Distinguishing Loss of Structure from Subunit Dissociation for Protein

Article pubs.acs.org/ac

Distinguishing Loss of Structure from Subunit Dissociation for Protein Complexes with Variable Temperature Ion Mobility Mass Spectrometry Kamila J. Pacholarz†,‡ and Perdita E. Barran*,‡ †

University of Edinburgh, School of Chemistry, West Mains Road, Edinburgh EH9 3JJ, United Kingdom University of Manchester, School of Chemistry, Manchester Institute of Biotechnology, Michael Barber Centre for Collaborative Mass Spectrometry, 131 Princess Street, Manchester M1 7DN, United Kingdom

‡

Downloaded by STOCKHOLM UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): June 3, 2015 | doi: 10.1021/acs.analchem.5b01063

S Supporting Information *

ABSTRACT: The thermal stability and strength of interactions in proteins are commonly measured using isothermal calorimetry and differential scanning calorimetry providing a measurement that averages over structural transitions that occur as the proteins melt and dissociate. Here, we apply variable temperature ion mobility mass spectrometry (VT-IMMS) to study the effect of temperature on the stability and structure of four multimeric protein complexes. VT-IM-MS is used here to investigate the change in the conformation of model proteins, namely, transthyretin (TTR), avidin, concanavalin A (conA), and human serum amyloid P component (SAP) at elevated temperatures prior, during, and after dissociation up to 550 K. As the temperature of the buffer gas is increased from 300 to 350 K, a small decrease in the collision cross sections (DTCCSHe) of protein complexes from the values at room temperature is observed, and is associated with complex compaction occurring close to the reported solution Tm. At significantly higher temperatures, each protein complex undergoes an increase in DTCCSHe and in the width of arrival time distributions (ATD), which is attributed to extensive protein unfolding, prior to ejection of a highly charged monomer species. This approach allows us to decouple the distinct gas phase melting temperature (Tm) from the temperature at which we see subunit dissociation. The thermally induced dissociation (TID) mechanism is observed to initially proceed via the so-called “typical” (CID) dissociation route. Interestingly, data collected at higher analysis temperature suggests that the TID process might be adapting more “atypical” dissociation route.

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other structural techniques, such as NMR or X-ray crystallography. The first reports of CID on protein multimeric complexes such as tetrameric streptavidin, dimeric cytochrome c or tetrameric TTR, showed asymmetrical charge partitioning among subunits upon complex activation.12−14 This asymmetrical charge partitioning results in a marginally destabilized subunit, resulting from intramolecular Coulombic repulsions.15 Upon collisional activation of the complex, one or more subunits begin to unfold. As the new surface area is exposed, charge migration occurs to that region and drives further unfolding that eventually results in release of highly charged monomer.16 This has been described as “typical” CID behavior.17 The conjunction of ion mobility and CID allows concurrent probing of conformational change during the CID process. Hall et al. recently delineated a second type of CID behavior, where dissociation routes without unfolding and termed it “atypical” CID17 where lower charged monomers, as well as higher order subunit complexes are ejected, potentially with more relation to the native solution topology.18

anoelectrospray ionization (nESI), coupled to mass spectrometry (MS), allows the analyst to isolate and analyze conformations and stoichiometries presented by protein complexes that would be averaged in a solution phase experiment, and further to elucidate intrinsic intramolecular interactions. Over the past two decades, mass spectrometry has become an important tool for characterization of proteins and protein complexes’ native structure.1−5 With a careful choice of solution and desolvation conditions, nESI-MS is capable of reflecting solvent conditions in the gas phase or probing changes induced in solution due to pH, solvent composition, or temperature.6−9 Information on subunit composition and topology of multimeric protein complexes can be obtained by activating protein ions in the gas phase to induce dissociation of these subunits. The most commonly used activation method is collision induced dissociation (CID) achieved via energetic collisions of the ion with neutral gas. Nevertheless, other activation methods exist, such as surface induced dissociation (SID),10 blackbody infrared radiative dissociation (BIRD),11 or via application of elevated injection or acceleration voltages in the mass spectrometer. Inclusion of the related technique of ion mobility to mass spectrometry (IM-MS), gives further conformational information in the form of mass selected collision cross sections (CCS). These can be later compared with theoretical CCS calculated from coordinates obtained via © 2015 American Chemical Society

Received: March 19, 2015 Accepted: May 20, 2015 Published: May 20, 2015 6271

DOI: 10.1021/acs.analchem.5b01063 Anal. Chem. 2015, 87, 6271−6279

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Analytical Chemistry

Figure 1. Mass spectra of (a) transthyretin (TTR) and (b) human serum amyloid P component (SAP) acquired at several discrete buffer gas temperatures illustrating thermally induced dissociation (TID) of complexes in the gas-phase. TID mass spectra of avidin and concanavalin A are available in the Supporting Information, Figure S2.

of the buffer gas temperature.34 Although other IM-MS instruments with temperature control exist, allowing work either at cryogenic or elevated temperatures, their mass working range is limited.35,36 In this study, we examine the effect of elevated drift cell buffer gas (He) temperature on four multimeric protein complexes sprayed from native solution conditions at temperatures from 300 K up to 550 K and report thermally induced dissociation (TID) of subunits in the gasphase. VT-MS experiments were performed on four protein complexes: tetrameric transthyretin (TTR, 55 kDa), avidin (64 kDa), and concanavalin A (conA, 103 kDa) and a pentameric human serum amyloid P component (SAP, 125 kDa) (Figure S1) on our in-house modified DT-IM-MS instrument (MoQToF).34

Wysocki et al. have employed surface induced dissociation (SID) and demonstrated conditions that can achieve dissociation of complexes without any significant unfolding of subunits prior to dissociation.10,19 Additionally, in IM-MS experiments, ions can be activated via higher energy collisions which occur during transfer into the drift cell regions against the positive buffer gas pressure. High injection energies can be used to effect structural changes and dissociation or fragmentation of the ions20 and has been demonstrated on several molecular systems.21−23 Williams and co-workers have widely investigated the dissociation of monomeric proteins using BIRD,24−26 while the first application of BIRD to multimeric protein complexes was reported by Klassen et al. on a pentameric Shiga-like Toxin I.27 Asymmetric dissociation into monomers and tetramers was observed along with a remarkably large Arrhenius parameter, suggested to be indicative of the dissociation mechanism proceeding via the unfolding of a monomeric subunit. Further BIRD experiments on both Shiga-like Toxin I and streptavidin, found that the dissociation pathway is dependent on temperature, with the loss of a single subunit dominant at higher reaction temperatures, and fragmentation of the backbone dominant at lower reaction temperatures and longer reaction times.28,29 For a 32 kDa protein homodimer (Ecotin) Klassen et al. report no fragments from the low native-like charge states with cell temperatures of up to 175 °C and reaction times as long as 300 s. In order to obtain fragmentation, the protein solution was acidified to provide solution disrupted structures.30 Jarrold et al. first reported the use of VT-IM-MS to probe the temperature dependent conformations adopted by proteins in the gas phase. We have recently extended that study to examine other monomeric systems.31,32 Moreover, Bowers and coworkers applied VT-IM-MS to study unfolding and dissociation of amyloid β-protein peptide multimers.33 Our in-house modified linear drift tube ion mobility mass spectrometer features heating and cooling capabilities and allows for control

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EXPERIMENTAL METHODS Preparation of the Protein Complex Solutions. Avidin from egg white (A9275) and concanavalin A from Canavalia ensiformis (C2010) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Human TTR was obtained from SCIPAC (Sittingbourne, U.K.); human SAP was obtained from CalBioChem (Darmstadt, Germany). Ammonium acetate (AmAc) was obtained from Fisher Scientific (Loughborough, U.K.). 30 μM protein complex solutions were prepared in 200 mM AmAc (pH 6.8), and the buffer was exchanged to 100 mM AmAc on the day of analysis, using Biospin-6 columns (BioRad, U.S.A.). Variable Temperature (Ion Mobility) Mass Spectrometry Experiments. Mass spectra were recorded on an inhouse modified Q-TOF (Waters, Manchester, U.K.) to include a 5.1 cm drift cell with buffer gas temperature control.34 The source conditions were adjusted to preserve noncovalent interactions (Figures 1 and S2). Each VT-MS experiment was performed in triplicate and each VT-IM-MS was performed in duplicate; the average values are reported. Further instrumental details, data analysis and DTCCSHe calculations are described in the SI and elsewhere.32 6272


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Analytical Chemistry Gas-Phase Protein Complex Dissociation Curves from VT-MS Data. The ratio of the summed intensities of charge states of the intact complex [Icomplex] over the sum of the summed intact complex charge state intensities and summed intensities of monomer charge states resulting from the thermal dissociation [Icomplex + Imonomer] was plotted as a function of buffer gas temperature and the data points fitted with a sigmoidal function. For complexes not fully dissociated at 550 K (conA and SAP), this fit was extended for the dissociation ratio to approach 0. From this approach we obtain a parameter termed TGPD, the apparent temperature at which 50% of the protein has dissociated. To establish if the complex had reached thermal equilibrium in our drift cell, we varied the drift voltage which altered the time each complex resides in the drift cell. The TGPD is seen to decrease slightly with longer drift times for all of the proteins (Figure S3). Data points were fitted with an exponential decay function to determine a dissociation equilibrium temperature (Teq) for each complex (Figure S4). Measurements taken at the lowest DV are the closest to the Teq as can be achieved with current instrumentation. Data acquired at the longest drift time available was used to construct the dissociation curves. Calculations of Interfaces and Noncovalent Interactions Based on X-ray Structures. Calculations of surface interface area, possible number of hydrogen bonds and salt bridges of TTR and avidin were carried out using the online ‘Protein interfaces, surfaces and assemblies’ service PISA at the European Bioinformatics Institute Web site (http://www.ebi. ac.uk/pdbe/prot_int/pistart.html).37,38 Three structures of TTR (PDB: 1BMZ, 1DVQ, 3U2I) and avidin (PDB: 1AVE, 1VYO, 1RAV) deposited in the Protein Data Bank were used and the average values for each protein are reported.

broader charge state distribution than TTR, centered on [P + 24H]24+ (∼m/z 5330; Figure 1b). Decameric species are also present (as previously reported40) and remain observable until above 550 K. Mass spectra acquired at temperatures between 300 and 500 K, show a gradual decrease of the decameric SAP population. Remarkably, no dissociation of SAP pentamer into monomers is observed below 500 K. Above 500 K, pentameric SAP begins to dissociate into monomeric species centered on [M + 11H]11+ (∼m/z 2320), as shown in the top spectrum in Figure 1b. As for TTR, asymmetric charge partitioning is observed, implying unfolding of the subunit(s) prior to complex dissociation. Similar unfolding and dissociation trends have been observed for tetrameric avidin and concanavalin A (data and further discussion in SI, Figure S2). Gas-Phase Protein Complex Dissociation Curves. From our thermal dissociation mass spectra (Figure 1), we can compare TGPD parameter found for each protein complex to Tm values from DSC measurements (Figure 2 and Table 1).

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RESULTS Variable Temperature Mass Spectrometry (VT-MS) of Protein Complexes. The mass spectra of each complexes under investigation were recorded at temperatures ranging from 300 to 550 K at 20−50 K intervals and present relatively narrow charge state distributions indicating a well preserved gas-phase structure (Figures 1 and S2). Mass spectra of TTR and SAP at progressively higher temperatures are shown in Figure 1. TTR, a 55 kDa homotetrameric protein, is present in a narrow charge state distribution centered on [T + 14H]14+ (∼m/z 3950). Upon increase of temperature to 400 K, the TTR complex remains intact as a tetramer during the ∼4.1 ms it spends in the drift cell. Above 450 K, dissociation of this tetrameric complex into monomers is observed. Several monomeric species are now present in the m/z 1200−2400 region of the mass spectrum, with [M + 8H]8+ (∼m/z 1735) being the most abundant. Above 550 K, only a small amount of tetrameric species remains as seen in the top mass spectrum in Figure 1a, dominated by TTR monomers with 6+ ≤ z ≤ 10+. Moreover, low charge trimeric species, centered on [Tr + 6H]6+ (∼m/z 5900), are present. We assume that charge is more or less evenly distributed among the subunits at 300 K. At elevated temperatures, when the dissociation occurs, the ejected monomer carriers over 50% of the total charge. This “asymmetric” charge partitioning suggests that the subunit undergoes unfolding prior to dissociation as observed in CID experiments.39 Pentameric SAP requires a higher temperature for the onset of dissociation to occur. The 125 kDa SAP presents a slightly

Figure 2. Temperature induced dissociation (TID) curves of avidin (gray), TTR (green), conA (red), and SAP (purple) for TGPD determination, determined near the equilibrium drift time (DV = 15 V for TTR, avidin, and conA; DV = 25 V for SAP).

Table 1. Literature in-Solution Melting Temperature Tm and Gas-Phase Dissociation Temperature TGPD Determined for the Protein Complexes Investigated protein complex

molecular mass (kDa)

number of subunits

Tm (K)

TGPD (K)

TTR Avidin ConA SAP

55 64 103 125

4 4 4 5

370.95 356.70 338.00 ± 6.5 359.2−362.2

467.8 474.0 520.9 521.6

The TGPD of TTR was determined to be 474.7 ± 1.6 K. The reported literature Tm of TTR is 370.95 K, determined by DSC,41 which is over 100 K lower than TGPD found via VT-MS experiments. The TGPD increases for the other complexes were found to be 474.3 ± 2.5 K, 536.1 ± 0.3 K and 534.1 ± 1.5 K for avidin, conA, and SAP, respectively. ConA exists in equilibrium between its dimeric and monomeric form, so for the purpose of calculating the dissociation ratios we have considered only the [M + 15H]15+ to [M + 11H]11+ (∼m/z 1715−2340) which are produced during the TID as opposed to the naturally occurring [M + 10H]10+ to [M + 8H]8+ monomers (∼m/z 2560−3200; Figure S2b). As we are unable to mass select parent ions prior 6273


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to DTCCSHe, determined at 300 K, implying a collapse from room temperature species. For TTR the median DTCCSHe decreases from 31.3−33.5 nm2 to 30.7−32.3 nm2 at 350 K and further to 30.5−32.3 nm2 at 400 K. The extent of the collapse is greater for higher charge states (Figure 4a bottom). At 450 K, the DTCCSHe of high charge states increases and is attributed to one or more subunits unfolding; while [T + 13H]13+ remains compact. Around TTR’s TGPD, a significant increase in DT CCSHe is observed and the median DTCCSHe range across charge states increases to 38.3−42.0 nm2 at 475 K and further to 39.5−47.0 nm2 at 500 K. The relative change in the median DT CCSHe with respect to the median DTCCSHe at 300 K is more extensive for higher charge states reaching +40.3% for [T + 15H]15+. The DTCCSHe of the dissociated highly charged monomer has been measured at temperatures near TGPD and above and found to be 16.8−22.6 nm2 at 475 K similar to the CCS previously reported for a CID dissociated monomer,10,42 implying extensive subunit unfolding prior to TID. The SAP pentamer follows a similar pathway of compaction, unfolding and dissociation. At 350 and 400 K a minor decrease in the median DTCCSHe is observed for the [P + 22H]22+ to [P + 25H]25+. The median DTCCSHe across charge states changes from 59.1−63.7 to 58.9−66.4 nm2 at 350 K, with −3.65% reduction in the median DTCCSHe for [P + 22H]22+ and only −0.39% reduction for [P + 25H]25+. The compaction progresses further at 400 K to 58.8−71.9 nm2. In contrast to TTR, lower charge states experience a greater degree of compaction; by contrast the [P + 26H]26+ exhibits a slight increase in DTCCSHe at 350 K (+2.71%). The behavior can be attributed to a collapse of the subunits of SAP into the central cavity.43 This compaction path could be attributed to stabilizing or destabilizing effects of charge interactions on the protein ion’s surface and collapse of high charge states may be also mitigated by Coulombic repulsion.43,44 The DTCCSHe for all


to VT-IM-MS due to instrument layout, the dissociation curve for SAP has been contracted assuming the dissociated monomer is originating from the SAP pentamer only. We find that the TGPD parameter is not only dependent on the mass of the complex, but is sensitive to noncovalent interactions between subunit interfaces as discussed later. Variable Temperature Ion Mobility Mass Spectrometry. IM-MS experiments were performed at temperature from 300 to 500 or 550 K, dependent on the TGPD determined earlier. We present DTCCSHe distributions (DTCCSDHe) derived from arrival time distributions (ATD) of TTR (Figure 3) and

Figure 3. DTCCSDHe of TTR ([T + 13H]13+, [T + 14H]14+, and [T + 15H]15+) acquired at buffer gas temperatures ranging from 300 to 500 K. DTCCSDHe for the other proteins systems investigated are available in the SI, Figure S5−S7.

SAP (Figure S5) acquired at range of temperatures. In Figure 4, median DTCCSHe (nm2) of TTR tetramer and TTR dissociated monomer (a), and SAP pentamer and SAP dissociated monomer (b) are plotted as versus buffer gas temperature (K). Ion mobility data acquired at 350 and 400 K show a minor decrease in the DTCCSHe of both TTR and SAP in comparison

Figure 4. Median DTCCSHe in nm2 as a function of the buffer gas temperature (K) for different charge states of (a) TTR and (b) SAP (top panel), and the percentage change in DTCCSHe with respect to the DTCCSHe at 300 K (bottom panel). VT-IM-MS measurements illustrate an initial decrease in the DTCCSHe associated with complex’s minor compaction and latter significant increase in DTCCSHe as protein subunits begin to unfold and subsequently dissociate. Data for avidin and conA is available in the SI, Figure S8. 6274


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Analytical Chemistry charge states increase above 450 K to 77.4−89.7 nm2 at 550 K. The DTCCSHe of the highly charged ejected monomer is again similar to the one determined through CID experiments on SAP, ranging from 25.7−34.2 nm2 at 500 K for [M + 10H]10+ to [M + 13H]13+.10 Similar behavior is found for both avidin and conA (Figures S6−S8). Both complexes experience compaction at 350 and 400 K and subsequent unfolding at temperatures above 450 K shown by an increase in the DTCCSHe. As for TTR, initial compaction is more extensive for the higher charge states, and so is unfolding. It is plausible that this phenomenon is dictated by the size of the cavity in-between protein subunits and this will be discussed later. Width of CCS Distribution (DTCCSDHe) Derived from the Experimental Arrival Time Distribution (ATD) vs Temperature. The distribution of CCS (CCSD) derived from the experimental ATD provides detail on protein flexibility and the amount of closely related or interconverting conformations. This is illustrated in Figure 5, and it is apparent that the higher Downloaded by STOCKHOLM UNIV on September 11, 2015 | http://pubs.acs.org Publication Date (Web): June 3, 2015 | doi: 10.1021/acs.analchem.5b01063

temperature near TTR TGPD at which 50% of the complex has dissociated, the DTCCSDHe of all three charge states increases significantly and nearly doubles for [T + 13H]13+. As the complex dissociates, the protein subunits unfold prior to their ejection. We anticipate various subunits to be unfolding and/or remaining compact to a diverse extent, resulting is many closely related conformational families, hence broad DTCCSDHe. After crossing the dissociation temperature barrier, the DTCCSDHe narrows. Unlike with TTR, the DTCCSDHe of SAP pentamer does not decrease from 350 to 400 K (Figure 5b). The DTCCSDHe for the [P + 22H]22+ and [P + 24H]24+ increases gradually with temperature and peaks around 550 K , that is, near SAP TGPD. The [P + 26H]26+ charge state behaves somewhat differently and a decrease in DTCCSDHe is observed at 450 K. Results for avidin and conA, which exhibit broadly similar trends, are shown in the SI (Figure S9). Noncovalent Interactions between Protein Subunits. As noted above, multimeric protein complex dissociation is highly controlled by the strength of noncovalent interactions between subunit interfaces.45 Here, for comparison we have chosen TTR and avidin, two protein complexes relatively close in mass with similar TGPD. The average interface surface area per subunit, average number of hydrogen bonds per subunit and average number of possible salt bridges per subunit have been calculated using the “Protein interfaces, surfaces and assemblies” service PISA at the European Bioinformatics Institute (Table 2). Despite a higher number of noncovalent Table 2. Comparison of TTR and Avidin Dissociation Temperature TGPD, Average Interface Surface per Subunit, Average Number of Hydrogen Bonds per Subunit, Average Number of Possible Salt Bridges per Subunit and the Percentage Increase in the DTCCSHe at 475 K protein complex mass (kDa) TGDP (K) avg interface surface per subunit (nm2) avg number of H-bonds per subunit avg number of salt bridges per subunit percentage increase in the median DT CCSHe at 475 K

Figure 5. Baseline width of DTCCSDHe of TTR SAP (b) acquired at buffer gas temperatures ranging from 300 to 550 K. It is shown how the DTCCSDHe width is dependent on the charge state with higher charge states having broader DTCCSDHe. The width of DTCCSDHe varies significantly with the temperature of the analysis environment. In the TTR plot (a), DTCCSDHe width data points at 475 K for [T + 13H]13+ and [T + 14H]14+ are overlapping; as are DTCCSDHe width data points at 500 K for all TTR charge states.

TTR

Avidin

55 474.7 ± 1.6 17.11 ± 2.28

64 474.3 ± 2.5 25.63 ± 0.50

20 ± 2

36 ± 8

1+1

7±5

[13+] [14+] [15+]

+22.5% +25.0% +25.4%

[14+] [15+]

+8.7% +15.9%

interactions between the subunits, avidin unfolds to a lesser extent near the TGPD compared to TTR. The most abundant charge state of TTR [T + 14H]14+ has increased its DTCCSHe by +25.0% at 475 K in comparison to avidin with only +15.9% increase in DTCCSHe for [T + 15H]15+. This suggests that the noncovalent interactions between TTR subunits are stronger, in comparison to avidin, as they are holding the subunit within the complex allowing for extensive unfolding prior to dissociation.

the charge state, the broader the DTCCSDHe becomes and also that the width also varies with the temperature. For the [T + 13H]13+ ion of TTR, the DTCCSDHe becomes narrower at temperatures between 350 and 450 K (Figure 5a). This correlates with a decrease in DTCCSHe. As the complex attempts to adopt the most compact structure, the limited range of conformational combinations is reflected by this narrow DT CCSDHe. The width of the other two charge states fluctuates slightly within the discussed temperature range. At 475 K, a

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DISCUSSION Loss of Structure is Decoupled from Dissociation. Insolution studies allow measurement of melting temperatures, that is, the onset of protein transformation; however, they cannot distinguish between loss of structure and complex dissociation, nor do they distinguish in detail the route by 6275


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Analytical Chemistry which structure is lost. The difference between Tm and TGPD was over 100 K for each of the protein complexes investigated here; moreover, dissection of the two processes has revealed a pathway for dissociation that proceeds via a collapsed state. Our high values for TGPD compare favorably with the findings of Klassen et al., who were unable to dissociate the low charge states of a 32 kDa protein Ecotin in the gas phase at 175 °C even with 300 s of irradiation.30 The TID charge state dissociation pathways for these complexes are similar to the dissociation profiles from CID experiments reported by others.8,17,42,44 We postulate that CID and TID activate the complexes in a very similar fashion. Correlating TID temperatures with CID energies could serve as a framework for thermal stability screening, especially useful when a limited amount of material is available to perform DSC measurements. As an illustration, about 725 eV (Elab, based on data reported by Pagel et al.42) is required to achieve 50% dissociation of the TTR tetramer, which compares to TGPD of ∼476 K. Upon Temperature Increase Multimeric Protein Complexes Generally Undergo Initial Compaction and Subsequent Unfolding Prior to Dissociation Similarly to the CID Process. CID of protein complexes proceeds by initial compaction of the complex and subsequent unfolding of subunits.43,46 For statistical reasons, one subunit might carry slightly more charge than the others. This asymmetrical charge partitioning results in a marginally destabilized subunit, resulting from intramolecular Coulombic destabilization.15 Upon collisional activation, one or possibly multiple subunits begin to unfold and as the new surface area is exposed, charge migration occurs to that region and drives further unfolding which eventually results in the release of highly charged monomer.16 We also observe release of highly charged monomers as a result of elevated buffer gas temperature, reminiscent of CID behavior. VT-IM-MS data shows a minor decrease in the DTCCSHe of the four protein complexes in comparison to the DTCCSHe determined at 300 K, suggesting a structural compaction. This decrease in the DTCCSHe is attributed to loss of structure and would arise from a collapse of the native structure into the intrasubunit cavities as well as collapse within each subunit. IMMS data shows that the naturally occurring monomer of conA (see SI) follows the same pathway of collapse (at 350 and 400 K) and unfolding above 450 K, suggesting that compaction is not only originating from the intrasubunit cavity collapse but also from significant collapse within each subunit attributable to a loss of hydrophobic effects. The temperature at which maximum compaction occurs is comparable to the Tm obtained from in-solution studies. The DTCCSHe of the ejected highly charged monomers have been obtained at temperatures near TGPD and above; for all four complexes, the large values are indicative of unfolded subunits as for CID.10 Complexes become more compact due to loss of structure, and as the subunits unfold, charge migration occurs and eventually results in the ejection of a highly charged unfolded monomer. Degree of Compaction and Unfolding Varies Across Charge States. Past compaction a gradual increase in DT CCSHe was noted across all charge states for all complexes investigated in this study. Some experience close to a 50% increase in DTCCSHe in comparison to those found at 300 K. A common trend was observed; higher charge states experience a greater degree of extension and require less energy (in the form

of collisions) for the onset of unfolding. This phenomenon is believed to be influenced by electrostatics but also suggests that each charge state represents different forms of the complex. The degree of compaction varied among the protein complexes. Low charge states of SAP, a complex with relatively large internal cavity (Figure S1b), experience greater compaction, in agreement with MD and CID experiments reported by Hall et al.,43 whereas the highest charge state does not undergo any compaction. The compaction pathway is believed to be suppressed due to competing electrostatic effects,44 whereas the lower charge states collapse, presumably into the internal cavity. The other three complexes studied here, display an opposite trend, that is, greater extent of compaction for higher charge states. These complexes have significantly smaller intrasubunit cavities compared to SAP. We speculate that the majority of compaction observed must be coming from a collapse/loss of structure within the subunits themselves. An additional explanation is that lower charge states are already quite compact at 300 K. Upon exposure to higher temperature, the total change for the lower charge state is relatively low whereas higher charge states with marginally larger DTCCSHe at 300 K, collapse to the same extent as lower charge states; however, the relative change observed is greater. Broadening of the DTCCSDHe Varies with Increasing Buffer Gas Temperature Suggesting the Presence of Multiple Closely Related Conformers Upon Complex Dissociation. The width of a CCSD can provide information on protein flexibility and on closely related or interconverting conformations.47 It was noted that the higher the charge state, the broader the DTCCSDHe and that the width also varies with temperature. At 350 and 400 K, the DTCCSDHe either slightly decreases or remains almost constant. At these temperatures, each protein complex is losing its quaternary structure (as indicated by the decrease in DTCCSHe), collapsing into its internal cavity, adopting a kinetically trapped gas phase conformation. The structure becomes “locked” and has a minimum number of conformers present (as shown by the decrease in the width of the ATDs). When the subunits begin to unfold at 450 K and continue to do so at higher temperatures, a significant increase in the DTCCSDHe is observed. Here subunits are both unfolding and remaining collapsed to a diverse extent, this results in many closely related conformational families. Pagel et al. have reported the presence of five intermediate resolvable conformers of TTR tetramer at various CID activation energies.42 After reaching the TGPD barrier and the widest DTCCSDHe, the values begin to decline. At this point, most of the lower energy intermediate conformations should have transformed into high energy conformers at the edge of the dissociation energy barrier. For the SAP [P + 26H]26+, the onset of the decrease in the DTCCSDHe width occurs at a lower temperature. As higher charge states are more prone to unfolding and require less energy for dissociation, this observation again confirms that the TGPD barrier is reached at a lower temperature. DTCCSDHe width analysis indicates that these protein complexes undergo a series of unfolding events prior to dissociation. TID Facilitates a More “Atypical” Dissociation Process at Very High Temperatures. Ejection of a highly charged monomer, initial collapse of the complex structure followed by subunit unfolding and presence of unfolding intermediates reflected by broad baseline DTCCSDHe are all supportive of the 6276


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“typical” dissociation route.17 Despite this an interesting observation was made, as the analysis temperature is increased, the average charge state (zavg) of the ejected monomer decreases accordingly. The zavg values for all four complexes were calculated across temperatures at which the dissociation into subunits is observed, that is, 450−550 K for TTR, avidin, and conA and 475−550 K for SAP and are shown in Figure 6.

DT

CCSDHe decreases, implying fewer monomeric conformers. Testing these hypotheses would gather additional support toward the TID mechanism being temperature dependent. Our work so far suggests that TID dissociation initially follows the “typical” dissociation route and then adopts a more “atypical” dissociation route as the analysis temperature in increased. Variable Temperature IM-MS Provides Information on Strength of the Noncovalent Interactions Between the Protein Subunits. The increase of TGPD with increasing molecular mass of the complex is a clear trend observed from VT-MS experiments. The size of the multimeric protein complex will correlate somewhat to the interface area inbetween subunits. To provide more insights into what governs the extent of unfolding of subunits, we have calculated the interface surface area, the amount of hydrogen bonds and salt bridges per subunit for TTR and avidin (complexes within reasonable size range and close TGPD values) based on X-ray crystallography structures. Avidin has a larger area of interface and more noncovalent interactions per subunit than TTR. Interestingly, the extent of unfolding was ∼10% less than TTR ions carrying the same amount of charge. More noncovalent interactions might hinder extensive unfolding and keep the subunit compact prior the dissociation energy barrier is reached. At the same time, fewer interactions and extensive unfolding of subunits TTR, suggests that the noncovalent interactions between the subunits must be stronger holding the subunit within the complex prior its ejection. Measuring the extent of unfolding with VT-MS experiments could provide insights into the strength of noncovalent interactions between subunits when high resolution data is not available or obtainable.

Figure 6. Average charge state (zavg) of the ejected monomer of the four protein complexes vs buffer gas temperature (K). zavg is noted to decrease at higher buffer gas temperature suggesting that the dissociation mechanism might be on a route to resembling “atypical” (SID-like) dissociation pathway at very high temperatures.

The zavg decreases from 8.8 ± 0.3 to 7.9 ± 0.1 for TTR (Δz = 0.9), from 10.6 ± 0.1 to 8.6 ± 0.1 for avidin (Δz = 2.0), from 18.8 ± 0.2 to 12.8 ± 0.1 for con A (Δz = 6.06) and from 13.3 ± 0.2 to 12.0 ± 0.1 for SAP (Δz = 1.3). The charge of conA monomers undergoes greatest change; however, this value may be affected by the presence of monomeric conA species existing already in the spray solution. Nevertheless, dissociation of all four complexes at increasing temperature results in decreased zavg, suggesting an “atypical” dissociation route.10,48 Lower zavg indicates less asymmetrical charge partitioning during the charge migration as the monomer experiences less unfolding. Additionally, the median DTCCSHe of high charge state monomers were found to be smaller at 500 K than median DT CCSHe at 475 K. It can be speculated that at the higher buffer gas temperatures, the dissociation process occurs on a shorter time scale leading to ejection of slightly less unfolded monomer with lower zavg, especially for highly charged species where the dissociation energy barrier is lower.17,43 Moreover, the baseline width of the DTCCSDHe for the ejected monomer decreases at higher temperature. Figure S10 presents DTCCSDHe of the [M + 7H]7+ to [M + 9H]9+ ejected TTR monomers at 475 and 500 K, where it can be seen that the DTCCSDHe becomes narrower at 500 K in comparison with what is found at 475 K, especially for the [M + 8H]8+ and [M + 9H]9+. A narrower DTCCSDHe indicates more conformationally related species are present: here this is likely to be unfolded monomers. Currently, the temperature working range of the our ion mobility instrument is limited to about 550 K, but it is tempting to speculate what might happen at buffer gas temperatures above 550 K, whether (a) the zavg of the ejected monomer would decrease further, suggestive of more symmetrical charge partitioning during charge migration, (b) the DT CCSHe of the ejected monomer also decreases, suggestive of ejection of slightly less unfolded monomer, (c) the width of the

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CONCLUSIONS In this study, we have applied VT-IM-MS methodology to study four multimeric protein complexes and to probe thermally activated dissociation and related conformational change at temperatures up to 550 K. The presented data pinpoints the importance of analysis temperature for protein structural and thermal stability studies and highlights the difference between gas-phase and solution thermal stability. We have applied VT-MS to probe large protein complex dissociation which allows us to define a gas phase dissociation parameter (TGPD), and we note how much higher this is than the solution melting temperature (Tm). VT-IM-MS was applied to track conformational changes as the dissociation occurs. Complexes were found to undergo initial compaction at 350 and 400 K subsequent unfolding and eventual release of a monomer (above 450 K). The TID pathway proceeds in a similar fashion to what has been termed “typical” CID, that is, a release of a highly charged monomer as proven by VT-MS dissociation profiles and extent of unfolding determined via VT-IM-MS. The unfolding process progresses via several conformational closely related intermediates as pictured by broad DTCCSDHe. Interestingly, TID at higher buffer gas temperatures was observed to adopt more “atypical”like dissociation pathways; nevertheless, further investigation is necessary to confirm this hypothesis. Correlating TID temperatures and CID energies could serve as a framework for thermal stability screening to be performed on widely available instruments with CID capabilities. The TID experiments provide us with fundamental insights into the protein unfolding pathways and to subunit interactions. 6277


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ASSOCIATED CONTENT

S Supporting Information *

Experimental details, supporting results, discussion, and Figures S1−S10 are available, as mentioned in the text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b01063.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.


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ACKNOWLEDGMENTS This work has been funded by the award of an MRC Industrial Case Studentship to K.J.P. in collaboration with UCB Pharma. Authors would like to thank Dr. Thomas Jowitt from the Faculty of Life Sciences at the University of Manchester for performing the DSC experiment on SAP. We also thank the British Mass Spectrometry Society for a grant that allowed us to purchase our nanospray tip pipet puller still going strong after 12 years of pulling.

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ABBREVIATIONS AmAc, ammonium acetate; ESI, electrospray ionization; CID, collision induced dissociation; CCS, collision cross section; DT CCS He , drift-tube IM based collision cross section determined in helium; CCSD, collision cross section distribution; DTCCSDHe, drift-tube IM based collision cross section determined in helium; conA, concanavalin A; D, dimer; M, monomer; P, pentamer; SAP, serum amyloid P component; SID, surface induced dissociation; TGPD, gas-phase dissociation temperature; T, tetramer; Tr, trimer; TID, thermally induced dissociation; Tm, melting temperature; TTR, transthyretin; VTMS, variable temperature mass spectrometry; VT-IM-MS, variable temperature ion mobility mass spectrometry

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Distinguishing Loss of Structure from Subunit Dissociation for Protein

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