Gas-Phase Compaction and Unfolding of ProteinStructures

Izhak Michaelevski,† Miriam Eisenstein,‡ and Michal Sharon*,†

Departments of Biological Chemistry and Chemical Research Support, Weizmann Institute of Science,Rehovot 76100, Israel

Ion-mobility mass spectrometry is emerging as a powerfultool for studying the structures of less established proteinassemblies. The method provides simultaneous measure-ment of the mass and size of intact protein assemblies,providing information not only on the subunit compositionand network of interactions but also on the overalltopology and shape of protein complexes. However, howthe experimental parameters affect the measured collisioncross-sections remains elusive. Here, we present anextensive systematic study on a range of proteins andprotein complexes with differing sizes, structures, andoligomerization states. Our results indicate that theexperimental parameters, T-wave height and velocity,influence the determined collision cross-section indepen-dently and in opposite directions. Increasing the T-waveheight leads to compaction of the protein structures, whilehigher T-wave velocities lead to their expansion. Thesedifferent effects are attributed to differences in energytransmission and dissipation rates. Moreover, by analyz-ing proteins in their native and denatured states, we couldidentify the lower and upper boundaries of the collisioncross-section, which reflect the “maximally packed” and“ultimately unfolded” states. Together, our results providegrounds for selecting optimal experimental parametersthat will enable preservation of the nativelike conforma-tion, providing structural information on uncharacterizedprotein assemblies.

One of the major challenges in the postgenomic era is therevelation of the dynamic interactions and structural propertiesof multiprotein complexes. Such information is critical for under-standing the biological roles and mechanism of action of molecularassemblies. However, the large size, asymmetric contour, hetero-geneous composition, and flexible conformations of proteincomplexes pose a considerable challenge to conventional struc-tural biology approaches. There is therefore a need to expandthe plethora of biophysical tools and develop complementaryapproaches that will enable the investigation of large proteinassemblies. Ion mobility (IM) separation coupled to mass spec-trometry (MS) is a recent addition to the structural biology tool

kit.1-3 The method offers simultaneous measurement in the gasphase of the mass and size of intact assemblies, providinginformation not only on the subunit composition and network ofinteractions but also on the overall topology and subunit packingof protein complexes.

Ion mobility is a method that measures the time taken for ionsto traverse through a region of neutral buffer gas under theinfluence of a weak electric field.4 The migration rate is dependenton the size of the ion. Large ions will experience more collisionswith the background gas in comparison with smaller ions andwill therefore traverse the mobility device at longer drift times.In general, the measured drift time is related to the collision cross-section (CCS) of the ion, and therefore, it is associated with itsstructural properties. While the method was established as a stand-alone technique,5-8 recently an ion mobility device was integratedwithin a quadrupole time-of-flight mass spectrometer.1-3 In thisinstrument (Synapt, Waters, UK Ltd., Manchester, U.K.) themobility chamber that is filled with neutral gas molecules, usuallynitrogen, is placed between two analyzers. By applying a low-voltage DC pulse across a series of stacked rings, a traveling wave(T-wave) of electropotential carries the ions through the driftchamber.1,2 This traveling wave technique does not provideabsolute measurements of the drift time; however, by applying acalibration approach using standard proteins, the transit time canbe converted into collision cross-section values.3,9,10 This tech-nological development expands the span of information that canbe revealed by mass spectrometry. Now, not only can thecomposition, stoichiometry, and the interaction network of aprotein complex be defined, but insight into the topological

* Corresponding author. E-mail: michal.sharon@weizmann.ac.il. Phone: 972-8-9343947. Fax: 972-8-9346010.

† Department of Biological Chemistry.‡ Department of Chemical Research Support.

(1) Giles, K.; Pringle, S. D.; Worthington, K. R.; Little, D.; Wildgoose, J. L.;Bateman, R. H. Rapid Commun. Mass Spectrom. 2004, 18, 2401–2414.

(2) Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.;Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. Int. J. MassSpectrom. 2007, 261, 1–12.

(3) Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.;Robinson, C. V. Science 2005, 310, 1658–1661.

(4) Mason, E. A.; McDaniel, E. W. Transport Properties of Ions in Gases; JohnWiley & Sons: New York, 1988.

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1485.(7) Wyttenbach, T.; Paizs, B.; Barran, P.; Breci, L.; Liu, D.; Suhai, S.; Wysocki,

V. H.; Bowers, M. T. J. Am. Chem. Soc. 2003, 125, 13768–13775.(8) Hoaglund-Hyzer, C. S.; Counterman, A. E.; Clemmer, D. E. Chem. Rev.

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C. V. Nat. Protoc. 2008, 3, 1139–1152.(10) Smith, D. P.; Knapman, T. W.; Campuzano, I.; Malham, R. W.; Berryman,

J. T.; Radford, S. E.; Ashcroft, A. E. Eur. J. Mass Spectrom. (Chichester,Engl.) 2009, 15, 113–130.

Anal. Chem. 2010, 82, 9484–9491

10.1021/ac1021419 2010 American Chemical Society9484 Analytical Chemistry, Vol. 82, No. 22, November 15, 2010Published on Web 10/22/2010

arrangement of subunits and the quaternary organization of theassembly can be gained as well.

The application of IM-MS for protein complex structuralanalysis holds a promising potential, especially for gainingstructural details on less established protein assemblies withunknown structures. However, like any new biophysical approach,its full range of abilities and limitations should be explored.Various fundamental studies have been carried out recently,demonstrating the principles of the IM-MS method. Initially acalibration method was established,3,9 and conditions for optimiz-ing CCS separation were screened.10 Moreover, the dependenceof the CCS values on the charge states3,11 and activation energieswas investigated.12,13 In addition, the promise the IM-MS methodholds for subunit architecture definition was explored.14,15 How-ever, little attention was directed toward understanding the effectof traveling wave parameters, height and velocity, on the measuredCCS values of native proteins and protein complexes.

Here we have performed a systematic study on the influencethe traveling wave profile has on the measured CCS values andon the inferred gas-phase protein structure. We have examined arange of model proteins and protein assemblies for which precisedetails of the three-dimensional structure are known. Our resultsdemonstrate that the traveling wave velocity and height haveopposite effects on the CCS values. While an increased T-waveheight prompts compaction of the protein structure, an increasedT-wave velocity triggers protein expansion, presumably due tounfolding. On the basis of these observations, we suggest astrategy of defining optimal traveling wave conditions that willpreserve the native structure. Overall, our investigation highlightsthe fundamental importance of finding the right balance betweenT-wave height and velocity to retain the native 3D architecture ofthe ion.

EXPERIMENTAL SECTIONCollision Cross-Section Measurements. IM-MS measure-

ments were performed on a Synapt HDMS system (Waters, UKLtd., Manchester, U.K.) as described elsewhere,16,17 with thesample introduced by electrospray from gold-coated borosilicatecapillaries prepared in-house.18,19 The instrument parameters wereoptimized to remove adducts while preserving noncovalentinteractions.17,19 The experimental conditions are described indetail in the Supporting Information. Drift times were convertedto CCSs using a calibration protocol reported elsewhere.9,19 All

the presented results are an average of at least three independentexperiments, which were conducted under exactly the sameconditions and followed by a CCS calibration procedure. Inaddition, when the effect of the T-wave height (TH) or T-wavevelocity (TV) for a given protein was examined, all themeasurements were conducted in the same day. Overall, theaverage relative precision of the drift time measurements isapproximately 2.1 ± 0.4%, and the average error of the CCScalculation is 4.3 ± 0.2%. The error associated with calibrantcross-sections is 1.4 ± 0.7%, and that associated with thecalibration curve is 2.7 ± 0.3%.

Theoretical Cross-Section Calculations. The CCSs of allmodel structures were calculated using the program MOBCAL,20,21

adapted for large all-atom coordinate sets.9 Gas-phase mini-mization was applied to all the structures after exclusion of thewater coordinates in the PDB files. CCS values were calculatedusing three models: projection approximation (PA), exact hardsphere collision (EHSC), and trajectory modeling (TM).

RESULTS AND DISCUSSIONMeasuring Collision Cross-Sections of Proteins and Pro-

tein Complexes. To validate the applicability of the IM-MSapproach for the investigation of protein complexes with unknownstructures, we have conducted a systematic study to understandthe influence of the different T-wave parameters on the generatedcollision cross-section values. To this end we have selected a setof proteins and protein complexes that fulfill the following criteria:(i) The protein or protein complex has a known high-resolutionstructure (solved by X-ray crystallography); consequently itstheoretical CCS value can be calculated. (ii) The PDB coordinatesinclude 96% of the protein amino acids or more. With such a lowpercentage of truncations the deviation of the theoreticallycalculated CCS from the measured CCS (for the full-lengthprotein) is negligible compared to the wide range of the theoreticalCCS values (particularly PA versus EHSC). (iii) Availability of theprotein or protein complex is.

In total our data set includes 7 proteins and 10 proteincomplexes, with diverse sizes, oligomerization states, and struc-tural properties (see the protein data set details in the SupportingInformation). Two of the analyzed monomeric proteins, cyto-chrome c (Cyt c) and myoglobin, were characterized in their nativeform, and the results were compared with the CCS obtained fortheir denatured state during the calibration procedure. Given thatthe CCS values of the target proteins are obtained via a calibrationapproach and not by direct measurement, it was important toinclude Cyt c and myoglobin in the data set, because for theseproteins there is no need for extrapolation and the errors relatedto the calibration approach are minimized.

Theoretical CCS values were calculated for all proteins andprotein complexes using the PA, EHSC, and TM models inMOBCAL.9,21,22 CCS values calculated via the EHSC and TMapproximations are in good agreement (the value from TM ishigher by 2.24 ± 0.3% than that from EHSC), and as expectedthese values are consistently higher than those calculated using

(11) Scarff, C. A.; Thalassinos, K.; Hilton, G. R.; Scrivens, J. H. Rapid Commun.Mass Spectrom. 2008, 22, 3297–3304.

(12) Hyung, S. J.; Robinson, C. V.; Ruotolo, B. T. Chem. Biol. 2009, 16, 382–390.

(13) Ruotolo, B. T.; Hyung, S. J.; Robinson, P. M.; Giles, K.; Bateman, R. H.;Robinson, C. V. Angew. Chem., Int. Ed. 2007, 46, 8001–8004.

(14) Leary, J. A.; Schenauer, M. R.; Stefanescu, R.; Andaya, A.; Ruotolo, B. T.;Robinson, C. V.; Thalassinos, K.; Scrivens, J. H.; Sokabe, M.; Hershey, J. W.J. Am. Soc. Mass Spectrom. 2009, 20, 1699–1706.

(15) Pukala, T. L.; Ruotolo, B. T.; Zhou, M.; Politis, A.; Stefanescu, R.; Leary,J. A.; Robinson, C. V. Structure 2009, 17, 1235–1243.

(16) Pringle, S. D.; Giles, K.; Wildgoose, J. L.; Williams, J. P.; Slade, S. E.;Thalassinos, K.; Bateman, R. H.; Bowers, M. T.; Scrivens, J. H. Int. J. MassSpectrom. 2007, 261, 1–12.

(17) Michaelevski, M.; Kirshenbaum, N.; Sharon, S. J. Visualized Exp. [Online]2010. DOI: 10.3791/1985. http://www.jove.com/index/Details.stp?ID)1985.

(18) Hernandez, H.; Robinson, C. V. Nat. Protoc. 2007, 2, 715–726.(19) Kirshenbaum, N.; Michaelevski, I.; Sharon, M. J. Visualized Exp. [Online].

(20) Mesleh, M. F.; Hunter, J. M.; Shvartsburg, A. A.; Schatz, G. C.; Jarrold,M. F. J. Phys. Chem. 1996, 100, 16082–16086.

(21) Shvartsburg, A. A.; Jarrold, M. F. Chem. Phys. Lett. 1996, 261, 86–91.(22) Mesleh, M. F.; Hunter, J. M.; Shvartsburg, A. A.; Schatz, G. C.; Jarrold,

M. F. J. Phys. Chem. 1996, 100, 16082–16086.

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the PA model by about 20%.10,11 Although, the PA, TM, and EHSCtheoretical CCS span a wide range, there is a strong correlationbetween these values (R2 ) 0.991). In general, experimental CCSvalues of native structures should fall between the PA and TM/EHSC values, as the PA approximation underestimates the ccswhile TM and EHSC overestimate them.11,23

Increasing the T-Wave Height Induced Protein StructureCompaction. To find whether there is a relation between CCSvalues and T-wave heights, we performed measurements of drifttime distributions over a range of T-wave heights, keeping theT-wave velocity fixed at 450 m/s. The maximal T-wave heightswere dictated by the ability to record well-resolved drift timedistributions for the calibrant proteins: myoglobin, Cyt c, andubiquitin. The minimal T-wave heights were defined by the lowestpotential that can be used without appearance of the rollover affect.In Figure 1A the influence of the T-wave height on the measureddrift time for ovalbumin and G�γ is shown. Both proteins have asimilar molecular mass (Table S1, Supporting Information);however, while ovalbumin is monomeric, G�γ is a heterodimer.The measurements revealed that in both cases increasing theT-wave height systematically increased the mobility rate and

reduced the drift time values by a factor of 4.85 ± 0.12 (TH range of14-22 V) and 9.05 ± 0.14 (TH range of 12-24 V) for ovalbuminand G�γ, respectively. Moreover, the decrease in drift time valuesfor both proteins was similar for all charge states.

Using the measured drift times and the standard calibrationapproach,9,17 CCS values were derived for each charge state as afunction of the T-wave height. Both ovalbumin and G�γ experiencea reduction in their CCS values as a function of the T-wave height(Figure 1B), suggesting that the proteins undergo compaction.At a relatively low voltage, both proteins have a larger CCS valuecompared to the calculated CCS; these values drop steadily asthe T-wave height increases (Figure 1B inset). Overall, a decreaseof 15 ± 1% (1.7 ± 0.1% per volt) and 16 ± 1% (1.3 ± 0.1% per volt)from the initial CCS value was observed for ovalbumin and G�γ,respectively. Unlike the drift time values, the extent of CCSdecrease varied among the different charge states. Moreover, theCCS versus T-wave height and charge state surface is rougherthan the drift time surface. This result probably reflects minorerrors in the process of extracting drift time values for individualcharge states (Figure S1, Supporting Information).

To probe whether the compaction of the protein structure asa function of the T-wave height is a general phenomenon, weextended the measurements to a series of proteins of differentsizes and compositions. Interestingly, a similar trend, which is

(23) Bush, M. F.; Hall, Z.; Giles, K.; Hoyes, J.; Baldwin, A. J.; Benesch, J. L. P.;Ruotolo, B. T.; Robinson, C. V. Abstr. Am. Soc. Mass Spectrom. Meet., inpress.

Figure 1. Increasing the T-wave height reduces the ion mobility drift time and the collision cross-sections. (A) Drift time distribution as afunction of the T-wave height. Results for ovalbumin are shown in the left panel and for G�γ in the right panel; curves are colored according tothe drift time values (see the keys). Measurements were performed at a T-wave velocity of 450 m/s, trap CE of 15 V, transfer CE of 12 V, andbias voltage of 25 V (ovalbumin) or 32 V (G�γ). Inset: Drift time dependence on the T-wave height taken for the centroid of the lowest chargestates, +11 and +12 for ovalbumin and G�γ, respectively. Data were fitted with the exponential equation td ) ae-bTH + c, where a, b, and c arereal number coefficients, TH is the T-wave height, and td is the drift time (ms). (B) Dependence of the collision cross-section of ovalbumin andG�γ on the T-wave height. CCSs were calculated for each of the charge states and colored according to their values (see the key in the center).The horizontal white planes represent the theoretically calculated CCS values based on MOBCAL calculations using the TM (upper) and PA(lower) models. In the inset the approximately linear dependence of the CCS values on the T-wave height is shown for the lowest charge statesof both proteins. The presented data are an average of three independent experiments.

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often linear, was observed for all proteins and protein complexes(Figure S2, Supporting Information), emphasizing that as theT-wave height increases more closely packed structures aredetected. The extent of compaction varies between differentcharge states and different proteins. Typically, the CCS valuesfor the lowest charge state are between the PA and TM values,while larger ccs are observed for the high charge states, sug-gesting reduced structural stability of the higher charge states.Notably, the measured peaks are narrower for shorter drift times(Figure S3, Supporting Information), suggesting that the morecompact states obtained upon the increase in T-wave height arepopulated by fewer discrete structures, representing a smallerconformational space. Such compaction of protein structuresduring IM-MS measurements was indicated in earlier studies.For example, the higher charge states of the Trp RNA bindingprotein were reported to have a collapsed protein quaternarystructure.3 In another study the compaction of the glutaminesynthetase complex was indicated.15 Moreover, differences be-tween CCS values measured for different charge states werereported,3,11,24,25 in which the lowest charge states are in the bestagreement with the calculated structure. In summary, althoughdifferent charge states of the same ion react differently to theapplied potential, on the whole we show that high T-wave valueslead to a pronounced CCS reduction.

In general, the desolvation process that accompanies thetransition from the solution to the gas phase supports theformation of new salt-bridge links between the charged side chainson the exterior of the native protein; this process leads to a globalcompaction of the structure.26-29 Our result indicates that inaddition to this initial side-chain collapse a second compactionstep occurs when the T-wave height is increased. Each time atraveling wave hits an ion, a packet of energy is absorbed. Thisenergy can be distributed between different modes of motion: rigidbody rotation (tumbling), translation, and internal energy. Mostof the energy will trigger tumbling and translation, leading toshorter drift times for higher T-waves. However, some of theenergy will be converted to internal energy, which will initiatestructural reorganization and compact packing of internal voidsand pockets. In general, proteins are tightly packed, yet theycommonly have internal cavities, which account for 1-2.3% of theprotein volume,30 and their number depends on the length of thepolypeptide chain.31 The degree of compaction detected here isin the range of 11-20%, larger than that expected. We attributethis deviation to the fact that the calibrants we use are in adenatured state, which is more sensitive to the effect of elevatedT-wave heights (as seen in Figure 2). As a consequence thecalibration curves may introduce errors in the CCS calculationsof native proteins.

Native and Unfolded Proteins Collapse to a Similar Size.To investigate whether CCS reduction is a general phenomenonor it characterizes the folded state of the protein or proteincomplex, we examined the influence of denaturing conditions ondrift time distributions. IM-MS spectra of unfolded and foldedhemoglobin were recorded at increasing T-wave heights, and theCCS values of tetrameric (R2�2) and dimeric (R�) hemoglobincomplexes were determined. The T-wave heights were in-creased in steps from 7 to 13 V (for the dimer) and from 7 to14 V (for the tetramer) at a T-wave velocity of 350 m/s;measurements below 7 V were impossible because of the“rollover” phenomenon.9,17 The results indicated that the ex-perimentally derived CCS values of the folded and unfolded states,at the lowest T-wave height, increased from 3964 ± 87 to 7013 ±249 Å2 and from 2660 ± 65 to 4524 ± 359 Å2 for the dimeric andtetrameric forms of hemoglobin, respectively (Figure 2A). This75% increase in CCS indicates expansion of the structure.Moreover, broader drift time peaks were measured, indicatingthat although the oligomeric state is preserved the subunits hadundergone partial unfolding.32

Upon increasing the T-wave height, the unfolded and foldedstructures started to collapse (Figure 2B,C). This observation wasdetected for tetrameric (left panel) and dimeric (right panel)

(24) Smith, D. L.; Zhang, Z. Mass Spectrom. Rev. 1994, 13, 411–429.(25) van Duijn, E.; Barendregt, A.; Synowsky, S.; Versluis, C.; Heck, A. J. J. Am.

Chem. Soc. 2009, 131, 1452–1459.(26) Breuker, K.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,

18145–18152.(27) Meyer, T.; de la Cruz, X.; Orozco, M. Structure 2009, 17, 88–95.(28) Steinberg, M. Z.; Elber, R.; McLafferty, F. W.; Gerber, R. B.; Breuker, K.

ChemBioChem 2008, 9, 2417–2423.(29) Patriksson, A.; Marklund, E.; van der Spoel, D. Biochemistry 2007, 46,

933–945.(30) Hubbard, S. J.; Gross, K. H.; Argos, P. Protein Eng. 1994, 7, 613–626.(31) Rother, K.; Preissner, R.; Goede, A.; Frommel, C. Bioinformatics 2003,

19, 2112–2121.(32) Benesch, J. L.; Ruotolo, B. T.; Simmons, D. A.; Barrera, N. P.; Morgner,

N.; Wang, L.; Saibil, H. R.; Robinson, C. V. J. Struct. Biol., in press.

Figure 2. The unfolded and folded forms of hemoglobin collapse toa similar collision cross-section value. (A) Relation between T-waveheights and CCS values of tetrameric (left panel) and dimeric (rightpanel) forms of hemoglobin, measured for five different charge states.The red surface represents a denatured conformation, whereas theblue surface corresponds to the native conformation. Each data pointis an average of three independent experiments. The horizontal whiteplanes mark the CCS value calculated using the TM (upper) and PA(lower) methods. The correlation between CCS values and the T-waveheight for the lowest charge states is shown for native (B) andunfolded (C) hemoglobin.

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hemoglobin forms, for all examined charge states. However, adifferent collapse pattern was observed for the different oligo-merization states (Figure 2B,C). At the highest T-wave heights,13 and 14 V, the CCS values dropped to the minimal values of3631 ± 125 and 2138 ± 112 Å2 for dimeric and tetramerichemoglobin, respectively. Interestingly, both the unfolded andfolded states converged to these minimal CCS values, deviatingby only 1.0-1.8%. Hence, although the unfolded hemoglobindimer and tetramer structures exhibited a larger extent of CCSdecrease, their size at a high T-wave height was identical tothat of the collapsed native structures. This result indicates thatthere is a minimal volume that the protein structures canassume within the mobility chamber, regardless of the startingstate.

Increased T-Wave Frequencies Have an Effect Oppositeto That of the T-Wave Height. The observation that the experi-mentally derived CCS decrease upon increasing the T-waveheights led us to examine whether changing the T-wave velocitiesinduces a similar effect. To this end, we acquired IM-MS data atdifferent T-wave velocities. At each T-wave velocity we recordedseveral spectra using different T-wave heights. Higher T-waveheights had to be used when the T-wave velocity was increasedto preserve the mobility separation capacity. Overall, our goal wasto cover as wide a range as possible of T-wave heights withoutmajor distortion in the ion mobility separation. Four velocities wereexamined: 100, 350, 480, and 650 m/s (Figures 3 and S4,Supporting Information).

We observed that irrespective of the type of protein or proteincomplex or its molecular weight, secondary structure composition,or oligomerization state, an increase of the T-wave velocity led toan increase of the CCS value for each charge state (Figure 3).Although the extent of CCS expansion was different for the variousproteins, a clear trend could be detected. Thus, at a low T-wavevelocity (100 m/s) the measured CCS values were often belowthe theoretical PA values, while at 350 and 480 m/s the experi-mental CCS values were between the PA and TM estimates. At ahigh velocity (650 m/s) the experimentally determined ccs wereconsistently larger than the theoretical values. This behavior wasalso observed for Cyt c and myoglobin, which served as calibrantsin their denatured state, suggesting that the trend that weobserved is not an artifact related to the indirect CCS determi-nation. Overall, we trust that the phenomena we observe hereare general, although the absolute degree of expansion andcompaction might vary with new calibration approaches that arecoming to the fore.

Our results indicate that the T-wave velocity and height haveopposite effects on the protein structure. While the velocity ofthe T-wave induces expansion of the protein, the T-wave heighttriggers its compaction. It can be seen in Figure S4 (SupportingInformation) that the velocity of the T-waves has a stronger impacton the protein structure than the T-wave height and a generaltrend of protein unfolding is observed as a function of the T-wavevelocity. However, for a given T-wave velocity, increased T-waveheights cause reduction of the CCS values for most of the studiedproteins, suggesting that the two effects are independent. Foralchohol dehydrogenase (ADH) and hemoglobin we notice thatat the highest T-wave height the CCS values start to increase

(Figures 3, S2, and S4). This result could perhaps point to adifferent mechanism of energy dissipation in these tetramericstructures.

To examine whether the phenomenon we observed is generaland whether it also characterizes denatured proteins, we derivedthe CCS values of transferrin as a function of the T-wave velocityfor the native and denatured states (Figure 4). In both states anincrease in CCS values is obtained, which reflects an expansionin structure. Given that denatured transferrin starts in a moreextended shape, the expansion effect is expected to be lesspronounced for this state. Indeed, at the highest measured T-wavevelocity, 800 m/s, the CCS values of the denatured and nativestates are larger by a factor of 1.16 ± 0.03 and 2.05 ± 0.18,respectively, compared to the CCS obtained at the lowest velocity(100 m/s). Moreover, our results show that this trend is generaland an increase in the T-wave velocity triggers a structuralexpansion of both natively folded and denatured proteins (FigureS5, Supporting Information). Denatured proteins experience arelatively small increase in their CCS values upon elevation ofthe T-wave velocity, indicating that they indeed started from anunfolded state, while native proteins undergo a pronouncedexpansion (Figure S5). Interestingly, the expansion patterns ofthe native and denatured forms suggest the existence of a pointat which the native and denatured states converge to an “ultimatelyunfolded” conformation. We estimate this point to be in the rangeof 800-1000 m/s for all tested proteins, except for hemoglobin.The denatured hemoglobin has an expansion pattern similar tothose of all the other proteins, but native hemoglobin, both thedimer and tetramer, is relatively slower to undergo denaturation;possibly, the quaternary assembly of this complex has an impacton its unfolding rate. Overall, the existence of an ultimatelyunfolded conformation falls in the same line as the finite compac-tion of denatured and native states (Figure 2). Thus, in additionto the most compact structure induced by the T-wave height, thereis an upper CCS limit generated by high T-wave velocities.

What is the reason for the observed effect of the increasedT-wave velocities? Increased velocity actually means that the ionsexperience shorter intervals between voltage pulses. Hence, lesstime is given for energy dissipation and relaxation between pulses,leading to a gradual increase in the internal energy of the ions.This excess of energy is distributed among the vibrational androtational/translational modes of motion and assists disorderingevents and local unfolding of the protein. These unfolding eventsare reflected in higher CCS values.

Isotropically and Anisotropically Shaped Proteins ShowSimilar Mobility Patterns. Although all tested proteins undergounfolding in response to the increase in the T-wave velocity, thedependency is different (Figures 3 and S4, Supporting Informa-tion). This observation led us to the hypothesis that the processesof energy absorption and dissipation might be different betweenproteins with more globular structures than those with anisotropicshapes. To test this assumption, we performed measurement ofdrift time distribution profiles for 17 proteins varying in theiroverall shape and mass (between 14 and 460 kDa) (Table S1).The degree of shape isotropy of the selected proteins wasestimated by an anisotropy index A, calculated from the inertia

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Figure 3. T-wave height and T-wave velocity have opposite effects on the collision cross-section values of proteins and protein complexes.The combined impact of increasing the T-wave height and velocity is demonstrated for the different charge states of seven proteins/protein complexes in their native form. Four colored surfaces are shown in each panel, one for each of the four fixed T-wave velocities:100 (blue), 350 (green), 480 (yellow), and 650 (red) m/s. As the T-wave velocity increases, the surfaces gradually shift from left to right,because higher T-wave velocities require the use of higher T-wave height voltages. The shift of the surface upward indicates that theincrease of the T-wave velocity leads to the expansion of the ion structure. Each point represents an average value of three or moreindependent experimental measurements. The white horizontal planes correspond to the theoretically calculated CCS value derived usingthe TM (upper) and PA (lower) methods.

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tensor (see the Supporting Information and Figure 5A). Proteinswith a generally spherical structure have an anisotropy index closeto 0, whereas elongated or disk-shaped structures have a largerA.

CCS values were obtained experimentally for all 17 proteinsat 11 T-wave velocities in the range of 100-800 m/s (Figure S6,Supporting Information). For each T-wave velocity, we adjustedthe T-wave height to the minimal value possible (without expe-riencing the rollover effect) to minimize its effect on the results(see the Supporting Information). As expected, a significantincrease in CCS values as a function of the T-wave velocity wasobserved for all tested proteins. At the highest T-wave velocity(800 m/s) the protein structures expanded by (2.1 ± 0.2)-fold onaverage, compared to those at the lowest T-wave velocity (100m/s) (Figure S6). The CCS increase in the T-wave velocity rangeof 300-600 m/s is nearly linear, while for higher velocities,650-800 m/s, most of the proteins showed slight saturation inthe CCS increase. We could not obtain CCS data for the largestproteins, ADH, lactate dehydrogenase (LDH), and �-galactosidase,at the highest T-wave velocity setting, due to instrumentallimitations; for these cases a plateau was reached in the CCSversus T-wave velocity graphs. It appears that for all tested proteinsthe CCS values change in response to an increase in the T-wavevelocity in a manner that generally complies with a sigmoidal fit,and there is a limited range of T-wave velocities, up to ∼300 m/s,in which the CCS values remain approximately constant (FiguresS5 and S6).

The dependency of the CCS on the T-wave velocity doesnot appear to be related to the molecular weight of the proteinsor their oligomerization state. To test whether there is a relationto the shape, we compared normalized graphs for all 17proteins; minimum-maximum normalization was used (see theSupporting Information). We found that the normalized curvesfor almost all the proteins overlap closely (Figure 5B). Incontrast to our expectations, we did not observe any differencein CCS dependency on the T-wave velocity for the mostisotropically and most anisotropically shaped subgroups ofproteins; on the contrary, the rate of change in CCS values is

clearly shape independent. Hence, the data collection experi-ments presented here do not allow extraction of the degree ofshape isotropy of proteins. The randomly sprayed ions do notgain an anisotropic separation either because of the tumblingof the ions or because of the limited time span in the mobilitychamber. To detect shape isotropy of proteins, we propose theuse of a nematic neutral gas, whose anisotropically shapedmolecules will entangle in the mobility chamber, slow theprotein ions, and possibly limit the angular range of theirtumbling.

Figure 4. The CCS values of the denatured and native forms oftransferrin increase as a function of the T-wave velocity. Relationbetween the T-wave velocity and CCS values of denatured (redsurface) and native (blue surface) forms of transferrin. Although anexpansion is seen for both states, the effect is more pronounced forthe native form of transferrin. The horizontal white planes designatethe theoretical CCS values calculated using the TM (upper) and PA(lower) methods. Each data point is an average of three independentexperiments.

Figure 5. CCS measurements cannot discriminate between isotro-pically and anisotropically shaped structures. (A) The protein’scoordinates were used to calculate the anisotropy index (see theSupporting Information). The proteins were then divided into threesubgroups on the basis of their anisotropy index, high (red), medium(gray), and low (green) anisotropy. (B) The obtained data (Figure S6,Supporting Information) were subjected to ternary data normalizationusing the minimum-maximum approach with 100% range and plottedas a function of the T-wave velocity, 100-800 m/s for all the presentedproteins except ADH and LDH, for which 750 m/s was the highestvelocity used. Interestingly, all analyzed proteins show the same trendirrespective of their structural symmetry.

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CONCLUSIONSHere we have systematically investigated the effect of the

T-wave height and velocity on the measured CCS values. A rangeof experimental conditions was tested on a series of proteins andprotein complexes with a wide span of masses, tertiary structures,and assembly states. The comparison with the theoreticallycalculated CCS enabled us to draw general conclusions on howthe T-wave profile influences gas-phase structures. In general, wecould identify an opposite effect of the T-wave height and velocity.Increasing the T-wave height triggered compaction of the proteinstructures, which adds to the initial collapse experienced duringthe transition from the solution to the gas phase.26 On the otherhand, raising the T-wave velocities induced the formation ofextended conformers. The two effects appear to be independent,but we noticed that the gas-phase structures are affected moreby the T-wave velocity change than by the T-wave height change.We also noticed that under the current experimental conditionsall proteins undergo a similar pattern of expansion regardless oftheir shape. In summary, ions propagating through the IMchamber are influenced by two opposing forces, one inducingcompaction and the other expansion; the combined contributionof these factors is represented in the measured CCS value.

The ultimate goal of IM-MS is to define the overall architec-ture and packing of protein complexes15,25 and to providestructural information on protein assemblies for which high-resolution structures are not available. Our observations highlightthe importance of measuring the CCS values at conditions thattruthfully reflect the native structure in solution. From thissystematic study we conclude that both the T-wave height andvelocity affect the conformation of the ion and for each proteinan optimal balance between these parameters should be found.In general, we noticed that the T-wave velocity has a strongerimpact on the conformation of the ions, and we propose to keepit between 300 and 400 m/s. As for the T-wave heights, we suggestthe use of a low voltage, about 1 V above the rollover potential.This value increases systematically as the size of the proteinsincreases; for example, at 300 m/s, the T-wave height for proteinsup to 100 kDa is 7 V, for proteins between 100 and 200 kDa weused 8.5 V, and for proteins above 400 kDa we used 11 V. Overall,we trust that the IM-MS technique holds great promise for theinvestigation of large and complex protein assemblies providedthat the experimental settings are carefully selected to maintainthe structure of the investigated complex.

The same trends of compaction and expansion that we describehere are relevant for both denatured and native states, althoughthe extent of the structural change for the two forms differed.

Thus, in the denatured state, a significant decrease in the CCS isobserved when the T-wave height is increased and a relativelysmall increase in the CCS is detected when the T-wave velocityis raised. Characterizing the two states, denatured and native,enabled us to determine the minimal and maximal packing a givenprotein can assume. These data can be exploited when proteinswith unknown structures are analyzed. For example, the foldingstate of a given protein can be assessed by the observed changein the CCS as a function of the T-wave parameters. Moreover,given that we can determine the “maximal packing” of a proteinand we can estimate the percentage of internal voids from thelength of the polypeptide chain,30,31 the CCS of the nativelikestructure can be calculated.

The key question that was addressed in this study is howthe IM-MS experimental conditions influence the outcome ofthe measurement. Experimental measurements always affectthe object being measured, and this limitation is reflected inour study through the T-wave’s energy accumulation anddissipation. The waves that propagate through the ion mobilitychamber transfer energy to the ions to impart axial velocityand reduce their residence time.33 The amount of energytransmitted and the dissipation rate influence the proteinstructure. If there is enough time between the energy transferevents, internal rearrangement and compression of internalvoids will take place, producing more compact structures.However, if the energy transmissions are frequent, dissipationand relaxation cannot effectively occur, and as a result theinternal energy increases and unfolding takes place. On thewhole, these experimental results, which shed light on theoptimal conditions in IM-MS studies, are also relevant to otherexperimental methods which involve the transfer of protein ionsfrom the solution phase to the gas phase, such as the manyimaging technologies that are coming to the fore.34

ACKNOWLEDGMENTM.S. and M.E. are grateful for the support of the Helen and

Milton A. Kimmelman Center for Biomolecular Structure andAssembly. M.S. acknowledges funding from the European Re-search Council (ERC) under the European Community’s SeventhFramework Programme (Grant FP7/2007-2013)/ERC Grant Agree-ment No. 239679. M.S. is the incumbent of the Elaine BlondCareer Development Chair.

SUPPORTING INFORMATION AVAILABLEAdditional information as noted in the text. This material is

available free of charge via the Internet at http://pubs.acs.org.

Received for review August 19, 2010. Accepted October11, 2010.

AC1021419

(33) Giles, K.; Pringle, S. D.; Worthington, K. R.; Little, D.; Wildgoose, J. L.;Bateman, R. H. Rapid Commun. Mass Spectrom. 2004, 18, 2401–2414.

(34) Neutze, R.; Wouts, R.; van der Spoel, D.; Weckert, E.; Hajdu, J. Nature2000, 406, 752–757.

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