Ion Mobility-Mass Spectrometry Reveals the Influence of Subunit

Nov 5, 2010 - University of Oxford. ..... circles; 10-mer, green circles; 9-mer, white ellipses; 8-mer, white circles; ... 2-mer, yellow ellipses; 1-m...
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Anal. Chem. 2010, 82, 9702–9710

Ion Mobility-Mass Spectrometry Reveals the Influence of Subunit Packing and Charge on the Dissociation of Multiprotein Complexes Elisabetta Boeri Erba,†,⊥ Brandon T. Ruotolo,†,‡ Daniel Barsky,†,§ and Carol V. Robinson*,†,| University Chemistry Department, University of Cambridge, Cambridge, United Kingdom, Department of Chemistry, University of Michigan, Ann Arbor, Michigan, Lawrence Livermore National Laboratory, Livermore, California, and Department of Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford, United Kingdom The composition, stoichiometry, and organization of protein complexes can be determined by collision-induced dissociation (CID) coupled to tandem mass spectrometry (MS/MS). The increased use of this approach in structural biology prompts a better understanding of the dissociation mechanism(s). Here we report a detailed investigation of the CID of two dodecameric, heat-stable and toroidally shaped complexes: heat shock protein 16.9 (HSP16.9) and stable protein 1 (SP-1). While HSP16.9 dissociates by sequential loss of unfolded monomers, SP-1 ejects not only monomers, but also its building blocks (dimers), and multiples thereof (tetramers and hexamers). Unexpectedly, the dissociation of SP-1 is strongly charge-dependent: loss of the building blocks increases with higher charge states of this complex. By combining MS/MS with ion mobility (IM-MS/MS), we have monitored the unfolding and dissociation events for these complexes in the gas phase. For HSP16.9 unfolding occurs at lower energies than the ejection of subunits, whereas for SP-1 unfolding and dissociation take place simultaneously. We consider these results in the light of the structural organization of HSP16.9 and SP-1 and hypothesize that SP-1 is unable to unfold extensively due to its particular quaternary structure and unusually high charge density. This investigation increases our understanding of the factors governing the CID of protein complexes and moves us closer to the goal of obtaining structural information on subunit interactions and packing from gas-phase experiments. The mass of protein complexes together with their stoichiometry and subunit interactions can be established by electrospray (ESI) mass spectrometry (MS).1-3 Obtaining these data contributes to the increasing use of ESI-MS in hybrid structural biology * To whom correspondence should be addressed. † University of Cambridge. ⊥ Current address: Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland. ‡ University of Michigan. § Lawrence Livermore National Laboratory. | University of Oxford. (1) Heck, A. J.; Van Den Heuvel, R. H. Mass Spectrom Rev 2004, 23, 368– 389. (2) Hernandez, H.; Robinson, C. V. Nat. Protoc. 2007, 2, 715–726. (3) Sharon, M.; Robinson, C. V. Annu. Rev. Biochem. 2007, 76, 167–193.

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approaches.3 In ESI-MS, protein complexes are ionized and maintained intact typically in a mass spectrometer which has been modified to analyze high mass ions.4 In order to determine the composition and organization of a protein complex, the protein assembly is subjected to tandem mass spectrometry (MS/MS) whereby the protein complex is made to dissociate into product ions.5 Several dissociation techniques have been developed for MS/MS and collision-induced dissociation (CID) is most commonly used due to its ease of set up and operation.6 In a CID experiment, protein complexes are accelerated in a collision cell containing neutral gas, such as argon or xenon, with which the complexes collide, raising their internal energy.7 If we consider the steps involved as the acceleration energy is raised, an ionized protein complex undergoes collision-induced desolvation, restructuring, and unfolding.8 During the collisioninduced desolvation, solvent and buffer salts bound to the protein complex are removed.9 This has the effect of improving the mass to charge resolution of the intact protein complex ions.9 Further increases in acceleration voltage, lead to a collision-induced restructuring whereby the structure of the protein complexes can be altered; in some cases becoming more compact.10 In many cases collision-induced unfolding takes place along with this restructuring.8,11-14 It is not clear whether unfolding occurs in one or several subunits.8,15 However, the majority of studies support the first hypothesis in which collision-induced activation leads to an increase in the internal energy of the protein complex and a single subunit (monomer) begins to unfold. Consequently, (4) Sobott, F.; Hernandez, H.; McCammon, M. G.; Tito, M. A.; Robinson, C. V. Anal. Chem. 2002, 74, 1402–1407. (5) van den Heuvel, R. H.; van Duijn, E.; Mazon, H.; Synowsky, S. A.; Lorenzen, K.; Versluis, C.; Brouns, S. J.; Langridge, D.; van der Oost, J.; Hoyes, J.; Heck, A. J. Anal. Chem. 2006, 78, 7473–7483. (6) Wells, J. M.; McLuckey, S. A. Methods Enzymol. 2005, 402, 148–185. (7) Loo, J. A. Mass Spectrom. Rev. 1997, 16, 1–23. (8) Benesch, J. L. J. Am. Soc. Mass Spectrom. 2009, 20, 341–348. (9) McKay, A. R.; Ruotolo, B. T.; Ilag, L. L.; Robinson, C. V. J. Am. Chem. Soc. 2006, 128, 11433–11442. (10) Ruotolo, B. T.; Giles, K.; Campuzano, I.; Sandercock, A. M.; Bateman, R. H.; Robinson, C. V. Science 2005, 310, 1658–1661. (11) Benesch, J. L.; Aquilina, J. A.; Ruotolo, B. T.; Sobott, F.; Robinson, C. V. Chem. Biol. 2006, 13, 597–605. (12) Felitsyn, N.; Kitova, E. N.; Klassen, J. S. Anal. Chem. 2001, 73, 4647– 4661. (13) Jurchen, J. C.; Garcia, D. E.; Williams, E. R. J. Am. Soc. Mass Spectrom. 2004, 15, 1408–1415. (14) Jurchen, J. C.; Williams, E. R. J. Am. Chem. Soc. 2003, 125, 2817–2826. (15) 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. 10.1021/ac101778e  2010 American Chemical Society Published on Web 11/05/2010

charges migrate to the newly available surface area.14,16,17 Further unfolding and charge migration take place until the Coulombic repulsion between charges on the unfolded monomer and the rest of the protein complex surmounts the interactions which keep the protein complex together.8 As a consequence, the unfolded monomer is ejected and a “stripped complex” remains. Because of the unfolding, the ejected monomer has a large surface area and it can accommodate many charges,11,16 yielding a highly asymmetric charge partitioning between the product ions.13,14 Along with the compositional information that can be gleaned from MS/MS of protein complexes, key structural information can be obtained by ion mobility mass spectrometry (IM-MS).18 IM is a validated technique for investigating the shape and conformation of small molecules and proteins.19-21 However, it has only recently been applied to protein complexes revealing their overall topology in the gas phase.10,22-25 IM-MS analysis has recently been used to demonstrate that the native-like topology of protein complexes can be retained in the absence of bulk solvent.10,23,26 Moreover, IM-MS can be used to monitor gross conformational changes and consequently can be used to assess the effects of point mutations and ligand binding on the dissociation and unfolding of protein complexes.27 IM-MS has also been used to study the CID of charge-reduced tetrameric transthyretin (TTR) ions, revealing alternate dissociation pathways that become available to complexes at low charge states.28 Although a monomer is ejected in a typical CID experiment, two protein complexes have been identified in which intact dimers were ejected in single dissociation steps: the hetero hexameric textilotoxin and the tetrameric 2-keto-3-deoxyarabinonate.5,29 However, it has not been possible to relate the dissociation properties of these complexes to their subunit architecture and propensity of monomeric subunits to unfold, since IM-MS was not employed in these studies. Here we report the use of IM-MS to investigate the gas-phase unfolding and dissociation of two large noncovalent homo-oligomeric complexes, the heat shock protein (HSP16.9) from wheat and the boiling stable protein 1 (SP-1) from (16) Csiszar, S. T., M. Can. J. Chem. 2004, 82, 1736–1744. (17) Sinelnikov, I.; Kitova, E. N.; Klassen, J. S. J. Am. Soc. Mass Spectrom. 2007, 18, 617–631. (18) 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. (19) Hoaglund-Hyzer, C. S.; Counterman, A. E.; Clemmer, D. E. Chem Rev 1999, 99, 3037–3080. (20) Jarrold, M. F. Annu. Rev. Phys. Chem. 2000, 51, 179–207. (21) von Helden, G.; Wyttenbach, T.; Bowers, M. T. Science 1995, 267, 1483– 1485. (22) Loo, J. A.; Berhane, B.; Kaddis, C. S.; Wooding, K. M.; Xie, Y.; Kaufman, S. L.; Chernushevich, I. V. J. Am. Soc. Mass Spectrom. 2005, 16, 998– 1008. (23) Teplow, D. B.; Lazo, N. D.; Bitan, G.; Bernstein, S.; Wyttenbach, T.; Bowers, M. T.; Baumketner, A.; Shea, J. E.; Urbanc, B.; Cruz, L.; Borreguero, J.; Stanley, H. E. Acc. Chem. Res. 2006, 39, 635–645. (24) van Duijn, E.; Barendregt, A.; Synowsky, S.; Versluis, C.; Heck, A. J. J. Am. Chem. Soc. 2009, 131, 1452–1459. (25) van Duijn, E.; Simmons, D. A.; van den Heuvel, R. H.; Bakkes, P. J.; van Heerikhuizen, H.; Heeren, R. M.; Robinson, C. V.; van der Vies, S. M.; Heck, A. J. J. Am. Chem. Soc. 2006, 128, 4694–4702. (26) Ruotolo, B. T.; Robinson, C. V. Curr. Opin. Chem. Biol. 2006, 10, 402– 408. (27) Hyung, S. J.; Robinson, C. V.; Ruotolo, B. T. Chem. Biol. 2009, 16, 382– 390. (28) Pagel, K.; Hyung, S. J.; Ruotolo, B. T.; Robinson, C. V. Anal. Chem., 82, 5363-5372. (29) Aquilina, J. A. Proteins 2009, 75, 478–485.

aspen plants (Populus tremula). These protein assemblies are dodecameric, heat-stable and toroidally shaped complexes with no intra- or intersubunit disulfide bonds (Figure 1). The HSP16.9 protein complex is a member of the small heat shock protein family of molecular chaperones that prevent protein aggregation during heat shock. The SP-1 protein complex is stable even above 100 °C and in vivo it is involved in response to various abiotic stresses, including desiccation, cold shock, and rehydration.30 In terms of its amino acid sequence, SP-1 is homologous to proteins in various other plants (including corn, wheat, and fern); structurally, it is in the “plant stress-induced protein” SCOP family of proteins, which also contains two proteins from Arabidopsis thaliana (fern). Despite their similarities, the two protein complexes display markedly different gas-phase dissociation behavior. Under CID conditions the HSP16.9 protein complex undergoes a typical dissociation via the ejection of monomers.11 By contrast, the SP-1 protein complex dissociates unusually via the ejection of its building blocks (dimers), and multiples thereof (tetramers and hexamers) as well as monomers. Unexpectedly we also found that by manipulating the charge state of the SP-1 precursor ions, we can control the oligomeric state of the product ions. Moreover, the laboratory frame energy (Elab) (defined as the product of the charge state of the parent ion and the accelerating voltage31-33) required to dissociate the SP-1 protein complex was found to decrease with increasing charge state. At low charge states the complex can unfold without significant dissociation and at high charge states unfolding and dissociation occur simultaneously. We speculate that the atypical dissociation of the SP-1 protein complex is due to its “supercharged” character as well as its quaternary structure. EXPERIMENTAL SECTION Protein Expression and Reagents. The HSP16.9 protein complex from wheat and the SP-1 protein complex from aspen plants (Populus tremula) were purified as described previously.30,34 Dimethylsulfoxide (DMSO), triethylammonium acetate and ammonium acetate were purchased from Sigma-Aldrich Company Ltd. (Gillingham, Dorset, UK); sulfolane (thiolane 1,1-dioxide) from Fluka production GmbH (Buchs, Switzerland). Sample Preparation for Mass Spectrometry. The HSP16.9 protein complex was buffer exchanged into 100 mM ammonium acetate (pH 7.0) using micro Biospin 6 columns (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK). The SP-1 protein complex was concentrated and desalted by centrifugation at maximum speed 10 000 g in Vivaspin concentrator tubes (exclusion limit 10 000 Da, Vivaspin, Sartorius Stedim UK Limited, Epsom, UK) to a final concentration of 40 µM of protein complex. The dilution of complex-containing solutions was carried out using ammonium acetate to a concentration 5-10 µM of the intact complex immediately prior to the mass spectrometry analysis. For the experiments in which the charge state is manipulated, the (30) Wang, W. X.; Pelah, D.; Alergand, T.; Shoseyov, O.; Altman, A. Plant Physiol. 2002, 130, 865–875. (31) Benesch, J. L.; Ruotolo, B. T.; Sobott, F.; Wildgoose, J.; Gilbert, A.; Bateman, R.; Robinson, C. V. Anal. Chem. 2009, 81, 1270–1274. (32) Laskin, J.; Futrell, J. H. Mass Spectrom. Rev. 2005, 24, 135–167. (33) Sievers, H. L.; Gru ¨ tzmacher, H.-F.; Caravatti, P. Int. J. Mass Spectrom. Ion Processes 1996, 158, 233–247. (34) van Montfort, R. L.; Basha, E.; Friedrich, K. L.; Slingsby, C.; Vierling, E. Nat. Struct. Biol. 2001, 8, 1025–1030.

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Figure 1. (A) The dodecameric HSP16.9 protein complex. Left panel: MS/MS spectrum of intact dodecameric HSP16.9 32+ ions. A schematic representation illustrates how this protein complex dissociates by forming lowly charged decamers and undecamer at high m/z and highly charged monomers at low m/z. This type of dissociation is defined as “typical” because there is sequential loss of monomeric subunits. 12-mer: blue; 11-mer: pink; 10-mer: green; and 1-mer: purple. Right panel: Crystal structure of the HSP16.9 protein complex (PDB code: 1gme). The Nand C-terminal amino acids of each protein within the protein complex are represented as spheres in order to clearly illustrate their positions. The N-termini of chains B, D, F, H, J, and L are disordered and not present in the crystal structure. (B) The dodecameric SP-1 protein complex. Left panel: MS/MS spectrum of intact dodecameric SP-1 27+ ions. 1-mers, 2-mers, 3-mers, 4-mers, 5-mers and 11-mers, 10-mers, 9-mers, 8-mers, 7-mers, 6-mers are present at low and high m/z in the spectrum, respectively. The Elab, used in order to dissociate the dodecameric SP-1 protein complex is 2106 eV. The dissociation of SP-1 is “atypical” due the ejection of dimers, tetramers. 12-mer, blue circles; 11-mer, pink circles; 10-mer, green circles; 9-mer, white ellipses; 8-mer, white circles; 7-mer, black ellipses; 6-mer, orange circles; 4-mer, brown circles; 2-mer, yellow ellipses; 1-mer, purple circles. Right panel: The crystal structure of the SP-1 protein complex (PDB code: 1tr0).

SP-1 protein complex in 100 mM ammonium acetate was diluted 1 to 4 in 1.2 M DMSO or 1 M triethylammonium acetate or 1.6 M sulfolane. Ion Mobility-Mass Spectrometry and calculations. Protein complex ions were generated using a nanoflow electrospray (nanoESI) source and nanoflow gold-coated borosilicate electrospray capillaries were prepared in-house as previously described.2 Mass spectrometry analyses were carried out on a quadrupole time-of-flight mass spectrometer (QSTAR, AB Sciex, Toronto, Canada). The instrument was modified for the detection of high masses.4 The following instrumental parameters were used: capillary voltage up to 1.5 kV, declustering potential 200 V, focusing potential 250 V, declustering potential-2 15 V, and collision energy up to 280 V, microchannel plate (MCP) 2350 V. For tandem mass spectra argon was used as a collision gas. Mass spectrometry and ion mobility measurements were performed using a Synapt HDMS (Waters Corporation, Manchester, UK) quadrupole-ion trap-IM-MS instrument. The following 9704

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instrumental parameters were used in MS mode: 1.5 kV; sample cone, 20 V; cone gas, 130 L hr-1; trap collision voltages, between 8 and 10 V; ion-transfer stage pressure, 5.0 mbar of Argon; ionmobility cell: 0.5 mbar of Nitrogen; time-of-flight analyzer pressure, 1.3 × 10-6 millibars. Under these conditions, no dissociation of the protein complexes was detected in the mass spectrum. For collision-induced dissociation experiments in MS/MS mode at a range of laboratory frame energies (Elab), all parameter settings were kept constant apart from the trap collision voltage which was varied stepwise during the acquisition of each spectrum. The Elab, applied to accelerate the dodecamer HSP16.9 and SP-1 in the collision-induced dissociation experiments in MS/MS mode, is defined as the product of the charge state of the parent ion and the trap collision voltage.31 All mass spectra were calibrated externally using a solution of cesium iodide (100 mg/mL in water) and were processed with Masslynx 4.1 software (Waters Corporation, Manchester, UK).

Traveling wave drift times were converted into collision cross sections.35 Drift times of protein ions and protein complexes ions of known collision cross sections (CCSs) were used to define the relationship between CCS and drift time, generating a calibration curve.35 This calibration curve was used to calculate CCS of the HSP16.9 and SP-1 protein complexes. In Figure 3, using IM data the relative abundance of the compact charge states of dodecamer HSP16.9 and SP-1 was calculated as a percentage of the total intensity of all peaks in the arrival time distribution plot. At each Elab value we calculated the areas under the curves representing the IM arrival time distributions for each conformational ensemble. Then we calculated the percentage of area under the curve representing the IM arrival time distribution of the folded dodecamer over the sum of the areas of all conformational ensembles present in the arrival time distribution plot. This percentage is a simple descriptor of dodecamer folded state and termed percentage “folded”. “folded” (%) ) area of IM arrival time distribution of folded dodecamer/area of IM arrival time distribution of all dodecameric conformers. In Figure 3, using MS/MS data the relative abundance of each charge state of the dodecamer HSP16.9 and SP-1 was calculated as a percentage of the total intensity of all mass peaks assigned to the intact protein complex relative to the peak intensity of dodecamer and all product ions (undecamer, decamer, etc.). This is defined as percentage “intact”. “intact” (%) ) peak intensity of the intact dodecamer/total peak intensity of dodecamer and all product ions. The program Mobcal was used to calculate the CCSs of the SP-1 and HSP16.9 protein complexes taking into account their crystallographic structures. Specifically, we used the projection approximation calculation.36,37 We and others have found that this calculation gives a good estimate of the experimentally measured CCSs in the absence of substantial conformational changes.35,38,39 The program MSMS was used to calculate the solvent excluded surface areas of the SP-1 and HSP16.9 protein complexes.40 RESULTS Typical and Atypical Dissociation Pathways Occur during Collision-Induced Dissociation. To investigate gas-phase dissociation behavior of the HSP16.9 and SP-1 protein complexes, we subjected both protein complexes to tandem mass spectrometry. The MS/MS spectrum of HSP16.9 indicates that this complex has a mass of 200 790 Da. Monomers are present at low m/z region in the spectrum and populations of undecamer and decamer are present at high m/z (Figure 1A).11 We note that at low m/z there are only monomers and no higher order oligomers (35) Ruotolo, B. T.; Benesch, J. L.; Sandercock, A. M.; Hyung, S. J.; Robinson, C. V. Nat. Protoc. 2008, 3, 1139–1152. (36) Mesleh, M. F. H., J. M.; Shvartsburg, A. A.; Schatz, G. C.; Jarrold, M. F. J. Phys. Chem. 1996, 100, 16082–16086. (37) Shvartsburg, A. A.; Jarrold, M. F. Chem. Phys. Lett. 1996, 261, 86–91. (38) Politis, A.; Park, A. Y.; Hyung, S. J.; Barsky, D.; Ruotolo, B. T.; Robinson, C. V. PLoS One, 5, e12080. (39) Williams, J. P.; Bugarcic, T.; Habtemariam, A.; Giles, K.; Campuzano, I.; Rodger, P. M.; Sadler, P. J. J. Am. Soc. Mass Spectrom. 2009, 20, 1119– 1122. (40) Sanner, M. F.; Olson, A. J.; Spehner, J. C. Biopolymers 1996, 38, 305–320.

are present. Therefore, it is likely that the decamer, like the undecamer, is formed by sequential ejection of monomers; that is, monomer ejection from the dodecameric HSP16.9 protein complex forms the undecamer, which subsequently can eject a second monomer, to form a decamer. Additional evidence for monomer ejection comes from consideration of the distribution of the available charge (32+ on the parent ion). For the undecameric ions ranging from 19+ to 25+ are formed while the monomers have between 7+ and 13+. Summation of the undecamer and monomer charges (25 + 7 or 13 + 19 ) 32) reveals the conservation of charge between the dissociation products. The mass spectrometric analysis of the SP-1 protein complex indicates that this complex has a mass of 147 348 Da. In contrast to what we observed for the HSP16.9 protein complex, MS/MS spectrum of the SP-1 27+ ion shows many oligomeric populations: monomers, dimers and tetramers at low m/z in the spectrum; and hexamers, heptamers, octamers, monomers, decamers, and undecamers at high m/z (Figure 1B). We reason that some of the oligomers in the spectrum are formed directly from the dissociation of the dodecameric complex, whereas others may be due to sequential dissociation of product ions. Our justification is that we note that as the laboratory frame energy (Elab) increases, the presence of monomers, ninemers, heptamers and hexamers increases (Supporting Information (SI) Figure S-1) implying that the nonameric, heptameric and hexameric species are not formed directly from the dissociation of the dodecameric complex. A more likely scenario is that these oligomers are due to further loss of monomers from product ions. For example, the SP-1 octamer, which is initially formed directly by ejection of a tetramer, can further dissociate by loss of monomers, generating heptameric product ions. Even though the decamers are present at low Elab (SI Figure S-1A), their presence increases and the presence of the undecamers decreases with the Elab (SI Figure S-1B). Therefore, it seems likely that decamers are not only formed directly from the dissociation of the dodecameric complex, through the loss of dimers, but also from the dissociation of the undecamers through the loss of monomers. Even though the HSP16.9 (PDB code 1gme) and SP-1 (PDB code 1tr0) protein complexes are both dodecameric, heat-stable and toroidally shaped complexes (Figure 1, right panels), they clearly dissociate through dissimilar pathways. Gas-Phase Unfolding of the Two Dodecamers Occurs under Very Different Conditions. To further characterize the factors controlling the dissociation of the HSP16.9 and SP-1 protein complexes, we recorded ion mobility (IM) arrival time distribution for both complexes at a range of laboratory frame energies (Figure 2). At an Elab of 2080 eV the arrival time distribution of the 32+ ion of the HSP16.9 protein complex is narrow, symmetrical and centered on 11 ms (A in Figure 2). At this energy, the experimental collisional cross section (CCS) (7255 Å2 +/- 5%) is consistent with the one calculated (7458 Å2) using crystallographic information and the program Mobcal.36,37 As the Elab increases, the drift time distribution of the 32+ ion is shifted to longer drift times, typical of an oligomer undergoing partial unfolding of one or more subunits (Figure 2A). At 11 680 eV, (five times the initial Elab) the drift time distribution is bimodal with peaks centered on 20.75 and 24 ms (F and G Figure 2A). Under these conditions the CCS of the dodecamer corresponds Analytical Chemistry, Vol. 82, No. 23, December 1, 2010

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Figure 2. (A) The drift time distributions of 32+ ions of the dodecameric HSP16.9 protein complex acquired at five different Elab (2080, 10 400, 10 880, 11 040, 11 680 eV) display a transition from compact to extended ion conformations. The centroid peaks are labeled from A to G and they correspond to the following CCSs. A, 7255; B, 8282; C, 8945; D, 9271; E, 10 954; F, 11 464; G, 12 759 Å2 +/-5%. A dashed line indicates the drift time, which corresponds to a 40% increment in the CCS.; (B) The drift time distributions of 27+ ions of the dodecameric SP-1 protein complex acquired at four different laboratory frame energies (270, 1620, 1755, 5400 eV) display a transition from compact to extended ion conformations. The centroid peaks are labeled from A to C and they correspond to the following collision cross sections. A, 6550; B, 6717; C, 7445 Å2 +/-6%. A dashed line indicates the drift time, which corresponds to a 40% increment of CCS.

to distribution centered on 24 ms, and correlates with a CCS of 12 759 Å2 (+/-5%) which is 75.9% larger than the initial CCS. Drift times for the 27+ ion of the dodecameric SP-1 protein complex, as a function of Elab, were also recorded (Figure 2B). At the lowest energy required for ion transmission (270 eV), the drift time profile is narrow and centered symmetrically on 8.9 ms (A in Figure 2B). This drift time corresponds to an experimental CCS of 6550 Å2 (+/- 6%) and is in agreement with the one calculated from the crystallographic coordinates (6200 Å2). At 1620 eV two additional peaks are observed (B and C in Figure 2B). At this energy the SP-1 dodecamers exist in only two discrete structural states, as opposed to the diffuse peaks observed for the HSP 16.9 protein complex. Further increases in energy lead to a drift time distribution of the SP-1 protein complex which is narrow and symmetrically centered on 14 ms (C in Figure 2B). Even though the Elab is 20-fold higher than the initial one (5400 eV), the arrival time distribution for the SP-1 protein complex is not shifted to significantly longer drift times. The corresponding CCS of the dodecamer is 7445 Å2 (+/-6%) which is only 13.7% larger than the initial CCS. This indicates that at an Elab of 5400 eV the dodecameric SP-1 protein complex has undergone only minimal unfolding. Interestingly, however, the complex has undergone significant dissociation, since 98% of the total ion current is assigned to product ions (data not shown). 9706

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Figure 3. Monitoring the unfolding and dissociation of the HSP16.9 and SP-1 protein complexes. (A) The percentages of 32+ ions of the intact HSP16.9 (black triangle) and of the folded HSP16.9 (circle) are plotted versus Elab. (B) The percentages of 27+ ions of the intact SP-1 (black triangle) and of the folded SP-1 (circle) are plotted versus Elab.

Dissociation and Unfolding Reveal Further Differences between the Dodecameric Complexes. To investigate the relationship between unfolding and dissociation within the two complexes we monitored simultaneously their conversion from a dodecameric, folded state to a dissociated, unfolded state. To do this we combined mass spectrometric and IM data as a function of increasing Elab (Figure 3). By calculating the percentage of the peak intensity of the dodecamer relative to the peak intensity of the dodecamer and all product ions (undecamer, decamer, etc.) we defined a value for the percentage “intact” complex. Simultaneously we monitored the population of the folded dodecamer by calculating areas under the curves representing the drift time distribution of each conformational ensemble (A-G in Figure 2A for dodecamer HSP16.9 and A-C in Figure 2B for dodecamer SP-1). By computing the percentage of the folded dodecamer (A) over the sum of the areas of all conformational ensembles we obtained a simple quantifier of the folded dodecamer. A plot of the resulting percentage folded dodecamer HSP16.9 (Figure 3A) indicates that at an Elab of 9760 eV, 85% of the dodecameric population remains intact, but only 61% of this population is folded. This means that the unfolding of the dodecamer HSP16.9 takes place at lower Elab than its dissociation into product ions. This observation is in agreement with a previous study on TTR where the wild type tetrameric complex significantly unfolded prior to its dissociation in the gas phase.27 Similarly, we monitored the population of the intact dodecameric SP-1 (27+ ion) and its foldedness as a function of Elab (Figure 3B). The plot shows that between the energies of 270 and 1620 eV the percentage of intact dodecamer SP-1 is very close to the percentage of folded population. This indicates that the gas-phase unfolding of the dodecamer SP-1 27+ ion requires the same energy as its dissociation into product ions. This unexpected result is the first indication that unfolding of the dodecameric SP-1 protein complex plays a pivotal role in its “atypical” dissociation.

Figure 4. MS and MS/MS spectra of the SP-1 protein complex from different solution conditions. (A) MS spectra of the SP-1 protein complex in three different solution conditions: 100 mM ammonium acetate (top panel), 6.25% sulfolane (middle panel), 2.5% DMSO (lower panel).; B) MS/MS spectra of three different charge states of the SP-1 protein complex: 26+ (top panel), 35+ (middle panel), 23+ (lower panel). 12-mer, blue circles; 11-mer, pink circles; 10-mer, green circles; 8-mer, white circles; 6-mer, orange circles; 4-mer, brown circles; 2-mer, yellow ellipses; 1-mer, purple circles. (C) Schematic representations illustrate the dominant dissociation pathways when the SP-1 protein complex is in the three different charge states. The SP-1 26+ ion dissociates via the ejection of 1-mers, 2-mers, and 4-mers, forming 8-mers, 10-mers, and 11-mers. The SP-1 35+ ion dissociates predominantly via the formation of 6-mers. The SP-1 23+ ion ejects 1-mers, generating 11-mers.

In summary, the laboratory frame energies necessary to unfold and dissociate 50% of the intact HSP16.9 (32+ ion) are 10037 and 11413 eV, respectively. For the SP-1 (27+ ion) these values fall to 1571 and 1552 eV. Consequently, the intact dodecamer HSP16.9 32+ ion is seven times more resistant to gas-phase unfolding and dissociation than the dodecamer SP-1 27+ ion. Manipulating Charge State Influences the Dissociative Behavior. To assess whether the number of charges of the SP-1 protein complex could govern its “atypical” dissociation, we manipulated the charge states observed for this protein complex and subjected them to IM-MS/MS at range of different Elab. Addition of dimethylsulfoxide (DMSO) or triethylammonium acetate to solutions of the protein complex reduces the number of charges on the intact SP-1 protein complex.41,42 By contrast, addition of sulfolane (i.e., thiolane 1,1-dioxide) was found to increase the number of charges on the SP-1 protein complex, (41) Lemaire, D.; Marie, G.; Serani, L.; Laprevote, O. Anal. Chem. 2001, 73, 1699–1706. (42) Tjernberg, A. M.; N. Griffiths, W. J.; Hallen, D. J. Biomol. Screening 2006, 11, 131–137.

consistent with previous reports.43 Mass spectra of the intact SP-1 protein complex recorded under these three different experimental conditions are shown (Figure 4A). In 100 mM ammonium acetate the SP-1 protein complex carried between 24+ and 28+ charges (top panel). In 6.25% sulfolane and 2.5% DMSO the SP-1 protein complex ions carried between 28+ and 36+ charges (middle panel) and 20+ and 26+ charges (lower panel), respectively. MS/MS spectrum of the SP-1 35+ ion shows that populations of monomers, dimers, tetramers, hexamers, octamers and decamer are present in the spectrum (Figure 4B, middle panel). Among these different oligomers high intensity hexamers are observed carrying between 17+ and 22+ charges. If we consider the dissociation of the 35+ ion into hexamers which carry 17+ and 18+ charges, these hexamers are half of the mass of the complex and hold half of the charge initially present on the intact protein complex. High charge states dissociating by a symmetrical charge partitioning process is in accord with previous studies of dimers.14 As a control experiment we verified that the charge state per se (43) Lomeli, S. H.; Peng, I. X.; Yin, S.; Loo, R. R.; Loo, J. A. J. Am. Soc. Mass Spectrom. 2010, 21, 127–131.

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and not the sulfolane influenced the dissociative behavior of the SP-1 protein complex by carrying out MS/MS of the same charge state in the presence and absence of sulfolane (SI Figure S-2). These results strongly imply therefore that with high charge a more symmetrical dissociation pattern is observed. Considering now a “reduced” charge state, the MS/MS spectrum of the SP-1 23+ ion shows that populations of monomers and undecamers are present at low and high m/z in the spectrum (Figure 4B, lower panel). Surprisingly at low m/z there are only monomers, no higher order oligomers are present. Dissociation of the 23+ ion results in the formation of monomers with between 5+ and 9+ charges and undecamers with between 15+ and 17+ charges. Interestingly the monomer, which is only 1/12 of the mass of the complex, carries approximately a third of the charges initially present on the intact protein complex. This behavior is often considered “typical” for the collision induced dissociation of protein complexes in the gas phase.11 Even though the SP-1 protein complex is able to dissociate atypically, the reduction of charges in this protein assembly induce it to behave typically, with asymmetric charge distribution between the product ions. We can conclude therefore that the number of charges of the SP-1 protein complex effectively governs its dissociation such that if the SP-1 protein complex has more than 24 charges (i.e., on average there are more than two charges per subunit), it dissociates atypically with ejection of higher oligomers. If it has less than 24 charge units (i.e., on average there are less than two charges per subunit) it dissociates typically through the ejection of monomers. Charge State of the Parent Ion Has a Marked Effect on the Dissociation and Unfolding of the SP-1 Protein Complex. To examine how the number of charges of the SP-1 protein complex influences its “atypical” dissociation, we measured CCSs for several charge states prior to activation and unfolding. For example, the CCS of the SP-1 21+ ion is 6754 Å2 (+/-7%) at 420 eV, the 27+ ion is 6521 Å2 (+/-6%) at 486 eV, the 35+ ion is 6674 Å2 (+/-6%) at 350 eV. All of the CCS measurements are within 9% of the calculated CCS (6200 Å2) obtained using the program Mobcal and taking into account the X-ray crystallographic structure where 1.85% of the amino acids are not present. Thus, the experimental CCS measurements correlate well with the calculated CCS across a range of different charge states prior to activation and unfolding. We also monitored the behavior of the different charge states during dissociation into product ions, monitoring their unfolding as well as the Elab necessary to convert the different charge state of SP-1 to dissociated and unfolded states (Figure 5). Through combining mass spectrometric and IM data as we did previously (Figure 3) we plotted charge states versus Elab required to induce dissociation of 50% of the dodecameric population and unfolding such that only 50% of the folded dodecameric SP-1 protein complex remains (Figure 5). The Elab at which 50% of the dodecamer remains intact, decreases monotonically as the charge state increases. By contrast, the energy at which 50% of the fully folded dodecamer remains, increases until the charge state reaches 24+. After this point, below 24+, the Elab decreases as apparent from the bell-shaped curve (Figure 5). Interestingly therefore we find that if the SP-1 protein complex has less fewer 24 charges (i.e., on average there are less than 9708

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Figure 5. Monitoring the unfolding and dissociation of the SP-1 protein complex as a function of charge state. The Elab, required to induce 50% intact dodecameric SP-1 (black triangles) and 50% folded dodecameric SP-1 (circles), are plotted as a function of the charge states of the complex. A dashed line divides the charge states in which the SP-1 protein complex dissociates typically from those in which the SP-1 protein complex dissociates atypically.

two charges per subunit) the Elab necessary to unfold subunits is less than that required for ejection. By contrast, if the SP-1 protein complex has more than 24 charges, the Elab is closely similar for ejection and unfolding of subunits. DISCUSSION We have shown that the gas-phase dissociation pathways of the dodecameric HSP16.9 and SP-1 protein complexes, which might be expected to behave similarly given their ring-shape topology, are in fact markedly different. The HSP16.9 protein complex dissociates in a typical way, through the sequential ejection of unfolded monomers with asymmetric charge partitioning. By contrast, the SP-1 protein complex dissociates atypically through ejection of monomers, dimers, tetramers, and hexamers with an almost symmetric charge partitioning. This atypical pathway was found to be highly dependent on the charge state of the parent ion. The charge state of the parent ion has been found to influence the dissociation behavior of tetrameric TTR.28 Specifically lowly charged TTR tetramers do not dissociate into subcomplexes (i.e., monomers and trimers), but fragment generating C-terminal peptide fragments. Furthermore, only two protein complexes have been reported that dissociate atypically in the absence of charge manipulation agents or other small molecule additives. In both cases, the intact complex produced dimeric product ions rather than the typical highly charged monomers.5,29 In the first case, tetrameric 2-keto-3-deoxyarabinonate dissociated into monomers, dimers, and trimers. It was suggested that this tetramer is relatively unstable in the gas phase, when compared to the tetramer arabinose dehydrogenase. It was proposed that the atypical dissociation pathway of this tetramer was due to one dimer-dimer interface being much smaller than the other and due to the gas-phase instability of the asymmetric structure of this protein complex.5 In the second case the heterohexameric textilotoxin complex was found to dissociate through the loss of noncovalently bound dimers with symmetric charge partitioning.29 It was suggested that the reason for this atypical dissociation is the inability of subunits to unfold, due to the presence of several intramolecular disulfide bonds. This hypothesis is in agreement with two previous studies of homodimers in which the use of intermolecular cross-linkers rendered monomers more resistant to unfolding and the presence of disulfide bonds, caused the

homodimers to dissociate in such a way that the charge partitioning is symmetric.14,44 Here we consider three possible explanations for the atypical dissociation of the SP-1 protein complex and its implications for the use of CID data for determining the structural details of multiprotein complexes. First, the SP-1 protein complex is in a “natural pre-unfolded state” which makes it ready for dissociation. Second, due to numerous charges per surface area, the SP-1 protein complex is apt to dissociate when it is in certain charge states; charge repulsion reduces the energetic gap between unfolding and ejection, such that it is not necessary for monomers to unfold prior to the ejection of subunits. Third, the SP-1 protein complex is unable to unfold physically due to a particular quaternary structure. The first, simple possible explanation is that this protein complex is “pre-unfolded” in the gas phase prior to activation and unfolding.45 To test this hypothesis we compared the experimental CCS measurements, made on several charge states of the SP-1 protein complex prior to unfolding and reported in the results section, to the CCS calculated by the program Mobcal. All the experimental CCS measurements of the charge states of the SP-1 protein complex are within a few percent of the calculated CCS. If unfolding takes place, the difference between measured and calculated CCS would be on the order of at least 30%.15 Therefore, we can rule out the hypothesis of a preunfolded state of the SP-1 protein complex. A second explanation for the atypical dissociative behavior of the SP-1 protein complex is related to the fact that the charge repulsion within the protein complex ion could be one of the most relevant driving forces for the dissociation in the gas phase. To explore this possibility we calculated the solvent excluded surface (SES) areas of both complexes.40 The SES areas of the HSP16.9 and SP-1 protein complexes are 67 151 Å2 and 43 993 Å2, respectively, and we calculated charges per unit area (Å2). The dodecameric SP-1 27+ ion has 0.6 10-3 charges per unit surface area; by contrast, the dodecameric HSP16.9 32+ ion has only 0.4 10-3 charges per unit surface area. Thus, the amount of charge per unit area is 50% greater for the SP-1 protein complex than for the HSP16.9 protein complex. This lends support to the notion that high surface charge can cause atypical dissociation. Consistent with this, the tetrameric 2-keto-3deoxyarabinonate (PDB code 3bqb), whose dissociation is atypical,5 has a SES area of 43 379 Å2 and the 27+ ion, analyzed in that study,5 has 0.6 10 -3 charges per unit surface area, which is also the value determined here for the SP-1 protein complex. That charge is an important factor in governing atypical dissociation of the SP-1 protein complex is further supported by our observation that without any additives this protein complex behaves atypically ejecting dimers and tetramers (Figure 4B, top panel). However, if we further increase the number of charges on the SP-1 protein complex, we promote atypical behavior such that hexamers are ejected with an almost symmetric charge distribution (Figure 4B, middle panel). By contrast, dramatically decreasing the number of charges on the SP-1 protein complex induces it to behave typically, ejecting monomers with asymmetric (44) Schenauer, M. R.; Leary, J. A. Int. J. Mass Spectrom. 2009, 287, 70–76. (45) Suckau, D.; Shi, Y.; Beu, S. C.; Senko, M. W.; Quinn, J. P.; Wampler, F. M., 3rd.; McLafferty, F. W. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 790–793.

charge partitioning (Figure 4B, bottom panel). In this way, the data on the SP-1 protein complex shown here are consistent with previously reported studies of protein homodimers, where lower charge states dissociate via the typical asymmetric process, whereas high charge states dissociate through a more-symmetric one.14 Furthermore, the findings presented here are consistent with recent data for the charge-reduced TTR,28 where the difference in ion energy required for unfolding and dissociation decreases as precursor ion charge is increased. It is also important to note that several aspects of this study contrast those of our previous study on charge-reduced TTR, illustrating the influence of complex-specific structural factors on the dissociation pathways. For example, low charged (e.g., 19+ and 20+) dodecameric SP-1 dissociate via the typical asymmetric charge partitioning mechanism, where low charged TTR precursor ions either dissociate more-symmetrically into “folded” monomers or undergo covalent fragmentation to release peptide ions from the intact assembly. This specific difference between the results on TTR and on the SP-1 protein complex is likely due to natively “supercharged” charge states observed for the latter complex. We further speculate that high charge repulsion on the SP-1 protein complex reduces the energetic gap between unfolding and ejection. This hypothesis is consistent with a theoretical study using constrained molecular dynamics (MD) calculations,46 which showed that the dissociation of a protein complex competes with the unfolding of a monomer, and that the charge partitioning within the protein complex contributes to determine which of these two pathways prevails. In summary, the data on the SP-1 protein complex indicate that charge state of this protein complex plays a pivotal role in controlling which dissociation pathway takes place. Finally, we consider the third interpretation of the atypical dissociation of the SP-1 protein complex and relate our findings to its quaternary structure. We note that the SP-1 protein complex is both compact and highly sensitive to acceleration in the gasphase, which induces dissociation under relatively mild conditions. We hypothesize that significant protein unfolding in any one subunit of the SP-1 protein complex is not physically possible if the protein complex is still retaining critical protein-protein contacts. Our hypothesis accords with that proposed for the atypical dissociation of heterohexameric textilotoxin which was attributed to inability to unfold.29 To test this hypothesis on resistance of the protein unfolding, we examined the crystal structures of the SP-1 and HSP16.9 protein complexes (PDB codes 1tr0 and 1gme, respectively, see SI). At first glance we observed several similarities between them since both are toroidal, dodecameric protein complexes formed by dimeric building blocks (Figure 1). If we consider the location of the N- and C-termini as key factors that initiate unfolding in the gas phase we find that the HSP16.9 protein complex could unfold by charge migration relatively easily since all the C-termini lie on the surface of the complex.11 Presumably they can be peeled away independently without perturbing the rest of the complex (Figure 1A). Furthermore, the C-terminus of each monomer is distant from N-terminus and, therefore, there is no interaction between the N- and C-termini of each monomer (SI Figure S-3A). Upon activation the HSP16.9 monomer could start to unfold, (46) Wanasundara, S. N.; Thachuk, M. J. Phys. Chem. A 2009, 113, 3814–3821.

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probably from its C-terminus. As it leaves the surface of the complex it will be able to accept migrating charges,17 and charge repulsion will then further drive the C-terminus away from the rest of the monomer. When the charge repulsion between the unfolded monomer and the rest of the complex surmounts the intersubunit stabilizing interactions, dissociation of the monomer from the complex will take place. Conversely, the structure of the SP-1 protein complex has two structural features that could prevent the ready unfolding of subunits. First, all the N-termini of the SP-1 protein complex are toward the central cavity formed by the dodecamer, and all the C-termini are buried inside the structure (Figure 1B). The N- and C-termini are woven into the protein in such a way (e.g., forming a middle strand in a β sheet) that they cannot be exposed to the surface without the near complete unfolding of the protein. Second, the N-terminus of each monomer is within a distance of 3.5 Å of the C-terminus, being at the ends of neighboring strands in the same beta-sheet and enabling strong interactions between the N-and C-terminus of each chain (SI Figure S-3B). Furthermore, the N- and C-termini of each monomer are in contact with those belonging to neighboring chains. For instance, Tyr-108 is the last amino acid of each polypeptide, and forms a very strong hydrogen bond between adjacent monomers, for example, Tyr-108 of chains A and D (SI Figure S-3B). In addition to being in contact with chain D, chain A also contacts chains B, K, and L. A very tight network of interactions within the SP-1 protein complex is therefore possible and does not allow the terminal residues to become exposed on the surface of this protein complex to initiate unfolding.

In particular, the structural properties of the SP-1 protein complex distinguish it from most other complexes that have been studied to date. Resistance to unfolding and high charge density likely contribute to the atypical gas-phase dissociation of the SP-1 protein complex. It is clear from this study that quaternary structure and charge density are two key factors that could be used to predict or alter the CID properties of macromolecular assemblies. An enhanced understanding of the mechanism of CID will undoubtedly pave the way for obtaining structural information on subunit interactions and packing from gas-phase CID data. Furthermore, the results presented here illustrate the clear analytical utility of charge manipulation (both “supercharging” and “charge-reduction”) in the analysis of multiprotein complexes by IM-MS. Based on the data presented here, it is likely that such charge manipulation will become a fundamental part of IM-MS protocols aimed at determining the quaternary structure of multiprotein complexes.

CONCLUSIONS The data presented here illustrate how the quaternary structure of protein complexes can strongly influence gas-phase dissociation.

Received for review July 5, 2010. Accepted October 15, 2010.

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ACKNOWLEDGMENT We gratefully acknowledge Elizabeth Vierling (University of Arizona), Orna Almog (Ben-Gurion University, Israel) for supplying purified HSP16.9 and SP-1. S. Teichmann and J. P. Benesch are acknowledged for critical reading of the manuscript. B.T.R acknowledges support from Waters Corp. in the form of a Waters Research Fellowship. C.V..R is a Royal Society Professor. SUPPORTING INFORMATION AVAILABLE Full description of the crystal structures of the SP-1 and HSP16.9 protein complexes, Figures S-1-S-3. This material is available free of charge via the Internet at http://pubs.acs.org.

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