Gas-Phase Compaction and Unfolding of Protein Structures

Oct 22, 2010 - Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues...
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Anal. Chem. 2010, 82, 9484–9491

Gas-Phase Compaction and Unfolding of Protein Structures 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 powerful tool for studying the structures of less established protein assemblies. The method provides simultaneous measurement of the mass and size of intact protein assemblies, providing information not only on the subunit composition and network of interactions but also on the overall topology and shape of protein complexes. However, how the experimental parameters affect the measured collision cross-sections remains elusive. Here, we present an extensive systematic study on a range of proteins and protein complexes with differing sizes, structures, and oligomerization states. Our results indicate that the experimental parameters, T-wave height and velocity, influence the determined collision cross-section independently and in opposite directions. Increasing the T-wave height leads to compaction of the protein structures, while higher T-wave velocities lead to their expansion. These different effects are attributed to differences in energy transmission and dissipation rates. Moreover, by analyzing proteins in their native and denatured states, we could identify the lower and upper boundaries of the collision cross-section, which reflect the “maximally packed” and “ultimately unfolded” states. Together, our results provide grounds for selecting optimal experimental parameters that will enable preservation of the nativelike conformation, providing structural information on uncharacterized protein assemblies. One of the major challenges in the postgenomic era is the revelation of the dynamic interactions and structural properties of multiprotein complexes. Such information is critical for understanding the biological roles and mechanism of action of molecular assemblies. However, the large size, asymmetric contour, heterogeneous composition, and flexible conformations of protein complexes pose a considerable challenge to conventional structural biology approaches. There is therefore a need to expand the plethora of biophysical tools and develop complementary approaches that will enable the investigation of large protein assemblies. Ion mobility (IM) separation coupled to mass spectrometry (MS) is a recent addition to the structural biology tool * Corresponding author. E-mail: [email protected]. Phone: 9728-9343947. Fax: 972-8-9346010. † Department of Biological Chemistry. ‡ Department of Chemical Research Support.

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kit.1-3 The method offers simultaneous measurement in the gas phase of the mass and size of intact assemblies, providing information not only on the subunit composition and network of interactions but also on the overall topology and subunit packing of protein complexes. Ion mobility is a method that measures the time taken for ions to traverse through a region of neutral buffer gas under the influence of a weak electric field.4 The migration rate is dependent on the size of the ion. Large ions will experience more collisions with the background gas in comparison with smaller ions and will therefore traverse the mobility device at longer drift times. In general, the measured drift time is related to the collision crosssection (CCS) of the ion, and therefore, it is associated with its structural properties. While the method was established as a standalone technique,5-8 recently an ion mobility device was integrated within a quadrupole time-of-flight mass spectrometer.1-3 In this instrument (Synapt, Waters, UK Ltd., Manchester, U.K.) the mobility chamber that is filled with neutral gas molecules, usually nitrogen, is placed between two analyzers. By applying a lowvoltage DC pulse across a series of stacked rings, a traveling wave (T-wave) of electropotential carries the ions through the drift chamber.1,2 This traveling wave technique does not provide absolute measurements of the drift time; however, by applying a calibration approach using standard proteins, the transit time can be converted into collision cross-section values.3,9,10 This technological development expands the span of information that can be revealed by mass spectrometry. Now, not only can the composition, stoichiometry, and the interaction network of a protein complex be defined, but insight into the topological (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. Mass Spectrom. 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; John Wiley & Sons: New York, 1988. (5) Jarrold, M. F. Annu. Rev. Phys. Chem. 2000, 51, 179–207. (6) von Helden, G.; Wyttenbach, T.; Bowers, M. T. Science 1995, 267, 1483– 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. 1999, 99, 3037–3080. (9) Ruotolo, B. T.; Benesch, J. L.; Sandercock, A. M.; Hyung, S. J.; Robinson, 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. 10.1021/ac1021419  2010 American Chemical Society Published on Web 10/22/2010

arrangement of subunits and the quaternary organization of the assembly can be gained as well. The application of IM-MS for protein complex structural analysis holds a promising potential, especially for gaining structural details on less established protein assemblies with unknown 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 a calibration method was established,3,9 and conditions for optimizing CCS separation were screened.10 Moreover, the dependence of the CCS values on the charge states3,11 and activation energies was investigated.12,13 In addition, the promise the IM-MS method holds for subunit architecture definition was explored.14,15 However, little attention was directed toward understanding the effect of traveling wave parameters, height and velocity, on the measured CCS values of native proteins and protein complexes. Here we have performed a systematic study on the influence the traveling wave profile has on the measured CCS values and on the inferred gas-phase protein structure. We have examined a range of model proteins and protein assemblies for which precise details of the three-dimensional structure are known. Our results demonstrate that the traveling wave velocity and height have opposite effects on the CCS values. While an increased T-wave height prompts compaction of the protein structure, an increased T-wave velocity triggers protein expansion, presumably due to unfolding. On the basis of these observations, we suggest a strategy of defining optimal traveling wave conditions that will preserve the native structure. Overall, our investigation highlights the fundamental importance of finding the right balance between T-wave height and velocity to retain the native 3D architecture of the ion. EXPERIMENTAL SECTION Collision Cross-Section Measurements. IM-MS measurements were performed on a Synapt HDMS system (Waters, UK Ltd., Manchester, U.K.) as described elsewhere,16,17 with the sample introduced by electrospray from gold-coated borosilicate capillaries prepared in-house.18,19 The instrument parameters were optimized to remove adducts while preserving noncovalent interactions.17,19 The experimental conditions are described in detail in the Supporting Information. Drift times were converted to CCSs using a calibration protocol reported elsewhere.9,19 All (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. Mass Spectrom. 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].

the presented results are an average of at least three independent experiments, which were conducted under exactly the same conditions and followed by a CCS calibration procedure. In addition, when the effect of the T-wave height (TH) or T-wave velocity (TV) for a given protein was examined, all the measurements were conducted in the same day. Overall, the average relative precision of the drift time measurements is approximately 2.1 ± 0.4%, and the average error of the CCS calculation is 4.3 ± 0.2%. The error associated with calibrant cross-sections is 1.4 ± 0.7%, and that associated with the calibration curve is 2.7 ± 0.3%. Theoretical Cross-Section Calculations. The CCSs of all model structures were calculated using the program MOBCAL,20,21 adapted for large all-atom coordinate sets.9 Gas-phase minimization was applied to all the structures after exclusion of the water coordinates in the PDB files. CCS values were calculated using three models: projection approximation (PA), exact hard sphere collision (EHSC), and trajectory modeling (TM). RESULTS AND DISCUSSION Measuring Collision Cross-Sections of Proteins and Protein Complexes. To validate the applicability of the IM-MS approach for the investigation of protein complexes with unknown structures, we have conducted a systematic study to understand the influence of the different T-wave parameters on the generated collision cross-section values. To this end we have selected a set of proteins and protein complexes that fulfill the following criteria: (i) The protein or protein complex has a known high-resolution structure (solved by X-ray crystallography); consequently its theoretical CCS value can be calculated. (ii) The PDB coordinates include 96% of the protein amino acids or more. With such a low percentage of truncations the deviation of the theoretically calculated CCS from the measured CCS (for the full-length protein) is negligible compared to the wide range of the theoretical CCS values (particularly PA versus EHSC). (iii) Availability of the protein or protein complex is. In total our data set includes 7 proteins and 10 protein complexes, with diverse sizes, oligomerization states, and structural properties (see the protein data set details in the Supporting Information). Two of the analyzed monomeric proteins, cytochrome c (Cyt c) and myoglobin, were characterized in their native form, and the results were compared with the CCS obtained for their denatured state during the calibration procedure. Given that the CCS values of the target proteins are obtained via a calibration approach and not by direct measurement, it was important to include Cyt c and myoglobin in the data set, because for these proteins there is no need for extrapolation and the errors related to the calibration approach are minimized. Theoretical CCS values were calculated for all proteins and protein complexes using the PA, EHSC, and TM models in MOBCAL.9,21,22 CCS values calculated via the EHSC and TM approximations are in good agreement (the value from TM is higher by 2.24 ± 0.3% than that from EHSC), and as expected these values are consistently higher than those calculated using (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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Figure 1. Increasing the T-wave height reduces the ion mobility drift time and the collision cross-sections. (A) Drift time distribution as a function of the T-wave height. Results for ovalbumin are shown in the left panel and for Gβγ in the right panel; curves are colored according to the 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, and bias voltage of 25 V (ovalbumin) or 32 V (Gβγ). Inset: Drift time dependence on the T-wave height taken for the centroid of the lowest charge states, +11 and +12 for ovalbumin and Gβγ, respectively. Data were fitted with the exponential equation td ) ae-bTH + c, where a, b, and c are real number coefficients, TH is the T-wave height, and td is the drift time (ms). (B) Dependence of the collision cross-section of ovalbumin and Gβγ 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 states of both proteins. The presented data are an average of three independent experiments.

the PA model by about 20%.10,11 Although, the PA, TM, and EHSC theoretical CCS span a wide range, there is a strong correlation between these values (R2 ) 0.991). In general, experimental CCS values of native structures should fall between the PA and TM/ EHSC values, as the PA approximation underestimates the ccs while TM and EHSC overestimate them.11,23 Increasing the T-Wave Height Induced Protein Structure Compaction. To find whether there is a relation between CCS values and T-wave heights, we performed measurements of drift time distributions over a range of T-wave heights, keeping the T-wave velocity fixed at 450 m/s. The maximal T-wave heights were dictated by the ability to record well-resolved drift time distributions for the calibrant proteins: myoglobin, Cyt c, and ubiquitin. The minimal T-wave heights were defined by the lowest potential that can be used without appearance of the rollover affect. In Figure 1A the influence of the T-wave height on the measured drift time for ovalbumin and Gβγ is shown. Both proteins have a similar molecular mass (Table S1, Supporting Information); however, while ovalbumin is monomeric, Gβγ is a heterodimer. The measurements revealed that in both cases increasing the T-wave height systematically increased the mobility rate and (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., in press.

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reduced the drift time values by a factor of 4.85 ± 0.12 (TH range of 14-22 V) and 9.05 ± 0.14 (TH range of 12-24 V) for ovalbumin and Gβγ, respectively. Moreover, the decrease in drift time values for both proteins was similar for all charge states. Using the measured drift times and the standard calibration approach,9,17 CCS values were derived for each charge state as a function of the T-wave height. Both ovalbumin and Gβγ experience a 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 value compared to the calculated CCS; these values drop steadily as the T-wave height increases (Figure 1B inset). Overall, a decrease of 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 Gβγ, respectively. Unlike the drift time values, the extent of CCS decrease varied among the different charge states. Moreover, the CCS versus T-wave height and charge state surface is rougher than the drift time surface. This result probably reflects minor errors in the process of extracting drift time values for individual charge states (Figure S1, Supporting Information). To probe whether the compaction of the protein structure as a function of the T-wave height is a general phenomenon, we extended the measurements to a series of proteins of different sizes and compositions. Interestingly, a similar trend, which is

often linear, was observed for all proteins and protein complexes (Figure S2, Supporting Information), emphasizing that as the T-wave height increases more closely packed structures are detected. The extent of compaction varies between different charge states and different proteins. Typically, the CCS values for the lowest charge state are between the PA and TM values, while larger ccs are observed for the high charge states, suggesting 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 more compact states obtained upon the increase in T-wave height are populated by fewer discrete structures, representing a smaller conformational space. Such compaction of protein structures during IM-MS measurements was indicated in earlier studies. For example, the higher charge states of the Trp RNA binding protein were reported to have a collapsed protein quaternary structure.3 In another study the compaction of the glutamine synthetase complex was indicated.15 Moreover, differences between CCS values measured for different charge states were reported,3,11,24,25 in which the lowest charge states are in the best agreement with the calculated structure. In summary, although different charge states of the same ion react differently to the applied potential, on the whole we show that high T-wave values lead to a pronounced CCS reduction. In general, the desolvation process that accompanies the transition from the solution to the gas phase supports the formation of new salt-bridge links between the charged side chains on the exterior of the native protein; this process leads to a global compaction of the structure.26-29 Our result indicates that in addition to this initial side-chain collapse a second compaction step occurs when the T-wave height is increased. Each time a traveling wave hits an ion, a packet of energy is absorbed. This energy can be distributed between different modes of motion: rigid body rotation (tumbling), translation, and internal energy. Most of the energy will trigger tumbling and translation, leading to shorter drift times for higher T-waves. However, some of the energy will be converted to internal energy, which will initiate structural reorganization and compact packing of internal voids and pockets. In general, proteins are tightly packed, yet they commonly have internal cavities, which account for 1-2.3% of the protein volume,30 and their number depends on the length of the polypeptide chain.31 The degree of compaction detected here is in the range of 11-20%, larger than that expected. We attribute this deviation to the fact that the calibrants we use are in a denatured state, which is more sensitive to the effect of elevated T-wave heights (as seen in Figure 2). As a consequence the calibration curves may introduce errors in the CCS calculations of native proteins. (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.

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

Native and Unfolded Proteins Collapse to a Similar Size. To investigate whether CCS reduction is a general phenomenon or it characterizes the folded state of the protein or protein complex, we examined the influence of denaturing conditions on drift time distributions. IM-MS spectra of unfolded and folded hemoglobin were recorded at increasing T-wave heights, and the CCS values of tetrameric (R2β2) and dimeric (Rβ) hemoglobin complexes were determined. The T-wave heights were increased in steps from 7 to 13 V (for the dimer) and from 7 to 14 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 experimentally 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 and tetrameric forms of hemoglobin, respectively (Figure 2A). This 75% increase in CCS indicates expansion of the structure. Moreover, broader drift time peaks were measured, indicating that although the oligomeric state is preserved the subunits had undergone partial unfolding.32 Upon increasing the T-wave height, the unfolded and folded structures started to collapse (Figure 2B,C). This observation was detected for tetrameric (left panel) and dimeric (right panel) (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.

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hemoglobin forms, for all examined charge states. However, a different collapse pattern was observed for the different oligomerization states (Figure 2B,C). At the highest T-wave heights, 13 and 14 V, the CCS values dropped to the minimal values of 3631 ± 125 and 2138 ± 112 Å2 for dimeric and tetrameric hemoglobin, respectively. Interestingly, both the unfolded and folded states converged to these minimal CCS values, deviating by only 1.0-1.8%. Hence, although the unfolded hemoglobin dimer and tetramer structures exhibited a larger extent of CCS decrease, their size at a high T-wave height was identical to that of the collapsed native structures. This result indicates that there is a minimal volume that the protein structures can assume within the mobility chamber, regardless of the starting state. Increased T-Wave Frequencies Have an Effect Opposite to That of the T-Wave Height. The observation that the experimentally derived CCS decrease upon increasing the T-wave heights led us to examine whether changing the T-wave velocities induces a similar effect. To this end, we acquired IM-MS data at different T-wave velocities. At each T-wave velocity we recorded several spectra using different T-wave heights. Higher T-wave heights had to be used when the T-wave velocity was increased to preserve the mobility separation capacity. Overall, our goal was to cover as wide a range as possible of T-wave heights without major distortion in the ion mobility separation. Four velocities were examined: 100, 350, 480, and 650 m/s (Figures 3 and S4, Supporting Information). We observed that irrespective of the type of protein or protein complex or its molecular weight, secondary structure composition, or oligomerization state, an increase of the T-wave velocity led to an increase of the CCS value for each charge state (Figure 3). Although the extent of CCS expansion was different for the various proteins, a clear trend could be detected. Thus, at a low T-wave velocity (100 m/s) the measured CCS values were often below the theoretical PA values, while at 350 and 480 m/s the experimental CCS values were between the PA and TM estimates. At a high velocity (650 m/s) the experimentally determined ccs were consistently larger than the theoretical values. This behavior was also observed for Cyt c and myoglobin, which served as calibrants in their denatured state, suggesting that the trend that we observed is not an artifact related to the indirect CCS determination. Overall, we trust that the phenomena we observe here are general, although the absolute degree of expansion and compaction might vary with new calibration approaches that are coming to the fore. Our results indicate that the T-wave velocity and height have opposite effects on the protein structure. While the velocity of the T-wave induces expansion of the protein, the T-wave height triggers its compaction. It can be seen in Figure S4 (Supporting Information) that the velocity of the T-waves has a stronger impact on the protein structure than the T-wave height and a general trend of protein unfolding is observed as a function of the T-wave velocity. However, for a given T-wave velocity, increased T-wave heights cause reduction of the CCS values for most of the studied proteins, suggesting that the two effects are independent. For alchohol dehydrogenase (ADH) and hemoglobin we notice that at the highest T-wave height the CCS values start to increase 9488

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(Figures 3, S2, and S4). This result could perhaps point to a different mechanism of energy dissipation in these tetrameric structures. To examine whether the phenomenon we observed is general and whether it also characterizes denatured proteins, we derived the CCS values of transferrin as a function of the T-wave velocity for the native and denatured states (Figure 4). In both states an increase in CCS values is obtained, which reflects an expansion in structure. Given that denatured transferrin starts in a more extended shape, the expansion effect is expected to be less pronounced for this state. Indeed, at the highest measured T-wave velocity, 800 m/s, the CCS values of the denatured and native states 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 general and an increase in the T-wave velocity triggers a structural expansion of both natively folded and denatured proteins (Figure S5, Supporting Information). Denatured proteins experience a relatively small increase in their CCS values upon elevation of the T-wave velocity, indicating that they indeed started from an unfolded state, while native proteins undergo a pronounced expansion (Figure S5). Interestingly, the expansion patterns of the native and denatured forms suggest the existence of a point at which the native and denatured states converge to an “ultimately unfolded” conformation. We estimate this point to be in the range of 800-1000 m/s for all tested proteins, except for hemoglobin. The denatured hemoglobin has an expansion pattern similar to those of all the other proteins, but native hemoglobin, both the dimer and tetramer, is relatively slower to undergo denaturation; possibly, the quaternary assembly of this complex has an impact on its unfolding rate. Overall, the existence of an ultimately unfolded conformation falls in the same line as the finite compaction of denatured and native states (Figure 2). Thus, in addition to the most compact structure induced by the T-wave height, there is an upper CCS limit generated by high T-wave velocities. What is the reason for the observed effect of the increased T-wave velocities? Increased velocity actually means that the ions experience shorter intervals between voltage pulses. Hence, less time 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 and rotational/translational modes of motion and assists disordering events and local unfolding of the protein. These unfolding events are reflected in higher CCS values. Isotropically and Anisotropically Shaped Proteins Show Similar Mobility Patterns. Although all tested proteins undergo unfolding in response to the increase in the T-wave velocity, the dependency is different (Figures 3 and S4, Supporting Information). This observation led us to the hypothesis that the processes of energy absorption and dissipation might be different between proteins with more globular structures than those with anisotropic shapes. To test this assumption, we performed measurement of drift time distribution profiles for 17 proteins varying in their overall shape and mass (between 14 and 460 kDa) (Table S1). The degree of shape isotropy of the selected proteins was estimated by an anisotropy index A, calculated from the inertia

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 the increase of the T-wave velocity leads to the expansion of the ion structure. Each point represents an average value of three or more independent experimental measurements. The white horizontal planes correspond to the theoretically calculated CCS value derived using the TM (upper) and PA (lower) methods. Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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Figure 4. The CCS values of the denatured and native forms of transferrin increase as a function of the T-wave velocity. Relation between the T-wave velocity and CCS values of denatured (red surface) and native (blue surface) forms of transferrin. Although an expansion is seen for both states, the effect is more pronounced for the native form of transferrin. The horizontal white planes designate the theoretical CCS values calculated using the TM (upper) and PA (lower) methods. Each data point is an average of three independent experiments.

tensor (see the Supporting Information and Figure 5A). Proteins with a generally spherical structure have an anisotropy index close to 0, whereas elongated or disk-shaped structures have a larger A. CCS values were obtained experimentally for all 17 proteins at 11 T-wave velocities in the range of 100-800 m/s (Figure S6, Supporting Information). For each T-wave velocity, we adjusted the T-wave height to the minimal value possible (without experiencing the rollover effect) to minimize its effect on the results (see the Supporting Information). As expected, a significant increase in CCS values as a function of the T-wave velocity was observed for all tested proteins. At the highest T-wave velocity (800 m/s) the protein structures expanded by (2.1 ± 0.2)-fold on average, compared to those at the lowest T-wave velocity (100 m/s) (Figure S6). The CCS increase in the T-wave velocity range of 300-600 m/s is nearly linear, while for higher velocities, 650-800 m/s, most of the proteins showed slight saturation in the CCS increase. We could not obtain CCS data for the largest proteins, ADH, lactate dehydrogenase (LDH), and β-galactosidase, at the highest T-wave velocity setting, due to instrumental limitations; for these cases a plateau was reached in the CCS versus T-wave velocity graphs. It appears that for all tested proteins the CCS values change in response to an increase in the T-wave velocity 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 (Figures S5 and S6). The dependency of the CCS on the T-wave velocity does not appear to be related to the molecular weight of the proteins or their oligomerization state. To test whether there is a relation to the shape, we compared normalized graphs for all 17 proteins; minimum-maximum normalization was used (see the Supporting Information). We found that the normalized curves for almost all the proteins overlap closely (Figure 5B). In contrast to our expectations, we did not observe any difference in CCS dependency on the T-wave velocity for the most isotropically and most anisotropically shaped subgroups of proteins; on the contrary, the rate of change in CCS values is 9490

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Figure 5. CCS measurements cannot discriminate between isotropically and anisotropically shaped structures. (A) The protein’s coordinates were used to calculate the anisotropy index (see the Supporting Information). The proteins were then divided into three subgroups 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 normalization using the minimum-maximum approach with 100% range and plotted as a function of the T-wave velocity, 100-800 m/s for all the presented proteins except ADH and LDH, for which 750 m/s was the highest velocity used. Interestingly, all analyzed proteins show the same trend irrespective of their structural symmetry.

clearly shape independent. Hence, the data collection experiments presented here do not allow extraction of the degree of shape isotropy of proteins. The randomly sprayed ions do not gain an anisotropic separation either because of the tumbling of the ions or because of the limited time span in the mobility chamber. To detect shape isotropy of proteins, we propose the use of a nematic neutral gas, whose anisotropically shaped molecules will entangle in the mobility chamber, slow the protein ions, and possibly limit the angular range of their tumbling.

CONCLUSIONS Here we have systematically investigated the effect of the T-wave height and velocity on the measured CCS values. A range of experimental conditions was tested on a series of proteins and protein complexes with a wide span of masses, tertiary structures, and assembly states. The comparison with the theoretically calculated CCS enabled us to draw general conclusions on how the T-wave profile influences gas-phase structures. In general, we could identify an opposite effect of the T-wave height and velocity. Increasing the T-wave height triggered compaction of the protein structures, which adds to the initial collapse experienced during the transition from the solution to the gas phase.26 On the other hand, raising the T-wave velocities induced the formation of extended conformers. The two effects appear to be independent, but we noticed that the gas-phase structures are affected more by the T-wave velocity change than by the T-wave height change. We also noticed that under the current experimental conditions all proteins undergo a similar pattern of expansion regardless of their shape. In summary, ions propagating through the IM chamber are influenced by two opposing forces, one inducing compaction and the other expansion; the combined contribution of these factors is represented in the measured CCS value. The ultimate goal of IM-MS is to define the overall architecture and packing of protein complexes15,25 and to provide structural information on protein assemblies for which highresolution structures are not available. Our observations highlight the importance of measuring the CCS values at conditions that truthfully reflect the native structure in solution. From this systematic study we conclude that both the T-wave height and velocity affect the conformation of the ion and for each protein an optimal balance between these parameters should be found. In general, we noticed that the T-wave velocity has a stronger impact on the conformation of the ions, and we propose to keep it between 300 and 400 m/s. As for the T-wave heights, we suggest the use of a low voltage, about 1 V above the rollover potential. This value increases systematically as the size of the proteins increases; for example, at 300 m/s, the T-wave height for proteins up to 100 kDa is 7 V, for proteins between 100 and 200 kDa we used 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 the investigation of large and complex protein assemblies provided that the experimental settings are carefully selected to maintain the structure of the investigated complex. The same trends of compaction and expansion that we describe here are relevant for both denatured and native states, although the extent of the structural change for the two forms differed.

Thus, in the denatured state, a significant decrease in the CCS is observed when the T-wave height is increased and a relatively small increase in the CCS is detected when the T-wave velocity is raised. Characterizing the two states, denatured and native, enabled us to determine the minimal and maximal packing a given protein can assume. These data can be exploited when proteins with unknown structures are analyzed. For example, the folding state of a given protein can be assessed by the observed change in the CCS as a function of the T-wave parameters. Moreover, given that we can determine the “maximal packing” of a protein and we can estimate the percentage of internal voids from the length of the polypeptide chain,30,31 the CCS of the nativelike structure can be calculated. The key question that was addressed in this study is how the IM-MS experimental conditions influence the outcome of the measurement. Experimental measurements always affect the object being measured, and this limitation is reflected in our study through the T-wave’s energy accumulation and dissipation. The waves that propagate through the ion mobility chamber transfer energy to the ions to impart axial velocity and reduce their residence time.33 The amount of energy transmitted and the dissipation rate influence the protein structure. If there is enough time between the energy transfer events, internal rearrangement and compression of internal voids will take place, producing more compact structures. However, if the energy transmissions are frequent, dissipation and relaxation cannot effectively occur, and as a result the internal energy increases and unfolding takes place. On the whole, these experimental results, which shed light on the optimal conditions in IM-MS studies, are also relevant to other experimental methods which involve the transfer of protein ions from the solution phase to the gas phase, such as the many imaging technologies that are coming to the fore.34

(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. Nature 2000, 406, 752–757.

Received for review August 19, 2010. Accepted October 11, 2010.

ACKNOWLEDGMENT M.S. and M.E. are grateful for the support of the Helen and Milton A. Kimmelman Center for Biomolecular Structure and Assembly. M.S. acknowledges funding from the European Research Council (ERC) under the European Community’s Seventh Framework Programme (Grant FP7/2007-2013)/ERC Grant Agreement No. 239679. M.S. is the incumbent of the Elaine Blond Career Development Chair. SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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