Digital Ion Trap Mass Spectrometer for Probing the Structure of

Apr 8, 2009 - To whom correspondence should be addressed. E-mail: [email protected] (F.L.B.), [email protected] (J.G.G.)., †...
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Anal. Chem. 2009, 81, 3392–3397

Digital Ion Trap Mass Spectrometer for Probing the Structure of Biological Macromolecules by Gas Phase X-ray Scattering Bryan J. McCullough,† Andrew Entwistle,‡ Ikuo Konishi,‡ Steven Buffey,§ S. Samar Hasnain,§ Francesco L. Brancia,*,‡ J. Gu¨nter Grossmann,*,§ and Simon J. Gaskell† Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, U.K., Shimadzu Research Laboratory (Europe) Ltd., Wharfside, Trafford Wharf Road, Manchester M17 1GP, U.K., and Molecular Biophysics Group, School of Biological Sciences, The University of Liverpool, Liverpool L69 7ZB, U.K. Small-angle X-ray scattering is a technique for the characterization and structural analysis of a variety of materials including biological macromolecules and polymers. For the conformational analysis of proteins, the interaction between sample and X-rays is generally performed when the proteins are present in solution. Here a threedimensional digital ion trap interfaced with a high intensity X-ray source is built to prove that X-ray scattering can be performed on ions isolated in gas-phase. Initial experiments on an unresolved ion population of multiply charged cytochrome C ions indicate that a small-angle X-ray scattering signal can be detected and that partial structural information can be extracted about the overall molecular structure of protein ions. With the availability of higher brilliance synchrotron radiation sources,1 as well as the imminent start-up of X-ray free electron lasers,2 X-ray experiments of unparalleled nature will become possible.3 One of the proposed “grand-challenge” type experiments aims to undertake structural analysis and imaging of single biological molecules by coherent X-ray scattering.4 A major experimental challenge for this kind of experiment is the sample manipulation and substrate-free delivery of macromolecular samples in vacuo at the interaction region. The sample delivery system needs to be optimized to achieve sufficient particle densities so as to improve sample selection as well as image acquisition rate in such experiments. Recently, Bogan et al.5 have reported pioneering work using a soft X-ray free electron laser (FEL) for the recording of singlepulse diffraction images of neutral DNA nanoparticles in vacuo. In these experiments, ions were generated by electrospray * To whom correspondence should be addressed. E-mail: francesco.brancia@ srlab.co.uk (F.L.B.), [email protected] (J.G.G.). † The University of Manchester. ‡ Shimadzu Research Laboratory (Europe) Ltd. § The University of Liverpool. (1) Diamond (www.diamond.ac.uk); NSLS2 (www.bnl.gov/nsls2); Petra3 (petra3.desy.de); SINAP China (www.sinap.ac.cn). (2) LCLS (lcls.slac.stanford.edu); SPRing-8 (www-xfel.spring8.or.jp); XFEL (xfel.desy.de). (3) Pellegrini, C.; Stoehr, J. Nucl. Instr. Meth. A 2003, 500, 33–40. (4) Miao, J. W.; Chapman, H. N.; Kirz, J.; Sayre, D.; Hodgson, K. O. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 157–176.

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ionization before being neutralized by an R-emitter source. The continuous beam of neutral nanoparticles was then focused and transmitted by a stack of aerodynamic lenses into a vacuum chamber where interaction with the pulsed FEL beam can occur. Using this technique the authors were able to demonstrate for the first time the possibility of measuring X-ray scattering patterns from “individual” macromolecules in the gas phase (i.e., without requiring substrate support in the X-ray illumination region). Structural characterization of neutral particles in the gas phase is challenging as they cannot be easily manipulated. Ions on the other hand can be straightforwardly controlled using electric and magnetic fields allowing for a less complicated delivery toward the X-ray illumination region. In recent years a number of techniques have been developed for the study of gas-phase ion structure using a variety of analytical approaches including spectroscopy,6,7 ion/molecule reactions,8,9 tandem mass spectrometry 10,11 and, most notably, ion mobility spectrometry (IMS).12-22 (5) Bogan, M. J.; Benner, W. H.; Boutet, S.; Rohner, U.; Frank, M.; Barty, A.; Seibert, M. M.; Maia, F.; Marchesini, S.; Bajt, S.; Woods, B.; Riot, V.; HauRiege, S. P.; Svenda, M.; Marklund, E.; Spiller, E.; Hajdu, J.; Chapman, H. N. Nano Lett. 2008, 8, 310–316. (6) Stearns, J. A.; Boyarkin, O. V.; Rizzo, T. R. J. Am. Chem. Soc. 2007, 129 (45), 13820–13821. (7) Polfer, N. C.; Bohrer, B. C.; Plasencia, M. D.; Paizs, B.; Clemmer, D. E. J. Phys. Chem. A 2008, 112 (6), 1286–1293. (8) Green, M. K.; Lebrilla, C. B. Mass Spectrom. Rev. 1997, 16, 53–71. (9) Mazurek, U.; Engeser, M.; Lifshitz, C. Int. J. Mass Spectrom. 2006, 249250, 473–476. (10) Zeller, L. C.; Farrell, J. T.; Kentta¨maa, H. I.; Kuivalainen, T. J. Am. Soc. Mass Spectrom. 1993, 4 (2), 125–134. (11) Benesch, J. L. P.; Aquilina, J. A.; Ruotolo, B. T.; Sobott, F.; Robinson, C. V. Chem. Biol. 2006, 13, 597–605. (12) Kanu, A. B.; Dwivedi, P.; Tam, M.; Matz, L.; Hill, H. H. J. Mass Spectrom. 2008, 43, 1–22. (13) Dugourd, P.; Hudgins, R. R.; Clemmer, D. E.; Jarrold, M. F. Rev. Sci. Instrum. 1997, 68, 1122–1129. (14) Shelimov, K. B.; Clemmer, D. E.; Hudgins, R. R.; Jarrold, M. F. J. Am. Soc. Mass Spectrom. 1997, 119, 2240. (15) McCullough, B. J.; Eastwood, H.; Clark, D. J.; Polfer, N. C.; Campopiano, D. J.; Dorin, J. A.; Maxwell, A.; Langley, R. J.; Govan, J. R. W.; Bernstein, S. L.; Bowers, M. T.; Barran, P. E. Int. J. Mass Spectrom. 2006, 252, 180– 188. (16) McCullough, B. J.; Kalapothakis, J.; Eastwood, H.; Kemper, P.; MacMillan, D.; Taylor, K.; Dorin, J.; Barran, P. E. Anal. Chem. 2008, 80, 6336–6344. (17) Wyttenbach, T.; Kemper, P. R.; Bowers, M. T. Int. J. Mass Spectrom. 2001, 212, 13–23. (18) Riba-Garcia, I.; Giles, K.; Bateman, R. H.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 2008, 19, 609–613. 10.1021/ac802662d CCC: $40.75  2009 American Chemical Society Published on Web 04/08/2009

In the latter case, the time taken for an ion to pass through a pressurized gas cell (usually to a few Torr) under the influence of an electric field is measured; this time can then be used to calculate the velocity of the ions and, in turn their mobility. Subsequently a rotationally averaged cross section (or in many cases, several different cross sections corresponding to different conformations) for the ion population can be calculated.12 Experimentally derived cross sections have been used in conjunction with molecular modeling by several groups to propose structures of biological molecules in the gas phase.14,15,17,20-22 For instance, Bowers and co-workers have extensively studied the structure and aggregation of the neuropathic protein amyloid-β using IMS and molecular modeling 20-22 allowing them to gain an insight into the protein structure changes which occur in the early stages of Alzheimer’s disease. However, to obtain detailed structural information from cross section data obtained using IMS, a large amount of complex computer modeling is often required which has obvious, inherent uncertainty. In contrast, small-angle X-ray scattering (SAXS) is a very valuable technique for the direct determination of three-dimensional (3D) morphology from noncrystalline particles sized between 1 and 100 nanometers, in particular biological macromolecules in solution.23,24 Over the last two decades the application of SAXS to issues in structural biology experienced a significant upturn primarily because of the availability of synchrotron radiation sources and advances in data analysis methods.25 The determination of consistent 3D shapes from scattering data and the rigid-body refinement of multidomain and multisubunit assemblies provide more meaningful structural details and images of biological macromolecules in solution26 than merely the extraction of their overall molecular parameters (such as radius of gyration or particle volume). Moreover, as a complementary method the effectiveness of SAXS emerges when applied as part of a multitechnique approach for complex questions in structural biology.27 SAXS measurements of proteins are typically performed in solution; the main drawback of this arises from the inherent high background caused by scattering from buffer molecules. The background generated from this can often mask important structural information. Starting from these premises, we designed a novel analytical platform incorporating a 3D digital quadrupole ion trap(DIT) to perform SAXS measurements of macromolecular ions isolated in gas-phase. In this paper we describe the design and performance of the mass spectrometer including initial proof of principle of gas-phase small angle scattering. (19) Garcia, I. R.; Giles, K.; Bateman, R. H.; Gaskell, S. J. J. Am. Soc. Mass Spectrom. 2008, 19, 1781-1787. (20) Bernstein, S. L.; Wyttenbach, T.; Baumketner, A.; Shea, J.; Bitan, G.; Teplow, D. B.; Bowers, M. T. J. Am. Chem. Soc. 2005, 127, 2075–2084. (21) Baumketner, A.; Bernstein, S. L.; Wyttenbach, T.; Bitan, G.; Teplow, D. B.; Bowers, M. T.; Shea, J. Protein Sci. 2006, 15, 420–428. (22) Teplow, D. B.; Lazo, N. D.; Bitan, G.; Bernstein, S.; Wyttenbach, T.; Bowers, M. T.; Baumketner, A.; Shea, J.; Urbanc, B.; Cruz, L.; Borreguero, J.; Stanley, H. E. Acc. Chem. Res. 2006, 39, 635–645. (23) Feigin, L. A.; Svergun, D. I. Structure Analysis by Small-Angle X-ray and Neutron Scattering; Plenum Press: New York and London, 1987. (24) Grossmann, J. G.; Hasnain, S. S. J. Appl. Crystallogr. 1997, 30, 770–775. (25) Svergun, D. I.; Koch, M. H. J. Rep. Prog. Phys. 2003, 66, 1735–1782. (26) Petoukhov, M. V.; Svergun, D. I. Curr. Opin. Struct. Biol. 2007, 17, 562– 571. (27) Robinson, C. V.; Sali, A.; Baumeister, W. Nature (London) 2008, 450, 973– 982.

EXPERIMENTAL SECTION DIT-SAXS Platform. The instrument consists of a 3D digital ion trap (DIT) mass spectrometer based on the design published previously.28-33 With respect to an amplitude driven quadrupole ion trap the DIT utilizes a rectangular waveform for both trapping and resonant ejection. In all commercial ion traps, mass scans are carried out by sweeping the trapping voltage at fixed frequency; as a consequence of this the upper mass range achievable (typically ∼4000 m/z) is determined by the maximum trapping voltage applied to the ring electrode for a given driving frequency. In principle ions can be scanned out of the trap by sweeping the frequency of the trapping waveform at a given constant voltage. In sinusoidal waveform-driven ion traps this is problematic because of inherent limitations of the electronics.29 If a digital waveform is used, however, a frequency scan can be carried out easily, allowing an almost limitless m/z range to be analyzed. An instrument schematic is shown in Figure 1a. It consists of an off-axis ESI source, followed by a “Q-array” (an RF ion guide consisting of three sets of electrode plates) which transports the ions (via a skimmer) to a segmented linear quadrupole in which ions can be stored prior to injection into the 3D trap. The two RF ion guides are also digitally driven using parameters which can be defined separately in the control software. The quadrupole is segmented in three distinct sections of 20 mm, 84 mm, and 6 mm, respectively. Voltages applied to each of these segments can be varied so that ions can be stored within the device. Ions are then pulsed into the 3D trap by lowering the potential on the gate electrode for a user defined time, typically a few microseconds. Selection of a specific ion population is obtained using an isolation technique known as digital asymmetric waveform isolation (DAWI; manuscript in preparation). As with the original DIT,29 this instrument comprises a theoretical, non-stretched geometry incorporating a field-adjusting electrode (FAE) located next to the entrance end-cap. Application of a direct current (DC) component on the FAE electrode allows correction of the distortions in the quadrupolar field within the trap caused by the apertures in the end-caps. Using this approach a resolution of ∼8000 has been reported.29 Ion detection is performed on a channel electron multiplier (Burle/Photonis, U.S.A.) with a conversion dynode. Figure 1b shows the 3D trap and surrounding lenses and housing. The key modification to the trap to allow X-rays to enter and exit was the addition of two 3.5 mm × 3.5 mm holes on opposite sides of the ring electrode. Additional two holes were machined in the ring electrode (at 90° to the X-ray holes) to allow the possibility of performing complementary experiments such as IRMPD; for the experiments described here the unused holes were blanked off on the outside of the ring electrode. X-rays enter the instrument through a mica window of 15 mm diameter and (28) Ding, L.; Sudakov, M.; Kumashiro, S. Int. J. Mass Spectrom. 2002, 221, 117–138. (29) Ding, L.; Sudakov, M.; Brancia, F. L.; Giles, R.; Kumashiro, S. J. Mass Spectrom. 2004, 39, 471–484. (30) Ding, L.; Brancia, F. L. Anal. Chem. 2006, 78, 1995–2000. (31) Berton, A.; Traldi, P.; Ding, L.; Brancia, F. L. J. Am. Soc. Mass Spectrom. 2008, 19, 620–625. (32) Brancia, F. L.; Giles, R.; Ding, L. J. Mass Spectrom. 2004, 39, 702. (33) Ding, L.; Brancia, F.; Giles, R.; Smirnov, S.; Nickolaev, E. Proceedings of the 55th ASMS Conference on Mass Spectrometry. 2005, 16 (5), 101S S1.

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Figure 1. Schematic diagrams of the instrument. 1a shows the instrument and mounting frame: (A) ESI probe; (B) sample cone; (C) quadrupole-array; (D) skimmer cone; (E) segmented linear quadrupole; (F) 3D ion trap (see 1b for zoom of this area); (G) conversion dynode; (H) electron multiplier; (I) X-ray entrance beampipe; (J) X-ray exit beampipe; (K) z-adjustment; (L) y-adjustment. 1b shows a zoom of the 3D ion trap: (a) final section of linear quadrupole; (b) gate electrode; (c) field adjust electrode; (d) entrance end-cap; (e) ring electrode; (f) 3.5 × 3.5 mm X-ray entrance/exit holes; (g) exit endcap; (h) extraction electrode; (i) beryllium entrance/exit windows. See text for details.

0.025 mm thickness and exit through a beryllium window of 50 mm diameter and 0.2 mm thickness. Both windows are located at a distance of 100 mm from the center of the ion trap in line with the holes in the ring electrode. This layout allows scattering angles of up to ±8° to be accessed. The instrument was developed for and first tested on beamline 2.1 of the Synchrotron Radiation Source (SRS) at the STFC Daresbury Laboratory (Warrington, U.K.). Its compact design enables installation of the ion trap at beamlines of other X-ray sources. The instrument electronics are housed in a separate box which can be located up to 2 m away from the core apparatus. Alignment of the ion trap with regards to the X-ray beam is crucial and can be achieved by horizontal and vertical translation relative to the instrument mounting platform. Moreover the instrument can be rotated (the center of rotation coincides with the center of the 3D ion trap) with respect to the incident X-ray beam (±10°) thus giving the option to access wider scattering angles which would allow to probe shorter molecular distances (the nominal spatial resolution also called Bragg resolution corresponds to 2π/qmax with qmax relating to the largest measured scattering angle). Mass Spectrometry Analysis. Angiotensins I, II, and II, and horse heart cytochrome c were obtained from Sigma Aldrich Ltd. (Poole, U.K.) and used without purification. Protein samples were prepared at 1-20 µM in 49.9:49.9:0.2 methanol/water/formic acid. The instrument software (developed in-house at Shimadzu Research Laboratory) allows the user detailed control of every 3394

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voltage and waveform applied to the instrument. In the scan table each operation (ion introduction, cooling, isolation, etc.) is controlled via a microsweep in which the applied potentials and waveforms applied to specific parts of the mass spectrometer (FAE, ring electrode, etc.) are defined. In a typical MS scan, ions are constantly being accumulated in the final section of the linear quadrupole, from where they are pulsed (typical pulse width 10-30 µs) into the 3D trap (with no trapping voltage applied). At a user specified time after the injection pulse the trap, the trapping voltage is applied to the ring electrode, together with the FAE voltage (typically 160% of the zero-to-peak trapping voltage); the timing of the application of these voltages can affect the ion population observed because of time-of-flight effects. Subsequently ions are cooled for a few milliseconds. Precursor ion isolation is performed typically by DAWI but can also be carried out by forward/reverse scanning.32 Mass scans can be performed with or without dipolar excitation of the ions. For the MS measurements the scan speed chosen was typically 750 Th s-1; ions were resonantly ejected at βz ) 1/2 with an excitation voltage of ±1.7 V. An interaction time of between 5 and 30 s was used for the SAXS experiments. To maximize signal-to-noise for synchrotron X-ray experiments, a key consideration relates to the number of ions which can be trapped in the DIT. SAXS studies on proteins in buffer solutions performed using a synchrotron X-ray source (such as the Daresbury SRS) are usually carried out on sample concentrations corresponding to approximately 1011 to 1013 particles in the volume where X-ray interaction takes place. To determine the trapping capacity of the DIT it is crucial to bear in mind that when the number of ions is too large, space charge effects are generated reducing the overall performance of an ion trap mass spectrometer. For a given set of analytical performances (mass resolution, mass accuracy, sensitivity, etc.) the definition of space charge limit, also known as trapping capacity, varies according to the operation mode used in the ion trap. There are several types of trapping capacity: (a) the storage trapping capacity (the total number of ions which can be stored), (b) the activation trapping capacity (the maximum number of ions which can be stored while maintaining the ability to activate ions with a given fragmentation efficiency), (c) the isolation trapping capacity (the maximum number of ions which can be stored while maintaining the ability to isolate ions with a specified resolution and efficiency), and more importantly, (d) the spectral trapping capacity (the maximum number of ions which can be stored while maintaining the ability to obtain a mass spectrum with a given specified mass resolution and mass accuracy).34 It is important to point out that the spectral trapping capacity does not correlate with the maximum number of ions which can be trapped (storage space charge limit) and probed with X-rays. However, for this design of DIT we determined that ∼1 × 103 ions are the spectral trapping capacity using the triply charged renin substrate isotopic ion envelope. The signal recorded at the detector for a single ion was used as reference to calculate the number of ions detected when the ion signal reaches saturation. For the purpose of this investigation we wish to store the largest number of ions as possible to gain the best possible (34) March, R. E.; Todd, J. F. J. In Quadrupole Ion Trap Mass Spectrometry; Wiley-Interscience: New York, 2005.

signal-to-noise ratio in our SAXS analyses. Assuming the X-ray entrance hole (3.5 mm × 3.5 mm) as the defining size constraint of the ion cloud, the trapping capacity can be calculated to be ∼2.5 × 106 ions. For experiments on electrosprayed proteins multiply charged ions must be considered. If we take as an example the +12 ion of cytochrome c, the maximum trapping capacity for only this ion is ∼2 × 105. To maximize the signalto-noise during the initial X-ray experiments, no charge state selection was carried out prior to X-ray exposure, and hence, the measurements were carried out on a mixture of protein ions bearing multiple charge states. SAXS Measurements. Initial SAXS measurements were carried out at an SRS station 2.1, 35,36 a beamline on a bending magnet of the STFC Daresbury Laboratory (Warrington, U.K.), using a collimated X-ray beam (2 mm horizontal × 0.75 mm vertical at sample position) with a fixed X-ray wavelength (1.54 Å). SAXS patterns were recorded as a function of the modulus of the momentum transfer q (q ) 4π sin Θ/λ where 2Θ corresponds to the scattering angle and λ is the X-ray wavelength) by means of a two-dimensional position sensitive X-ray detector.37 The distance between the mass spectrometer and the SAXS detector was set at 1m. The scattering data were calibrated using a low-angle diffraction standard, silver behenate (AgC22H43O2) powder (based on a diffraction spacing of 58.38 Å), positioned at a location equivalent to the center of the ion trap to match the distance to the X-ray detector. For X-ray measurements, MS analysis was performed as normal but with an added “trapping” microsweep (positioned after ion cooling and selection but before ion ejection) during which X-rays are allowed into the trap. This interaction time can be user defined but was typically 5 s although ions have been successfully trapped in the instrument for several minutes without any detectable losses [data not shown]. A signal from the mass spectrometer triggers the start of the X-ray data acquisition which in turn provides a stop signal when the collection time is over. The trigger signal also activates a fast shutter which stays open during the specified time window so that X-rays can interact with the ions in the 3D trap. After the X-ray exposure a mass scan was carried out to confirm which species were present during the interaction with the X-ray radiation and to monitor possible X-ray induced fragmentation. For each experiment several hundred MS scans are accumulated, and the corresponding X-ray scattering profiles recorded individually. A semitransparent beamstop allows the transmitted direct beam intensity to be measured. The systematic data reduction includes the radial integration of the 2D images, normalization of the subsequent one-dimensional data to the intensity of the transmitted direct beam, averaging of individual profiles, and subtraction of background scattering measured and accumulated from an empty 3D trap (the latter was collected using the same MS scan parameters yet without any sample flow). (35) Towns-Andrews, E.; Berry, A.; Bordas, J.; Mant, P. K.; Murray, K.; Roberts, K.; Sumner, I.; Worgan, J. S.; Lewis, R. Rev. Sci. Instrum. 1989, 60, 2346– 2349. (36) Grossmann, J. G. In Scattering; Sabatier, P., Pike, E. R., Eds.; Academic Press: London and San Diego, 2002; pp 1123-1139. (37) Lewis, R. J. Synchrotron Radiat. 1994, 1, 43–53.

Figure 2. Typical ESI-DIT mass spectrum of a three peptide mixture (angiontensin I, II, and III) with zoom of singly protonated angiotensin I inset.

Data analysis in view of the overall molecular size, that is, the radius of gyration, was accomplished using the Guinier approximation38 as well as the indirect Fourier transform method as implemented in the program GNOM.39 The latter also provides an evaluation of the maximum particle dimension (Dmax). The program CRYSOL40 was used to compare the experimental result with the theoretical profile based on a known crystal structure. RESULTS AND DISCUSSION General MS Performance. Figure 2 shows a typical mass spectrum for the peptides angiotensin I, angiotensin II and angiotensin III recorded with the ion optics optimized to transmit optimally at 1000 m/z. The spectrum shows, primarily, the singly protonated species of each analyte. The inset figure shows a zoom of singly protonated angiotensin I demonstrating the resolving power obtained for the type of scan outlined above which is ∼500. Although the obtained resolution is rather lower than that previously obtained for this design,29 the DIT analytical performance is still acceptable for trapping ions prior to X-ray irradiation. We attribute the reduction in the maximum attainable resolution to the perturbations in the electric trapping field caused by holes in the ring electrode. To ascertain whether an interaction can be observed between X-rays and trapped ions, initial experiments were performed with xenon gas (BOC Special Gases, Grimsby, U.K.) on the SRS station 2.1 beamline in Daresbury. The high atomic number for Xe has the advantage of providing a significantly larger cross section for elastic X-ray scattering than for atoms such as H, C, N, O, the main constituents of biological molecules and therefore produces a strong scattering signal. Xe was pulsed into the 3D trap at ∼0.5 s intervals throughout the X-ray acquisition time using the pulse valve normally used to pulse He into the trap during ion injection. Scattering patterns were collected in time frames of 60 s and accumulated over 30 min. Background measurements were performed in an equivalent manner but without using the xenon (38) Guinier, A. Ann. Phys. (Paris) 1939, 12, 161–237. (39) Semenyuk, A. V.; Svergun, D. I. J. Appl. Crystallogr. 1991, 24, 537–540. (40) Svergun, D. I.; Barberato, C.; Koch, M. H. J. J. Appl. Crystallogr. 1995, 28, 768–773.

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Figure 3. Experimental X-ray scattering profile of xenon gas showing the scattered intensity as a function of the magnitude of the momentum transfer q which is linked to the scattering angle 2Θ via the given relationship. For size comparison the calculated scattering profile of a uniform sphere of radius R ) 20 Å is also displayed.

gas stream. Figure 3 shows the background subtracted low angle X-ray scattering profile of xenon gas atoms which corresponds to the atomic scattering or form factor. The analysis gives a radius of gyration (Rg) of 1.35 Å ± 0.10 Å which for a spherical shape can be converted for a homogeneous sphere into an atomic radius R ) (5/3)1/2 Rg24 yielding R ) 1.68 Å ± 0.13 Å. This experimental value agrees well with data in the published literature,41 and lies between the calculated atomic radius (1.08 Å) and the van der Waals radius (2.16 Å) for xenon. Thus, this experimental outcome strengthens the prospect of being able to record X-ray scattering data from trapped ion populations within the 3D trap. For comparison Figure 3 also presents the theoretical form factor of a sphere with uniform density and radius R ) 20 Å (Rg ) 25.8 Å) calculated according to I(q) ) (3 ⁄ ξ3)(sin ξ - ξ cos ξ)2 with ξ ) qR. In general for q < 1/Rg (which corresponds to q ∼ 0.74 Å-1 for Xe and q ∼ 0.04 Å-1 in the case of a sphere with 20 Å radius, respectively) the spatial resolution is too crude to distinguish any structural characteristics as the scattered intensity is almost independent from q. From the latter it is obvious that the accessible scattering regime available with the geometrical constraints of the instrument is sufficient to provide information on size and shape of particles such as biological macromolecules (that are larger than ∼20 Å in size) and details on the relative arrangement of their internal constituents such as subunits or domains. Initial experiments on protein ions carried out at the Daresbury SRS showed to be inconclusive because of a low signal-to-noise ratio. Hence subsequent studies were perfomed on I22, the noncrystalline diffraction beamline located at an insertion device of the Diamond Light Source (Oxfordshire, U.K.) using a wavelength of 0.83 Å and a detector distance of 5 m from the mass spectrometer. This experimental station is expected to provide an X-ray flux that is 2 to 3 orders of magnitude superior to beamline 2.1 at the Daresbury SRS. Data collection and analysis followed the protocol as described in the Experimental Section. (41) Frank, D. G.; Hubbard, A. T. J. Phys. Chem. A 1997, 101, 894–901; see also http://www.webelements.com/xenon/atom_sizes.html.

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Figure 4. Background subtracted experimental X-ray scattering profile of cytochrome c confined inside the 3D ion trap (a) and corresponding mass spectrum (b). The solid red curve is a smooth representation of the experimental scattering data whereas the solid curve corresponds to a simulated scattering profile calculated from the known compact crystal structure (pdb code 1akk). The inset to (a) shows the Guinier plot of the small angle region alone from which the radius of gyration (Rg) is deduced. According to Guinier’s approximation38 the scattering curve at low q-values follows I(q) ) I0 exp(-Rg2q2/3) so that by linear regression Rg can be determined from the slope defined by the linear region in a ln I(q) versus q2 plot (data points highlighted in red are included in the linear fit and satisfy the necessary condition qRg < 2).

The data recorded on I22 represents the first outcome of preparatory experiments carried out on this recently commissioned SAXS beamline on U.K.’s third generation synchrotron source. A background corrected scattering signal for horse heart cytochrome c was generated and is shown in Figure 4a along with the mass spectrum (Figure 4b) obtained after X-ray exposure. Clearly, significant scattering signal is observed in these early measurements demonstrating the validity of the approach. Analysis of the small angle scattering features suggest an elongated conformation rather than a compact, globular conformation when comparing the measured (Rg ) 26.3 Å ± 0.5 Å) and calculated (Rg ) 13 Å) radius of gyration. The latter was computed from the crystal structure of cytochrome c (Protein Database

accession code 1akk, available at www.pdb.org) and the resulting theoretical scattering profile is superimposed on the experimental data in Figure 4a. An analogous discrepancy is observed when the experimentally derived maximum particle dimension (Dmax ) 71 ± 4 Å) is compared with the value (Dmax ) 43 Å) determined from the crystal structure. Thus a comparison of the experimental and theoretical results would clearly underline a substantial difference between the conformation of cytochrome c in the crystalline state and in vacuo. However, it is worthy of note that solution denaturation studies monitored with CD spectroscopy and SAXS yields a Rg value of 31.7 Å for alcohol denatured (60% methanol) cytochrome c.42 This molecular state is described as an extended chain-like conformation retaining high R-helical content. Further work on this and other larger macromolecular systems is necessary to establish such differences with increased confidence and to draw some general observation on the molecular behavior in the gas phase. From the mass spectrum shown in figure 4b, it is clear that the scattering profile was generated by interaction with a mixture of different charge states of cytochrome c (predominantly +9 and +8). Previous work on the gas phase structure of cytochrome c ions, obtained using ion mobility,14 has shown that ions at these charge states have significantly larger collision cross sections (i.e., are more unfolded) than would be expected for the native, compact structure. This unfolding is due to two factors: first, the minor rearrangement of protein structure to minimize Coulombic repulsion; and second, ionization of titratable groups within the protein with a resulting disruption of salt bridges. Our observation that the Rg of these ions is larger than that of the native conformation is therefore consistent with previous results. The measurement of scattering profiles from individual charge states of proteins would represent an exciting follow-up experiment thus allowing us to probe conformational characteristics of individual charge states. The scattering data generated from preliminary experiments on individual charge states suffered from a low signal-to-noise ratio because of the current limitations of the SR sources and their instrumentation. In this context, X-ray free electron lasers offer an exciting prospect. In the meantime, an obvious way to increase signal-to-noise is to increase the scattering power of the species being studied, that is, the examination of larger macromolecular species (Mw > 100 kD). The increased scattering power offered by larger molecules links up with the trapping capacity and available photon flux at which the X-ray experiments are carried out. Assuming X-ray studies are carried out on higher brilliance synchrotron sources (such as Diamond) then the trapping capacity of the DIT will play a decisive role in allowing us to accumulate a reasonable signal-to-noise within a few minutes of data collection time. In contrast, imaging experiments proposed on future high power X-ray free electron lasers are intended to be performed on single particles.4 Nevertheless, the image acquisition rate (or hit ratio) will be a limiting factor in recording sufficient diffraction images in a given time to allow for a complete 3D (42) Kamatari, Y. O.; Konno, T.; Kataoka, M.; Akasaka, K. J. Mol. Biol. 1996, 259, 512–523.

reconstruction. The authors of the pioneering DNA nanoparticle imaging study5 using focused neutral particle beams noted that with the FEL pulse rate used (135 pulses/s) the average hit rate was approximately one particle per minute, a hit rate which should increase linearly with rising pulse rate. At one of the first future hard X-ray FELs, such as the Linac Coherent Light Source (LCLS),2 the planned pulse rate is lower requiring further improvements in sample-handling to enhance image acquisition rates significantly. In this case an experimental setup as described here will be able to provide sufficient ions in the trap to improve the hit ratio significantly. However, the instrument and its trapping capacity would have to be finetuned so as to avoid two or more particles being hit by individual X-ray pulses at the same time. CONCLUSIONS Here a 3D digital ion trap interfaced with a high intensity X-rays source was built to discover whether X-ray scattering experiments can be performed on trapped ions in the gas-phase. Initial experiments on an unresolved mixture of multiply charged cytochrome c ions represent the first SAXS data obtained for protein ions isolated in gas-phase. Structural parameters such as the radius of gyration, Rg, and the maximum particle dimension, Dmax, could be directly extracted from the X-ray scattering profile in this first demonstrative experiment. It is clear that scattering data can be collected on macromolecular ions with the view of obtaining a three-dimensional shape of the macromolecule in the gas phase. In principle, the technique can allow a straightforward analysis of the ion conformation in the 3D trap, and therefore the data obtained are complementary to IMS mass spectrometry. The ability of SAXS to provide molecular dimensions and shape information, albeit at low resolution, is especially appealing and fascinating for establishing global information about partially folded or conformationally mobile states of macromolecular ions in the gas phase. With continual improvements being made to the mass spectrometer and to X-ray sources, we envisage this new combined technique presented here to become a vital and versatile tool for the analysis of biomolecular structure in the gas phase. ACKNOWLEDGMENT This work is funded by the European Union FP6 Design Study “Small-Angle X-ray Scattering Initiative for Europe” (SAXIER), RIDS 011934 (see also www.saxier.org). We thank STFC Daresbury Laboratory for beamtime at the SRS and Dr David Holland (STFC Daresbury Laboratory) for the provision of Xenon gas. Part of this work was carried out with the support of the Diamond Light Source. We also thank Dr Mark Malfois of the Diamond Light Source for his continual support on beamline I22 and George Jennings, Frank Barber and Phil Albericci from SRL.

Received for review December 17, 2008. Accepted March 22, 2009. AC802662D

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