrumentation in this work is supported by a grant from the NIH (NIH-5R01GM93322-2); other support was provided by the Indiana University METACyt initiative that is funded by a grant from the Lilly Endowment.
[15]
[16]
[17]
[18]
[19]
[20] [21]
[22] [23]
[24]
[25]
[26]
[27]
[28]
References
[29]
[1] S.J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry, University Science Books, CA, 1994. [2] R.H. Holm, P. Kennepohl, E.I. Solomon, Structural and functional aspects of metal sites in biology, Chemical Reviews 96 (1996) 2239–2314. [3] T. Dudev, C. Lim, Principles governing Mg, Ca, and Zn binding and selectivity in proteins, Chemical Reviews 103 (2003) 773–787. [4] E.A. Permyakov, Metalloproteomics, Wiley, NJ, 2009. [5] J.A. Loo, Studying noncovalent protein complexes by electrospray ionization mass spectrometry, Mass Spectrometry Reviews 16 (1997) 1–23. [6] P. Hu, M.L. Gross, Strong interactions of anionic peptides and alkaline earth metal ions: metal-ion-bound peptides in the gas phase, Journal of the American Chemical Society 114 (1992) 9153–9160. [7] A. Reiter, J. Adams, H. Zhao, Intrinsic (gas-phase) binding of Co2+ and Ni2+ by peptides: a direct reflection of aqueous-phase chemistry, Journal of the American Chemical Society 116 (1994) 7827–7838. [8] J.A. Loo, P. Hu, R.D. Smith, Interaction of angiotensin peptides and zinc metal ions probed by electrospray ionization mass spectrometry, Journal of the American Society for Mass Spectrometry 5 (1994) 959–965. [9] P. Hu, J.A. Loo, Gas-phase coordination properties of Zn2+ , Cu2+ , Ni2+ , and Co2+ with histidine-containing peptides, Journal of the American Chemical Society 117 (1995) 11314–11319. [10] O.V. Nemirovskiy, M.L. Gross, Determination of calcium binding sites in gasphase small peptides by tandem mass spectrometry, Journal of the American Society for Mass Spectrometry 9 (1998) 1020–1028. [11] B.A. Cerda, L. Cornett, C. Wesdemiotis, Probing the interaction of alkali and transition metal ions with bradykinin and its des-arginine derivatives via matrix-assisted laser desorption/ionization and postsource decay mass spectrometry, International Journal of Mass Spectrometry 193 (1999) 205–226. [12] S.P. Gaucher, J.A. Leary, Influence of metal ion and coordination geometry on the gas phase dissociation and stereochemical differentiation
[30]
[31]
[32] [33]
[34]
[35]
[36] [37]
[38]
[39] [40]
of N-glycosides, International Journal of Mass Spectrometry 197 (2000) 139–148. M.D. Leavell, J.A. Leary, Stabilization, linkage analysis of metal-ligated sialic acid containing oligosaccharides, Journal of the American Society for Mass Spectrometry 12 (2001) 528–536. J.D. Bridgewater, J. Lim, R.W. Vachet, Transition metal–peptide binding studied by metal-catalyzed oxidation reactions and mass spectrometry, Analytical Chemistry 78 (2006) 2432–2438. S.J. Ye, P.B. Armentrout, Absolute thermodynamic measurements of alkali metal cation interactions with a simple dipeptide and tripeptide, Journal of Physical Chemistry A 112 (2008) 3587–3596. M. Roˇzman, S.J. Gaskell, Non-covalent interactions of alkali metal cations with singly charged tryptic peptides, Journal of Mass Spectrometry 45 (2010) 1409–1415. K.K. Morishetti, S.C. Russell, X. Zhao, D.B. Robinson, J. Ren, Tandem mass spectrometry studies of protonated and alkali metalated peptoids: enhanced sequence coverage by metal cation addition, International Journal of Mass Spectrometry 308 (2011) 98–108. H.M. Watson, J.B. Vincent, C.J. Cassady, Effects of transition metal ion coordination on the collision-induced dissociation of polyalanines, Journal of Mass Spectrometry 46 (2011) 1099–1107. J. Lee, L.P. Jayathilaka, S. Gupta, J.-S. Huang, B.-S. Lee, Gold ion-angiotensin peptide interaction by mass spectrometry, Journal of the American Society for Mass Spectrometry 23 (2012) 942–951. J.A. Taraszka, J. Li, D.E. Clemmer, Metal-mediated peptide ion conformations in the gas phase, Journal of Physical Chemistry B 104 (2000) 4545–4551. B.T. Ruotolo, C.C. Tate, D.H. Russell, Ion mobility-mass spectrometry applied to cyclic peptide analysis: conformational preferences of gramicidin S and linear analogs in the gas phase, Journal of the American Society for Mass Spectrometry 15 (2004) 870–878. M. Kohtani, M.F. Jarrold, S. Wee, R.A.J. O’Hair, Metal ion interactions with polyalanine peptides, Journal of Physical Chemistry B 108 (2004) 6093–6097. T. Wyttenbach, D. Liu, M.T. Bowers, Interactions of the hormone oxytocin with divalent metal ions, Journal of the American Chemical Society 130 (2008) 5593–6000. Y. Berezovskaya, C.T. Armstrong, A.L. Boyle, M. Porrini, D.N. Woolfson, P.E. Barran, Metal binding to a zinc-finger peptide: a comparison between solution and the gas phase, Chemical Communications 47 (2011) 412–414. L. Chen, Y.Q. Gao, D.H. Russell, How alkali metal ion binding alters the conformation preferences of gramacidin A: a molecular dynamics and ion mobility study, Journal of Physical Chemistry A 116 (2012) 689–696. J.M. Dilger, S.J. Valentine, M.S. Glover, M.A. Ewing, D.E. Clemmer, A database of alkali metal-containing peptide cross sections: influences of metals on size parameters for specific amino acids, International Journal of Mass Spectrometry 330-332 (2012) 35–45. S.-J. Hyung, A.S. DeToma, J.R. Brender, S. Lee, S. Vivekanandan, A. Kochi, J.-S. Choi, A. Ramamoorthy, B.T. Ruotolo, M.H. Lim, Insights into antiamyloidogenic properties of the green tea extract (−)-epigallocathechin-3-gallate toward metal-associated amyloid- species, Proceedings of the National Academy of Sciences of the United States of America 110 (2013) 3743–3748. J.M. Dilger, S.J. Valentine, M.S. Glover, D.E. Clemmer, A database of alkalineearth-coordinated peptide cross sections: insight into general aspects of structure, Journal of the American Society for Mass Spectrometry 24 (2013) 768–779. D.E. Clemmer, M.F. Jarrold, Ion mobility measurements and their applications to clusters and biomolecules, Journal of Mass Spectrometry 32 (1997) 577–592. T. Wyttenbach, P.R. Kemper, M.T. Bowers, Design of a new electrospray ion mobility mass spectrometer, International Journal of Mass Spectrometry 212 (2001) 13–23. B.C. Bohrer, S.I. Merenbloom, S.L. Koeniger, A.E. Hilderbrand, D.E. Clemmer, Biomolecule analysis by ion mobility spectrometry, Annual Review of Analytical Chemistry 1 (2008) 293–327. S.A. McLuckey, D.E. Goeringer, Slow heating methods in tandem mass spectrometry, Journal of Mass Spectrometry 32 (1997) 461–474. N.A. Pierson, S.J. Valentine, D.E. Clemmer, Evidence for a quasi-equilibrium distribution of states for bradykinin [M+3H]3+ ions in the gas phase, Journal of Physical Chemistry B 114 (2010) 7777–7783. S.L. Koeniger, S.I. Merenbloom, S.J. Valentine, M.F. Jarrold, H.R. Udseth, R.D. Smith, D.E. Clemmer, An IMS–IMS analogue of MS–MS, Analytical Chemistry 78 (2006) 4161–4174. S.I. Merenbloom, S.L. Koeniger, S.J. Valentine, M.D. Plasencia, D.E. Clemmer, I.M.S–I.M.S, IMS–IMS–IMS/MS for separating peptide and protein fragment ions, Analytical Chemistry 78 (2006) 2802–2809. Protein Prospector. http://www.prospector.ucsf.edu/prospector/ (last accessed May 2013). K. Tang, A.A. Shvartsburg, H.-N. Lee, D.C. Prior, M.A. Buschbach, F. Li, A.V. Tolmachev, G.A. Anderson, R.D. Smith, High-sensitivity ion mobility spectrometry/mass spectrometry using electrodynamic ion funnel interfaces, Analytical Chemistry 77 (2005) 3330–3339. C.S. Hoaglund, S.J. Valentine, C.R. Sporleder, J.P. Reilly, D.E. Clemmer, Threedimensional ion mobility/TOFMS analysis of electrosprayed biomolecules, Analytical Chemistry 70 (1998) 2236–2242. E.A. Mason, E.W. McDaniel, Transport Properties of Ions in Gases, Wiley, New York, 1988. A.P. Arnold, D.M. Stanley, J.G. Collins, Zn2+ -induced deprotonation of a peptide nitrogen in angiotensin I, FEBS Letters 289 (1991) 96–98.
M.S. Glover et al. / International Journal of Mass Spectrometry 354–355 (2013) 318–325 [41] M. Babor, H.M. Greenblatt, M. Edelman, V. Sobolev, Flexibility of metal binding sites in proteins on a database scale, Proteins 59 (2005) 221–230. [42] H. Zheng, M. Chruszcz, P. Lasota, L. Lebioda, W. Minor, Data mining of metal ion environments present in protein structures, Journal of Inorganic Biochemistry 102 (2008) 1765–1776. [43] H. Sigel, R.B. Martin, Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands, Chemical Reviews 82 (1982) 385–426.
325
[44] R.C. Dunbar, N.C. Polfer, G. Berden, J. Oomens, Metal ion binding to peptides: oxygen or nitrogen sites? International Journal of Mass Spectrometry 330–332 (330) (2012) 71–80. [45] S.W. Ragsdale, Nickel-based enzyme systems, Journal of Biological Chemistry 284 (2009) 18571–18575. [46] N.A. Pierson, L. Chen, S.J. Valentine, D.H. Russell, D.E. Clemmer, Number of solution states of bradykinin from ion mobility and mass spectrometry measurements, Journal of the American Chemical Society 133 (2011) 13810–13813.