Native-Like and Denatured Cytochrome c Ions Yield Cation-to-Anion Proton Transfer Reaction Products with Similar Collision Cross-Sections
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The relationship between structures of protein ions, their charge states, and their original structures prior to ionization remains challenging to decouple. Here, we use cation-to-anion proton transfer reactions (CAPTR) to reduce the charge states of cytochrome c ions in the gas phase, and ion mobility to probe their structures. Ions were formed using a new temperature-controlled nanoelectrospray ionization source at 25 °C. Characterization of this source demonstrates that the temperature of the liquid sample is decoupled from that of the atmospheric pressure interface, which is heated during CAPTR experiments. Ionization from denaturing conditions yields 18+ to 8+ ions, which were each isolated and reacted with monoanions to generate all CAPTR products with charge states of at least 3+. The highest, intermediate, and lowest charge-state products exhibit collision cross-section distributions that are unimodal, multimodal, and unimodal, respectively. These distributions depend strongly on the charge state of the product, although those for the intermediate charge-state products also depend on that of the precursor. The distributions of the 3+ products are all similar, with averages that are less than half that of the 18+ precursor ions. Ionization of cytochrome c from native-like conditions yields 7+ and 6+ ions. The 3+ CAPTR products from these precursors have slightly more compact collision cross-section distributions that are indistinguishable from those for the 3+ CAPTR products from denaturing conditions. More broadly, these results indicate that the collision cross-sections of ions of this single domain protein depend strongly on charge state for charge states greater than ~4.
KeywordsIon mobility Ion/ion chemistry Charge reduction Proteins Structural biology Mass spectrometry
Ion mobility (IM), mass spectrometry (MS), and allied methods have emerged as powerful tools for characterizing the structures of biological molecules and their noncovalent complexes [1, 2, 3, 4]. IM separates ions in a neutral gas based primarily on their charge and shape, which can be quantified by determining the collision cross-section (Ω) of the ion-neutral pair . For example, Ω of complexes that contain 18, 40, 60, and 80 copies of the capsid protein of norovirus are consistent with sheet-like intermediates that are capable of forming capsids, rather than assembly-incompetent aggregates . These approaches can also be extended to study unfolded states, intermediates, and other forms of structural heterogeneity . For example, IM spectra of [Pro13 + 2H]2+ ions generated from a sample that was transferred from mostly propanol to mostly water exhibit a total of eight features with different Ω, the abundances of which depend on time since solvent transfer . The time-dependence of the IM results is strongly correlated with that for an orthogonal analysis using capillary electrophoresis, which suggests that both experiments are sensitive to the same structural transitions in solution .
Despite the effort and progress in using gas-phase measurements to probe the structures of biological molecules in solution, a robust understanding of the relationship between the charge state of the gas-phase ion, the structure of the gas-phase ion, and the original structure in solution remains elusive. Electrospray ionization of large, globular proteins and protein complexes typically yields ions that have a relatively narrow range of charge states relative to their denatured counterparts. The Ω of those ions can depend weakly on charge state [10, 11, 12, 13] and polarity , but the range of Ω values for each analyte usually exceeds the precision of those measurements. Electrospray ionization of proteins from acidic, denaturing solutions [14, 15, 16], as well as prion  and intrinsically disordered  proteins from neutral, aqueous solutions, yields ions with a broad range of charges states, the Ω of which depend strongly on their charge state. An improved understanding of the phenomena underlying these observations is important for maximizing the structural information that can be drawn from IM experiments. Two of the ongoing challenges include (1) the relationship between the structure(s) in solution and the resulting charge-state distribution after ionization [12, 19], and (2) the extent to which structure(s) in solution are retained or evolve in the corresponding gas-phase ions [20, 21].
Relative to ion/neutral reactions, ion/ion reactions benefit from more favorable kinetics and thermodynamics [25, 26, 27]. Ion/ion proton transfer reactions were pioneered by McLuckey and coworkers [26, 27], who perform these reactions under pseudo first-order kinetics with effectively constant anion abundance [26, 27, 28]. Recently, we reported an approach that we refer to as cation-to-anion proton transfer reactions (CAPTR) , in which the abundance of anions depletes during individual experiments and, as a result, a wide range of product ion charge states are formed from different numbers of sequential proton transfer events . Electron transfer can also be used to reduce the charge states of protein ions (electron transfer no dissociation) , but electron transfer can also result in bond cleavage [31, 32].
Recently, we reported the analysis of the CAPTR products of m/z-selected ions of denatured ubiquitin . In those experiments, each subsequent CAPTR event resulted in the formation of a charge-reduced product ion that had a more compact Ω. The Ω of the CAPTR product ions depended on their charge state, and were independent of the charge state of the precursor ion. One particularly interesting observation from that work was that the lowest charge-state product ion observed (3+) exhibited a Ω similar to that determined experimentally for native-like ions of ubiquitin [34, 35] and determined computationally for an energy-minimized version of an NMR structure . One limitation of that work was that Ω of some precursor ions depended on the temperature of the MS interface, which is heated during CAPTR experiments to prevent the buildup of byproducts of glow-discharge ionization on electrodes in the atmospheric pressure interface. Those changes in Ω were attributed to heat transfer from the MS interface to the sample capillary .
Here we use IM-MS to characterize the CAPTR products of ions of cytochrome c, which is a single domain protein bound to a heme prosthetic group. Ions were formed using a new temperature-controlled, nanoelectrospray ionization source, which enables independent temperature control of the sample capillary and MS interface. In these experiments, the sample capillary was set to 25 °C, and ions of cytochrome c were generated from either denaturing or native-like conditions. These results show that the CAPTR product ions with the lowest charge state (3+) all have similar Ω distributions, regardless of the Ω distribution of the precursor ion or whether the precursors were formed from denaturing or native-like conditions.
Samples and Ionization
Cytochrome c from equine heart (Sigma-Aldrich, St. Louis, MO, USA) was dissolved into either a denaturing solution of 70:30 water:methanol with 0.1% trifluoroacetic acid at pH 2 (denaturing conditions) or 200 mM aqueous ammonium acetate at pH 7 (native-like conditions). Ubiquitin (Boston Biochem, Cambridge, MA, USA) was prepared using denaturing conditions. All cations were generated by electrokinetic nanoelectrospray ionization from borosilicate glass capillaries that were pulled to a 1 to 3 μm tip on one end using a Sutter Instruments Model P-97 micropipette puller (Novato, CA, USA) .
CAPTR and IM-MS Experiments
All experiments were performed on a Waters Synapt G2 HDMS instrument equipped with a radio frequency (rf) confining ion mobility drift cell  and ion/ion reaction capabilities . CAPTR was performed as described previously . Briefly, for 0.1 s the [M-F]– fragments of perfluoro-1,3-dimethylcyclohexane (PDCH) were produced, quadrupole-selected, and accumulated in the Trap Cell of the instrument. [PDCH−F]− reacts exclusively through proton transfer (Reaction 2) [29, 39, 40]. Following anion accumulation, the instrument was switched into positive mode for 5 to 10 s, during which time a single charge state of cytochrome c was quadrupole-selected and transferred into the Trap Cell for CAPTR. Every 22 ms, CAPTR products and unreacted precursor ions were injected into the rf-confining drift cell  with a 212 V drift voltage and filled with 2.0 mbar helium. Based on the measured arrival-time distributions, the apparent Ω distributions were calculated using a method described in the Electronic Supplementary Material that is identical to that used previously for the CAPTR products of denatured ubiquitin .
For the native model, Chimera  was used to add missing side chain and hydrogen atoms to an X-ray crystal structure of cytochrome c (PDB: 1HRC ). The linear and α-helical models lack the heme group and were built using Chimera and the expected dihedral angles. Ω Values were calculated using the projection approximation (PA)  and the exact hard-sphere scattering approximation (EHSS)  as implemented in EHSS2/k . These approximations and their relationship to momentum transfer in IM have been discussed elsewhere [5, 44].
Previously, we reported the analysis of the CAPTR products of m/z-selected ions of ubiquitin from denaturing conditions . The Ω values of the CAPTR product ions depended strongly on the charge state of the product ion. Furthermore, the Ω of the lowest charge state product ions were similar to those for native-like ions of ubiquitin, implying that although the CAPTR product ions had folded in the gas phase, they had a similar size to the native-like structure of ubiquitin in the gas phase. One limitation of those experiments was that Ω of the precursor ions depended on the temperature of the atmospheric pressure interface to the mass spectrometer. In order to decouple the temperature of the interface and the liquid sample, we developed a new temperature-controlled nanoelectrospray ionization source. Using that source, we generated ions of cytochrome c from both denaturing and native-like conditions, which were m/z-selected prior to CAPTR and IM-MS analysis. These results provide detailed insights into how protein ions from denaturing and native-like conditions respond to changes in their charge state.
Effect of the Temperature of the Atmospheric Pressure Interface on the Temperature of the Sample
Performance of the Temperature-Controlled Source
In order to control the temperature of samples prior to ionization, we constructed a temperature-controlled source that is described in the Methods section, shown using a diagram in Figure 1, and shown using photographs in Supplementary Figure S1. Figure 3a also shows the temperature of samples using the temperature-controlled source as a function of the temperature of the MS interface and the Peltier device (colored circles). Unlike those of the original source, the temperature of the sample in the temperature-controlled source is independent of the temperature of the MS interface.
Figure 3b shows the temperature of the sample as a function of the temperature of the Peltier device, using interface temperatures of 28, 60, and 120,°C. As indicated in Figure 3a, the temperature in the capillary depends strongly on the temperature of the Peltier device and is independent of that of the MS interface. Supplementary Figure S2 shows that the differences between the temperatures of the Peltier device and the sample capillary are less than 1 °C for Peltier device temperatures from 20 to 30 °C and all MS-interface temperatures. Over the full range of Peltier device temperatures of 5 to 70 °C, the magnitude of the differences between the set temperatures of the Peltier device and the sample capillary span from +2 to −3 °C with increasing temperature. These small differences are consistent with inefficient heat transfer between the sample and the Peltier device.
Several approaches for controlling the temperature of samples for nanoelectrospray ionization have been reported, including the use of forced air in proximity to the sample capillary [46, 47] or flowing the sample through a temperature-controlled device . The present implementation is most similar to those reported by Robinson and coworkers  and later by Heeren and coworkers , in which a gold-plated sample capillary was enclosed in a stainless-steel capillary sleeve. The capillary sleeve was in thermal and electrical contact with the gold-plated capillary, which was electrically biased to provide the electrospray potential and was in thermal contact with the Peltier device. The present design uses thermally conductive and electrically insulating elastomer to establish thermal contact with the capillary, and a separate platinum wire inserted into the capillary to provide the electrospray potential. This approach provides a facile solution to ensuring independent thermal and electrical contact with the sample.
In order to further evaluate this temperature-controlled source, we measured the Ω distributions of 7+ ubiquitin from a denaturing solution as a function of the temperature of the interface using a Peltier device temperature of 25 °C (Figure 2b). With the interface at ambient temperature, the Ω distribution is bimodal and similar to that measured using that interface temperature and the original source (bottom distribution in Figure 2a). As the interface temperature was increased up to 120 °C, the more compact feature centered around 12 nm2 persisted at approximately the same relative intensity. The retention of the compact form of 7+ ubiquitin ions when using an interface temperature of 120 °C suggests that the elevated interface temperature for CAPTR is decoupled from the remainder of the experiment when using this temperature-controlled source. Note that controlling the temperature of the sample does not preclude structural isomerization in the gas phase aided by elevated gas temperatures in the interface, although no evidence for that was observed here.
Ω of Cytochrome c Ions
The Ω of cytochrome c ions from water/methanol/acetic acid (49/49/2%)  and water/acetonitrile/acetic acid (75/25/0–4%)  have been reported previously. Compared with the former study , the Ω determined here are 0.7% to 3.0% larger. Although these differences are comparable to the absolute errors estimated for rf-confining drift cells [10, 37], there may also be contributions from the CDF data analysis used here versus the centroid of the best-fit Gaussian functions used previously. Compared with the latter study , the Ω determined here range from 5.5% smaller to 3.4% larger. Those differences may be attributable to some combination of differences in instrumentation, solution conditions, and data analysis.
Using this approach, we also measured the arrival-time distributions of cytochrome c ions from 200 mM aqueous ammonium acetate at pH 7 (Figure 4b, black squares), which will be referred to as native-like ions. The 50% value of the CDF for the 7+ and 6+ native-like ions are 14.0 and 12.7 nm2. In comparison to previous results for native-like cytochrome c (Figure 4b, magenta diamonds ), these values are +2.4% and −7.2% different, respectively. The 2.4% difference for the 7+ ions may not be significant, given the absolute errors estimated for rf-confining drift cells [10, 37] and differences in data analysis. The −7.2% difference of the 6+ ion reported here is much more compact than observed previously, which may be the result of differences in the ionization and extents of activation in the two experiments. Furthermore, the apparent resolving powers for the ion mobility analysis of native-like ions of cytochrome c were previously found to be low relative to other native-like ions , which may accentuate differences in data analysis.
Ω Values Calculated for Models of Cytochrome c
Ω / nm2
CAPTR of Cytochrome c Ions from Denaturing Conditions
The Ω distributions for the 9→C ions from denaturing conditions are shown in Supplementary Figure S3b. There are similarities between the distributions for the 18→C and 9→C ions of a given C, particularly for the P→4 and P→3 products. The Ω distributions for the intermediate charge state products from P = 18 and 9 depend on the identity of the precursor. For example, Figure 5c and d show the Ω distributions (black solid lines) of 18→9 and 9→9 ions from denaturing conditions, respectively. These Ω distributions span similar ranges and have features centered around similar Ω values, but the relative intensities of the features are clearly different. The differences in the Ω distributions are also apparent in the corresponding critical Ω values (black dashed lines) calculated from the CDFs (red lines).
CAPTR of Cytochrome c Ions from Native-Like Conditions
The precursor ions generated using native-like conditions were each m/z-selected and subjected to CAPTR, using the same procedure described for ions from denaturing conditions. The Ω distributions for the native-like 7→C ions are shown in Figure 5b and reproduced in Supplementary Figure S3c. The Ω distribution for 7+ ions from native-like conditions is smaller in magnitude and narrower in width than the distributions for the P→7 ions from denaturing conditions (Supplementary Figure S3a and b). This trend continues for the 7→C ions from native-like conditions and the P→C ions from denaturing conditions, for C = 5 and 6. However, the Ω distributions for the 7→3 ions from native-like conditions are indistinguishable from those for the P→3 ions from denaturing conditions (Supplementary Figure S3a and b). Relative to the Ω distributions for the ions from denaturing conditions (Supplementary Figure S3a and b), the distributions for the ions from native-like conditions span a far narrower range of values.
The critical Ω values of native-like 7→C and 6→C ions were determined using the same procedure described for the ions from denaturing conditions and are shown in Figure 6b. Just as with the 7→C ions, the 6→C ions compact with decreasing C. The most compact products for both precursors are the P→3 ions, whose median Ω values are 12.4 and 12.5 nm2 for the 7→3 and 6→3 ions, respectively. These values are 10% and 3% smaller than those for the 7+ and 6+ precursor ions, respectively. The 6→C ions are more compact than the corresponding 7→C ions, but those differences decrease with decreasing C until those for the two P→3 ions are effectively indistinguishable. This may be due to the ions compacting to similar structural populations, or different structural populations with indistinguishable Ω distributions.
The critical Ω values for the 18→C ions from denaturing conditions are also shown in Figure 6b. Although the critical Ω values for the 18→7 and 18→6 ions from denaturing conditions are significantly larger than the corresponding values for the P→7 and P→6 ions from native-like conditions, respectively, those differences decrease with decreasing C. Therefore, all P→4 and P→3 ions have similar critical Ω values, regardless of the Ω distribution of the precursor ion or whether the precursors were formed from denaturing or native-like conditions.
These experiments used CAPTR and IM to investigate the relationship between the Ω distributions and charge states of cytochrome c ions generated at ambient temperature from denaturing and from native-like conditions. To enable these experiments, we first developed a temperature-controlled, nanoelectrospray ionization source to control the temperature of the liquid samples, which are in close proximity to the heated atmospheric pressure interface of the mass spectrometer in these experiments. We characterized this source using measurements of both the temperature of a liquid in the sample capillary (Figure 3) and the Ω distributions of 7+ ubiquitin ions from denaturing conditions (Figure 2b), which were sensitive to the temperature of the atmospheric-pressure interface in previous experiments using the original source (Figure 2a, ). These results all show that this temperature-controlled source decouples the temperature of the liquid sample from the temperature of the atmospheric pressure interface.
Comparisons between the experimental (Figure 6) and calculated (Table 1) Ω indicate that CAPTR results in the formation of a diverse set of structures, from those that are compact and native-like to those that are still very extended and have minimal interactions between neighboring amino acids. The Ω of the products depend most strongly on C. Most notably, the Ω distributions of all P→3 ions are very similar and are all centered near 12.4 nm2, a value that is bracketed by the two estimates for the native model of cytochrome c (Table 1) and is slightly smaller than the Ω measured for native-like ions of cytochrome c without CAPTR.
Several aspects of the IM results for the CAPTR products of cytochrome c ions from denaturing solutions (Figure 5a, Supplementary Figure S3b, and Figure 6a) share similarities with those reported for ubiquitin from denaturing solutions . Most notably, the magnitude of the Ω distribution decreases by ~50% from the highest C observed to the lowest C observed in both cases. Furthermore, in both cases the products with the highest and lowest values of C exhibit Ω distributions that are unimodal, whereas those with intermediate values of C exhibit multiple features. However, although the relative intensities of the features observed for the P→6 ions of ubiquitin depend on P, similar ranges of Ω were observed for all C. In contrast, the ranges and relative intensities of the features in the Ω distributions for the CAPTR products of cytochrome c depend more strongly on P (Figures 5c, d, and 6a). Finally, the Ω for the lowest-C CAPTR products of both proteins from denaturing solutions are all consistent with the formation of compact structures. These compact ions have Ω that are similar to those measured for the corresponding native-like ions and those calculated using the corresponding native structure. Future experiments will focus on probing the similarities and differences between these compact protein ions formed through such different mechanisms, which will require complementary probes of ion structure (e.g., ion chemistry [7, 20, 53], alternative dissociation techniques [20, 31, 32, 54], and spectroscopy ).
Research reported in this publication was supported by Eli Lilly and Company (Young Investigator Award in Analytical Chemistry to M.F.B.) and the National Institute of General Medical Sciences of the National Institutes of Health under Award number T32GM008268 (support to K.J.L.)
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