Identification of Unfolding and Dissociation Pathways of Superoxide

Oct 31, 2014 - Identification of Unfolding and Dissociation Pathways of Superoxide Dismutase in the Gas Phase by Ion-Mobility Separation and Tandem Ma...
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Identification of Unfolding and Dissociation Pathways of Superoxide Dismutase in the Gas Phase by Ion-Mobility Separation and Tandem Mass Spectrometry Xiaoyu Zhuang,†,‡ Shu Liu,† Ruixing Zhang,†,‡ Fengrui Song,*,† Zhiqiang Liu,*,† and Shuying Liu† †

National Center of Mass Spectrometry in Changchun, Jilin Province Key Laboratory of Chinese Medicine Chemistry and Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, China ‡ University of the Chinese Academy of Sciences, Beijing 100039, China S Supporting Information *

ABSTRACT: Cu, Zn-superoxide dismutase (SOD1) is a homodimeric enzyme of approximately 32 kDa. Each monomer contains one Cu2+ and one Zn2+ ion, which play catalytic and structural roles in the enzyme. Dimer formation is also essential to its functionality. The spatial structure of this metalloenzyme is also closely related to its bioactivities. Here we investigate the structural and conformational changes of SOD1 in the gas phase by electrospray ionization mass spectrometry (ESI-MS) and ionmobility (IM) separation combined with tandem mass spectrometry (MS/MS). First, the composition and forms of SOD1 were analyzed by ESI-MS. The dimer, monomer, and apomonomer were observed under different solvent conditions. The dimer was found to be stable, and could retain its native structure in neutral buffer. Ion-mobility separation combined with MS/MS was used to reveal the conformational changes and dissociation process of SOD1 when it was activated in the gas phase. Three different dimeric and two monomeric conformers were observed; three unfolding and dissociation pathways were also identified. The results from this study demonstrate that IM-MS/MS could be used to obtain spatial structural information on SOD1 and that the technique could therefore be employed to investigate the conformational changes in mutant SOD1, which is related to amyotrophic lateral sclerosis and other neurodegenerative disorders.

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monomers, which then nucleate the formation of aggregates.13,14 Wild-type SOD1 may also play a role in the etiology of sporadic ALS, as suggested by the presence of misfolded SOD1 in the spinal cord extracts of patients with sporadic ALS.11,15,16 The determination of protein spatial structure is also very important to the fundamental understanding of biochemical pathways. Some classic bioanalytical methods such as X-ray crystallography and nuclear magnetic resonance (NMR) have been used to characterize the stoichiometry and shape of such protein complexes. However, it is very difficult to obtain the high-quality crystals of biocomplexes required for X-ray diffraction (XRD) or the substantial amounts of high-purity sample needed for NMR analysis.17−20 Therefore, it is necessary to establish a more convenient method to investigate the three-dimensional structure of SOD1. The gentle analysis conditions of electrospray ionization (ESI) mass spectrometry (MS) preserve the structure of protein complexes upon their ionization. This technique has been used extensively to study

he homodimer Cu, Zn-superoxide dismutase (SOD1, approximately 32 kDa) is found in almost all eukaryotic organisms, where it plays an important role in antioxidant defense. Biochemical and biophysical studies suggest that wildtype SOD1 dimer is exceptionally stable due to the coordination of the metal ions.1−4 Dimer formation reduces the solvent-accessible surface area, greatly increasing the stability of SOD1; dimerization is also essential to its functionality.5,6 The different species of SOD1 have very similar native structures. Investigations of structural changes in SOD1 have attracted recent research attention, because the enzyme is involved in the pathogenesis of several central nervous system disorders, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis (ALS).7 ALS is the most intensely studied superoxide-dismutase-associated disease. It is a fatal neurodegenerative disease that attacks the nerve cells responsible for controlling voluntary muscle movement. Many studies have demonstrated that familial ALS, sporadic ALS, or both are possibly caused by the aggregation of SOD1, which could be triggered by the misfolding and unfolding of the enzyme owing to a toxic gain of function mutations.2,7−12 It is proposed that some mutations can cause a small perturbation of the enzyme’s interface, leading the dimer to dissociate into © 2014 American Chemical Society

Received: June 19, 2014 Accepted: October 31, 2014 Published: October 31, 2014 11599

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Conditions for IM-MS and IM-MS/MS. For IM-MS experiments, ESI was set in mobility TOF mode using positive ion sensitivity mode. To study the behavior of SOD1 upon collision-induced activation, the instrumental parameters in the source region were kept constant with the MS experiments apart from the sample cone voltage, which ranged stepwise from 20 to 100 V during the acquisition of each spectrum. All ion-mobility separations were performed at 800 ms−1 of traveling wave velocity (WV) and 40 V of wave height (WH). In IM-MS mode, collision energies of 6 and 4 V were applied to the trap and transfer collision cells, respectively. IM-MS/MS experiments were performed to investigate the unfolding and dissociation of SOD1 dimers upon CID activation. The SOD1 [dimer]11+ ion was selected for further investigation, because up to three dimer conformers could be observed at this charge state. Ions were fragmented before or after the IM separation to derive complementary structural information. For CID before ion-mobility separation, the collision energy (CE) of the trap cell was varied within 10−70 V. Alternatively, the precursor ion can be first separated in the ion-mobility cell, and then fragmented in the transfer region. The transfer CE was varied within 50−90 V. DriftScope version 2.1 (Waters Corp., Manchester, U.K.) was used to visualize and process the data and display two-dimensional (2D) contour plots of drift time versus m/z ratio. The plots used a black background and the hot metal color code from the DriftScope settings. Collision Cross Sections. Unlike standard drift tube mobility systems in which the measured drift-time values are linearly related to the CCS, in the traveling-wave IMS system it has been proposed that the CCS is proportional to tDX. The exponential factor X depends upon many variables, including the height and velocity of the voltage “waves” used to propel the ions through the IM separation region.31,32 The correlation between the measured drift times and CCSs was calculated using a calibration curve generated from calibrant proteins of known CCS (denatured myoglobin and cytochrome c). The calibration was conducted based on an established protocol.28,33 IM-MS data of the calibrant ions and SOD1 were recorded over a range of wave heights and velocities to separate the ions. Under each condition, a calibration curve was established to calculate the experimental CCSs. The drift times of the [Di]11+ ion of SOD1 were converted into CCS data for detailed analysis. The calculation results are presented in Table 1.

the molecular mass and stoichiometry of protein complexes. ESI-MS is particularly useful when coupled with ion-mobility (IM) separation. ESI-IM-MS can provide collision cross-section (CCS) data and depict the spatial information on biocomplexes, making it a powerful tool to investigate the conformational changes and dissociation of these complexes in the gas phase.21−27 Several researchers have employed ESI-IM-MS to investigate the mechanisms of dissociation of large protein assemblies.28−30 In this paper, we report the use of ESI-MS to investigate the conformation and composition of SOD1 under different solvent conditions: neutral buffer solutions, pure water, and low-pH acid solution. The neutral buffer was shown to favor retention of the fully metalated SOD1 dimer, the native form of SOD1, thus providing the conditions for further investigation of the structure of the dimer ions. To elucidate the mechanism of dimer unfolding and dissociation, collision-induced dissociation (CID) was employed to explore the spatial conformational changes of SOD1 when it was activated in the gas phase. IM-MS/MS proved SOD1 to be a dimer composed of two identical subunits. This method provides a simple, dynamic means to assay directly the unfolding and dissociation of SOD1 under different conditions. This method not only facilitates analysis, but also has a great practical value. It can be used to explore the abnormal dissociation and unfolding behaviors of SOD1 mutants in a way comparable to, and possibly more convenient and more rapid than, the classic methods of NMR and XRD.

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MATERIALS AND METHODS Materials. The ultrapure water used in all experiments was prepared by Milli-Q (Millipore, Bedford, MA). Superoxide dismutase from bovine erythrocytes and ammonium acetate (NH4OAC) were from Sigma-Aldrich (St. Louis, MO). Methanol and acetic acid (glacial) were from TEDIA Company (Fairfield, OH). Formic acid (FA, HPLC grade) was from Roe Scientific Inc. Sample Preparation. Standard bovine SOD1 was dissolved in ultrapure water to prepare a 50 μM SOD1 stock solution. The stock solution of SOD1 was subpackaged and sealed with Parafilm, and then stored at −20 °C. On the day of each experiment, a stock solution of SOD1 was thawed, diluted to 5 μM by 10 mM ammonium acetate (pH 6.8), ultrapure water, or 0.1% FA aqueous solution, and subjected to MS analysis. Conditions for ESI-MS. Mass spectrometry analyses were carried out using a quadrupole ion-mobility time-of-flight (QIM-TOF) mass spectrometer (Synapt G2, Waters Corp., Manchester, U.K.) in positive ion ESI mode. The temperatures of the source and desolvation gas were set at 80 and 150 °C, respectively. The instrument was operated under conditions specifically chosen to maintain the native structure of the protein dimer ions produced by ESI. To minimize artificial dissociation of the SOD1 dimer and to improve the signal-tonoise ratio (without compromising the transmission of ions), the voltages of the capillary and sample cone were optimized to 2.35 kV and 20 V, respectively. The flow rates of the cone gas and desolvation gas were set to 30 and 450 L h−1, respectively. Samples were introduced at a flow rate of 5 μL min−1. Data acquisition and processing were conducted using Masslynx 4.1 software (Waters Corp., Manchester, U.K.). The average molecular mass of each form of SOD1 was calculated using MaxEnt software (Waters Corp., Manchester, U.K.).

Table 1. CCSs of SOD1 [Di]11+ Ions

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RESULTS AND DISCUSSION Different Forms of SOD1. SOD1 was analyzed in different solutions. Its composition and form in the solutions (i.e., native or denatured, monomeric or diametric, and the metal ion coordination) could be readily probed by ESI-MS (the corresponding mass spectra are shown in Supporting Information Figures S1 and S2). As labeled in Supporting

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Figure 1. IM-MS experimental data for SOD1 acquired at different cone voltages. (A) 2D contour plot of drift time (ms) vs m/z. Ion intensity is represented by varying shades of color, with dark blue representing lower intensity and bright yellow higher intensity. (B and C) Drift-time distributions of SOD1 [monomer]7+ and [dimer]11+ ions displaying the transition from compact to extended ion conformations. The centroid peaks (dashed lines) for the monomer (Mo-A and Mo-B) and dimer (Di-A, Di-B, and Di-C) are labeled.

Information Figure S1A, ions at m/z 2858 were exclusively detected for Cu2, Zn2-dimeric SOD1 ([Di]11+); the ions at m/z 2246 were attributed to monomeric Cu, Zn-SOD1 ([Mo]7+). The ions at m/z 2620 ([Mo]6+ and [Di]12+) and 3144 ([Mo]5+ and [Di]10+) were assigned to joint contributions of metallized monomer and dimer ions. Consistent with previous works,34−37 the fully metalated dimeric and monomeric species of SOD1 could be observed by ESI-MS using 10 mM ammonium acetate buffer (pH 6.8). The mass spectrum was dominated by dimer ions (charge states 10+ to 13+); monomer ions (carrying 6+ to 8+ charges) were produced in low abundance. In contrast, an acidic solution (0.1% FA aqueous solution) allowed the detection of apomonomer SOD1. The use of ultrapure water as electrospray solvent caused most of the SOD1 dimers to dissociate into monomers, which remained associated with the Cu2+ and Zn2+ ions during the ionization process. The ESI charge-state distribution of a protein generally reflects its solution-phase conformation;19,38,39 the presence of monomer ions with higher charges (6+ to 11+) in the spectrum indicate that the monomers existed in more extended structures than that formed using neutral buffers. Various studies have reported the correlations between the gas-phase and solution-phase properties of proteins and protein complexes.17,19,40−42 Some support that gas-phase proteins tend to retain a memory of the solution from which they are formed, while others find that proteins unfold after their transfer to the gas phase.43 In this work, adequate ion strength of the spray solvent should help retain the solution-phase structural properties of the SOD1 once it is in the gas phase. IM-MS and IM-MS/MS experiments were performed aiming to investigate the behavior of SOD1 dimer ions; thus, a 10 mM ammonium acetate buffer solution was used in these experiments.

The average molecular mass of each of the three forms of SOD1 was obtained using MaxEnt software. SOD1 from eukaryotic sources is acetylated on the amino-terminus to increase the protein mass by 42.1 Da; the average molecular masses of Cu2, Zn2-dimer SOD1, Cu, Zn-monomer SOD1, and apomonomer SOD1 are 31432, 15716, and 15591 Da, respectively. The calculations are consistent with those reported elsewhere.5,34,44 Influence of Activation Voltage on the Gas-Phase Structures of SOD1. In this study, the sample cone voltage was found greatly to influence the profile of the mass spectra and the relative abundance of the various charge states of SOD1. To assess the stability of the SOD1 conformers and to understand their unfolding and dissociation behaviors, SOD1 was deliberately activated by gradually increasing the sample cone voltage. Increasing this voltage from 10 to 100 V (some of the data not shown) led to clear increases of the intensity of the ions at m/z 2620 and 3144, while only the intensity of the [Di]+11 ion at m/z 2858 decreased. This suggests that part of the Cu2, Zn2-dimer SOD was converted to Cu, Zn-monomer SOD by in-source fragmentation. Further investigations were undertaken by IM-MS analysis. This technique was used to explore the nature of the dissociation process, to find whether CID would cause all the dimer ions to dissociate into monomer ions, and to assess the effects, if any, of the activation voltage on the conformation of the SOD1 dimer and monomer. Figure 1 shows the great influence of sample cone voltage on the drift-time distributions of SOD1. At low voltages all the ions exhibit short drift times, while at higher voltages the drift times are longer and much more broadly distributed. As before, ions at m/z 2858 are exclusively detected for the Cu2, Zn2dimer SOD1 ([Di]11+). Any spectrum lacking a signal from the [Di]13+ ion also does not show any signal attributable to 11601

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Figure 2. IM-MS/MS data for [Di]11+ ions acquired at transfer CEs of 50 and 90 V. The sample cone voltage was consistently 50 V. The precursor ions at m/z 2858 are marked with asterisks. For each collision energy, there is a 2D plot of drift time vs m/z snapshot and the corresponding tandem mass spectra: (1A and 2A) Di-A and the product ions; (1B and 2B) Di-B and the product ions; (1C and 2C) Di-C and the product ions.

[Di]14+, thus indicating that ions at m/z 2246 are exclusively attributable to the Cu, Zn-monomer SOD1. At high activation voltages (above 60 V) very few ions corresponding to the dimer were detected, which is consistent with the previous results. Low activation voltages were required to observe compact states of the SOD1 dimer, and the ions corresponding to the dimer increased in size dramatically at higher voltages, which is indicative of its unfolding.45 DriftScope software was used to understand better the conformation changes of the SOD1 ions in the gas phase. The software allowed the extraction of m/z and drift-time information for any ion or inset region of interest and gave a much better insight into the drift-time separation. The extracted drift-time distributions for the individual and [Mo]7+ and [Di]11+ ions of SOD1 are shown in Figure 1, parts B and C. Native-like SOD1 ions should have compact structures and short drift times, whereas unfolded SOD1 ions are expected to show long drift times owing to their increased CCS.28 IM data showed that, at low sample cone voltages (10−35 V, data for 10 V not shown), both monomeric and dimeric species exhibited short and narrowly distributed drift times indicative of a single compact conformation. As activation energy was increased beyond a threshold value, a much broader distribution of the drift times for the intact protein complex emerged, indicating the unfolding of the ion. At a sample cone voltage of 40 V, the drift-time distribution of the [Mo]7+ ion of SOD1 was bimodal, with peaks centered on 9.65 and 14.48 ms. Drift times for the [Di]11+ ion were also recorded, with peaks centered on 11.2, 14.7, and 17.3 ms. Ruotolo et al. attributed this increase in drift time to the generation of multiple unfolded states of the protein complex owing to the internal energy of the protein complex being altered by collisional activation.29 In total, two different

monomeric and three dimeric conformers of SOD1 were observed in the gas phase. The native-like compact dimer of SOD1 is termed “Di-A”. “Di-B” represents the unfolded intermediate dimer, and “Di-C” refers to the much unfolded intermediate dimer. Similarly, the compact and extended monomers are, respectively, termed “Mo-A” and “Mo-B”. When the sample cone voltage was set below 20 V, the Di-A ions remained intact and compact, and it was the only dimeric species at m/z 2858. Increasing the activation voltage resulted in the formation of monomer ions and intermediate states of the SOD1 dimer. At 45 V, the partially unfolded state (Di-B) was also observed, and the absolute abundance of Di-A ions started to decay. This suggests that the Di-B ions formed from the Di-A ions and, in turn, were the originating state of the DiC ions. This indicates that the conformation changes occurred in a continuous manner. The gradual change in the relative amounts of Di-A and Di-B also reflects the corresponding stability of each conformer. That the monomer ions exist in only two discrete structural states suggests that Mo-B must be formed from the dissociation of dimeric ions, not directly from the unfolding of Mo-A. Increasing the activation voltage led the compact conformation of dimeric SOD1 (Di-A) to increase its drift time from 11.2 to 11.4 ms, while Di-B and Di-C remained stable and unaffected. Further increases of the activation energy did not cause the drift-time distribution of SOD1 [Di]11+ ions to expand. A stable unfolded intermediate (drift time 17.3 ms) was reached when its CCS did not further increase upon raising the activated voltage of CID. It is possible that in this conformation the dimeric SOD1 was elongated to the maximum extent allowable for its charge state. Note that Supporting Information Figure S2 shows a band circled with a dashed line that contains two plots corresponding to m/z 2246 and 2858. This resulted from two overlapping traces (Mo-A and Di-B). The m/z ratios and IM 11602

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drift times for the 12+ and 10+ charge states of SOD1 Di-B overlap with those of Mo-A in 6+ and 7+ charge states; the two traces were inseparable under the experimental conditions. The traveling wave drift times of the 11+ charge SOD1 dimers were converted into collision cross sections. Table 1 shows the unfolded intermediate state Di-B of the SOD1 dimer correlates with a CCS of 2855 Å2, which is 22% larger than that of original state Di-A (2340 Å2). Very few dimer ions remained at higher activation voltage. The experimental CCS of the greatly unfolded state Di-C was 3245 Å2, 39% larger than that of the original state. Although a stretched structure can accommodate more charges, the three conformers of the SOD1 dimer tended to carry 11+ or 12+ charges, with no obvious difference in charge-state distribution. Pathways of Collision-Induced Unfolding and Dissociation of SOD1. The collision-induced unfolding and dissociation of SOD1 dimers were investigated through a series of MS/MS experiments. The quadrupole mass analyzer was set to transmit the [Di]11+ ion at m/z 2858 into the Tri-Wave region of the instrument. For CID after ion-mobility separation, the precursor ions are fragmented in the transfer cell; any fragmentation products generated will appear at the same drift time as the original parent ion, which allows the relationship between the precursor and the product ions to be easily inferred. Figure 2 shows the drift time versus m/z contour plots for each collision energy together with the MS/MS spectra corresponding to Di-A (Figure 2, parts 1A and 2A), Di-B (Figure 2, parts 1B and 2B), and Di-C (Figure 2, parts 1C and 2C). A sample cone voltage of 45 V was used for each acquisition, as this condition allows the coexistence of the three dimer conformers before their propulsion to the Tri-Wave region. It should be pointed out that the relative abundance of [Di-A]11+ was nearly equal to that of [Di-B]11+ before their fragmentation. The figure shows that, at a transfer collision energy of 50 V, the product ion spectrum of the Di-A precursor ion (Figure 2, part 1A) exhibits a significant abundance of product ions in the presence of undissociated precursor ions, whereas the product ion spectra of the Di-B (Figure 2, part 1B) and Di-C (Figure 2, part 1C) precursor ions show low abundances of product ions. The abundance of undissociated Di-B precursor ions at a transfer CE of 90 V (Figure 2, part 2B) was about 50% of the total abundance. However, almost all of the Di-A precursor ions (Figure 2, part 2A) had already dissociated, implying that the SOD1 dimers with compact structures dissociate more easily than the extended structures. It is noteworthy that at a sample cone voltage of 45 V and transfer CE of 90 V, one metal-deficient monomer and another monomer with one additional metal ion were detected, indicating that fragmentation could occur at the coordinate bonds of the SOD1 dimer. Tandem MS experiments were also achieved by dissociation in the trap region to obtain conformational information about the product ions. The [Di]11+ precursor ion was selected and activated before ion-mobility separation. Experiments were performed at a sample cone voltage of 20 V to maintain the intact and compact structure of the dimeric SOD1 precursor before its fragmentation. We monitored simultaneously the conversion from a dimeric, folded state to a dissociated, unfolded state by the gradual increasing of the voltage applied to the trap cell (Figure 3). At the default trap CE (6 V), the dimer existed mainly as Di-A. When the trap CE was increased to 20 V, the fragment ions at m/z 2620 and 3144 were observed, which are easily recognized

Figure 3. Two-dimensional IM-MS/MS contour plots for [Di]11+ ions at m/z 2858. CID took place in the trap cell before ion-mobility separation at different collision energies (trap CE 20, 30, and 50 V). The precursor ions are circled with a dashed box.

as Mo-A from the drift times. Unfolded Di-B also appeared under this condition, indicating that the compact dimer of SOD1 was undergoing dissociation and was in the initial stages of unfolding. A further increase of the trap CE to 30 V led Di-C and product ion Mo-B to be also observed along with a decrease in the intensities of Di-A and Di-B. At a trap CE of 50 V, only Di-C was observed from the precursor ions at m/z 2858; all the Di-A and Di-B ions had dissociated. As mentioned before, the 2D plot of Mo-A ion was superimposed with that of the Di-B ion. At a trap CE of 50 V, in the absence of [Di-B]11+ precursor ion, only Di-C ions remained together with Mo-A (5+ and 6+) and Mo-B (6+ and 7+) as product ions. Note that the loss of the 4+ monomer from the spectrum was possibly due to its relatively low intensity and the slightly reduced detection efficiency at higher m/z. The greater abundance of Mo-A than Mo-B ions suggests that the Di-A ion mainly dissociated into the Mo-A ion, resulting primarily in a symmetric division of charge with respect to mass (generating a complementary 5+ and 6+ monomer pair from the precursor 11+ dimer).40 By contrast, as a partially unfolded intermediate state of SOD1 dimer, the Di-B ion probably dissociated into Mo-A and Mo-B through an asymmetric route. According to the generally accepted model of noncovalent protein complex dissociation,40,46,47 the asymmetric route can be explained as follows. A subunit gradually unfolds upon collisional-induced activation; proton transfer to this newly exposed surface area results in the unfolded subunit carrying away a disproportionate amount of charge for its relative mass. In the case of native-like SOD1 dimer, the unfolding barrier is greater than that of dissociation; thus, the dimer dissociates promptly and predominantly by a charge-symmetric pathway, with little structural changes in the products.48,49 These conclusions also agree well with the previous results that the dimers with compact structures dissociate preferentially. The 5+ and 6+ monomer product ions of [Di-C]11+ show evidence of a charge-symmetric dissociation pathway. Given the much unfolded structure of Di-C, the resulting product ions should obviously have more extended structures than the compact form of the monomer (Mo-A). We suggest that these product ions may have a similar structure to Mo-B, because no 11603

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other conformations corresponding to the monomeric SOD1 were observed in addition to Mo-A and Mo-B. The dissociation of all the investigated SOD1 [Di]11+ conformers resulted in a narrow distribution of product ions. This was possibly due to the presence of intramonomer disulfide bonding formed by Cys55 and 144, which decreased the gas-phase conformational flexibility of the proteins.40,47 To conclude the results of the MS/MS experiments, the unfolding and dissociation pathways are illustrated in Figure 4.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-431-85262044. Fax: +86-431-85262044. E-mail: [email protected]. *Phone: +86-431-85262236. Fax: +86-431-85262236. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 21175128, 81373952).

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Figure 4. Dissociation and unfolding pathways observed for the [Di]11+ ion at m/z 2858. Di-A represents the native-like compact dimer of SOD1, Di-B, an unfolded intermediate dimer, and Di-C, a greatly unfolded intermediate dimer. Mo-A denotes the compact monomer of SOD1, and Mo-B represents its unfolded monomer.

Note that the structures displayed here are only meant to demonstrate the trend of the structural changes, rather than to represent precisely the experimentally observed gas-phase conformers. Three different unfolding and dissociation pathways are depicted: (1) the intact dimer directly dissociates into two identical monomers without obvious unfolding, (2) common asymmetric dissociation involving the unfolding of one monomer,50 and (3) the dimer unfolds to reach its maximum elongated structure before following a symmetric dissociation pathway.

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CONCLUSIONS Through a series of ESI-MS assays, the composition and conformation of SOD1 were shown to vary under different solution conditions. A neutral buffer favored retention of the form of wild-type SOD1. It provided the conditions for further study of the structural changes of SOD1 in the gas phase. In the second part of this study, IM-MS and IM-MS/MS illustrated the way in which activation voltage strongly influences the quaternary structure of SOD1 and the dimer dissociation. It thus served ideally for the characterization of unfolding and dissociation behaviors of SOD1. This work’s findings will be instructive to the further study of the stability of SOD1. The methodology is also practically applicable to the comparative structural analysis of other homologous proteins and may be further used to identify populated monomeric and oligomeric species in neurodegenerative diseases.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed descriptions of ESI-MS experiments results and corresponding figures (Figures S1 and S2). This material is available free of charge via the Internet at http://pubs.acs.org. 11604

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Analytical Chemistry

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dx.doi.org/10.1021/ac502253t | Anal. Chem. 2014, 86, 11599−11605