Abstract
The dominant gas-phase conformer of [M+3H]3+ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. The [M+3H]3+ peptide ions are observed predominantly as a relatively compact conformer type. Upon subjecting these ions to electron transfer dissociation (ETD), the level of protection for each amino acid residue in the peptide sequence is assessed. The overall per-residue deuterium uptake is observed to be relatively more efficient for the neutral residues than for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison, the N-terminal and C-terminal regions of the serine peptide show greater relative protection compared with interior residues. Molecular dynamics (MD) simulations have been used to generate candidate structures for collision cross section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for select structural candidates from MD simulations has been used to suggest conformer types that could contribute to the observed HDX patterns. The results are discussed with respect to recent studies employing extensive MD simulations of gas-phase structure establishment of a peptide system.
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Introduction
Since the discovery of the structure of the α-helix in polypeptides [1], significant research has been devoted to determining factors that affect the formation of elements of secondary structure and in particular those effecting helix formation [2]. Initial efforts, boosted by the expanding available protein structure data, focused on the relationship between the presence of α-helices and the frequency of occurrence of specific amino acid residues [3,4,5,6,7]. Shortly later, Chou and Fasman proposed a method for predicting this secondary structure element using amino acid sequence information [8]. Over the next several decades, interest in helix structure prediction progressed to the point where a relatively high degree of accuracy was obtained [9, 10]. This progress coincided with efforts to refine helix propensities of the individual amino acid residues [2, 11, 12] and the concept has even been extended to structure destabilization imparted by D-amino acid residues [13, 14].
The rapid increase in the number of experimentally determined protein structures over the last three decades [15, 16] has significantly enabled protein structure prediction capabilities. Perhaps, the simplest conception is to use sequence homology based on comparisons to known structures to infer structural elements for proteins of interest [17,18,19]. Over the years, other computational techniques have advanced significantly and protein structure prediction efforts can currently be grouped into those using ab initio methods (or ab initio-guided approaches) and template-based methods (fold recognition and threading, comparative modeling) [20]. Currently, protein structure prediction remains a highly active and rapidly evolving area of research [21,22,23].
A challenge in the characterization of polypeptide structure is species that have been designated as intrinsically disordered [24,25,26]. Although efforts have been developed for predicting unstructured regions of proteins [27, 28], recent developments regarding the nature of species classified as intrinsically disordered have called into question whether or not structural assessments are possible. One area of interest is the study of peptide structure. For example, the peptide bradykinin was examined by NMR many years ago and only a small portion of the molecule was assigned to a β-turn structural type [29]. Recent experiments that employed ion mobility spectrometry (IMS) combined with mass spectrometry (MS) have shed some light on the inability of solution-phase measurements to determine prevailing structural motifs for peptides; for bradykinin, these experiments revealed the presence of multiple, co-existing solution structures [30, 31]. Although the exact relationship of gas-phase structures to antecedent solution states is still under investigation, such efforts suggested that gas-phase analyses could provide insight into the various structures of polypeptides that exist in solution. Interestingly, extending the approach has suggested the presence of multiple protein structures for species for which well-defined native conformations predominate [32].
Results from the early IMS-MS experiments lead to questions regarding the degree to which elements of secondary structure are preserved in the gas-phase for such dynamic systems and whether or not solution structures can be derived from high-quality gas-phase structures. To study this, recent experiments have examined the conformations of [M+3H]3+ ions of the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK (hereafter referred to as the A-containing peptide) using a combination of IMS-MS and gas-phase hydrogen/deuterium exchange (HDX) measurements and molecular modeling techniques [33,34,35]. This peptide was selected because of its relatively large propensity to form a helix in solution [2, 36, 37]. In general, studies suggested that the presence of multiple conformer types of varying degree of helicity can contribute to the [M+3H]3+ ion conformers that were studied [33]. In many respects, this work was similar in nature to early efforts aimed at designing gas-phase peptide ion structure [38] as well as studies that have characterized peptide structural motifs with gas-phase HDX [39, 40].
The present work examines the [M+3H]3+ ions of a the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK (hereafter referred to as the S-containing peptide). This peptide has been designed to exhibit less helical propensity in solution [2]. Indeed, because the hydroxyl oxygen of serine (S) can hydrogen bond with the carbonyl oxygen of the third residue, S is known to destabilize α-helix structure [41]. Additionally, regions of high S density are associated with helix-breaking structure [42]. The model peptide was also designed because of the potential to experience a different process of gas-phase conformation establishment when compared with the previously studied A-containing peptide. Previous work has suggested that the non-polar A residues may strongly associate with the apolar vacuum environment during the final stages of ESI [35, 43]; it may be expected that the S residues will more strongly favor solvent interaction during the final stages of ESI. These differences offer the opportunity to compare the ion structure types formed by very different solution conformers and possibly by different processes of gas-phase conformer establishment.
Overall, ion collision cross section values are nearly the same compared with the A-containing peptide. However, because the S-containing peptide has a larger molecular weight, one may conclude the overall structure is more compact. Additionally, there are significant differences in the relative accessibility of exchange sites of terminal and middle amino acid residues with the latter being more accessible to the S-containing peptide. These characteristics are discussed below using structures obtained from simulated annealing to help provide insight into relative accessibility. By providing information about gas-phase ion conformer establishment from different solution states, these efforts may help to enhance protein structure prediction tools in the future. Finally, this work adds to the growing repertoire of studies that employ IMS-MS, gas-phase HDX, and/or MD simulation techniques for peptide/protein ion structure characterization [44,45,46,47,48,49,50,51,52,53,54].
Experimental
Sample Preparation
The model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK (97.3% purity) was purchased from GenScript (Piscataway, NJ, USA) and used without further purification. To prepare the 1 mg/mL stock solution, the peptide was dissolved in the appropriate amount of Milli-Q water. The stock solution was then diluted 10-fold with a 100 mM solution of ammonium acetate in water to prepare the ESI solutions. This solution was infused (0.5 μL/min) through a pulled-tipped capillary which was biased ~ + 2200 V relative to the entrance of the hybrid IMS-MS instrument.
IMS-HDX-MS Measurements
For the ion mobility measurements conducted here, a dual-gate drift tube similar to others described in the literature was used [55, 56]. The details of the drift time (tD) and MS measurements have been described in detail previously [57, 58]. Briefly, for this study, ions generated by ESI were periodically (50 Hz) pulsed into a ~ 1-m-long drift tube that was filled with ~2.5 Torr of 300 K helium buffer gas. For the dual ion gate approach, the second gate was set to transmit at specific delay times in order to record mobility-resolved mass spectra. This delay time was scanned (200-μs increments) across the mobility distribution of the [M+3H]3+ ions. Mass spectra were recorded by a linear ion trap (LTQ Velos; ThermoScientific, San Jose, CA, USA) instrument. A mass-to-charge (m/z) range of 400 to 1000 was employed. For each tD, increment mass spectra were recorded for 0.5 min. For mobility selection, the second ion gate was set to transmit for 200 μs.
The HDX measurements conducted here are modeled after foundational work in which the reactivities of different biomolecular ions were examined upon reaction with gaseous deuterating reagent [59,60,61,62,63,64,65,66]. For HDX measurements, ~0.02 Torr of D2O was added to the buffer gas. With the addition of D2O, the tD distributions shifted to longer times as shown in Fig. 1. Therefore, for the tandem MS (MS/MS) measurements, the delay times were scanned across the tD distributions obtained when employing D2O to determine the mobility selection time corresponding with the dominant, compact conformer type for the [M+3H]3+ ions. As it was desired to examine the HDX behavior of the dominant, most compact ion conformer type, mobility selection was initiated 200 μs prior to the peak maximum time point in order to minimize contamination from larger ions (Fig. 1).
Peptide Ion Dissociation by ETD and Per-residue Deuterium Uptake Calculations
The MS/MS analyses described here have also been influenced by foundational work demonstrating the utility of MS/MS to determine deuterium uptake from gas- and solution-phase experiments [67,68,69]. Briefly, mobility-selected [M+3H]3+ ions were subjected to electron transfer dissociation (ETD) [70] to determine the per-residue deuterium incorporation as described previously [57, 71]. To perform ETD measurements, mobility-selected ions were isolated by m/z and the ion injection time was maintained at 200 ms (5 microscans). Because ETD did not generate a number of lower m/z ions, the deuterium uptake value for each residue was obtained using both z- and c-type ions. The per-residue deuterium uptake values for the fragment ions were obtained by first subtracting the average mass of the fragment ions obtained in the absence of D2O from the average mass of those generated upon using D2O reagent gas. For the triplicate measurements, the amount of deuterium incorporated in each residue was calculated by subtracting the deuterium uptake values of the appropriate adjacent fragment ions. Here, it is noted that mass spectrometer conditions including all focusing optics were maintained at conditions that have been shown to not induce deuterium scrambling [72].
Molecular Dynamics Simulations
Details regarding the in vacuo molecular dynamics (MD) simulations can also be found in the prior, related work [73]. Briefly, the initial structures of the [M+3H]3+ ions for simulated annealing were generated using the Visual Molecular Dynamics (VMD). The CHARMM36 force field was employed for structural parameterization. All of the parameters were defined in this force field. The CNEU patch in the top_all36_prot topology file were used to protonate the C-terminal K residue resulting in a +1 formal charge. The LSN patch was used to protonate the more N-terminal K residues. Acetyl groups were attached to the N-terminus portion of the P residue by using the ACP patch. Five thousand steps of geometry and energy minimization were carried out using the Nanoscale Molecular Dynamics (NAMD) software suite for the [M+3H]3+ peptide ions with charge arrangements of K(6)-K(11)-K(16), K(6)-K(11)-K(21), K(6)-K(16)-K(21), and K(11)-K(16)-K(21). Then, the structures were subjected to cyclic simulated annealing (SA) for conformational space sampling. For SA runs (timestep of 1 fs), the Berendsen temperature coupling algorithm was used. For the SA algorithm, the temperature of the in vacuo system was suddenly increased to 1010 K over 10 steps. The heated structure was maintained at that temperature for 40 ps and then cooled to a lower temperature (10 K) over two different timescales (40 and 1200 ps). The annealed structures were subsequently energy minimized to 0 K to generate candidate structures. The energy minimized structure of each cycle was imported as the starting point for the next heating-cooling cycle. Each minimized structure was gradually heated to 300 K and equilibrated. The structures then undergo 5 ns of MD simulations with no non-bonded cutoffs for long-range interactions while the temperature was constant. The Mobcal software suite [74] was used to calculate theoretical collision cross-section values using the trajectory method [75].
Hydrogen Accessibility Scoring and HDX Kinetics Modeling
Hydrogen accessibility scoring (HAS) scores were calculated for in silico structures with matching collision cross sections. Two candidate structures which contained CCS values matching experimental measurements were selected for discussion. The selection was based on a first-pass examination of HAS scores for which one ion structure displayed increased proximity of charge sites to middle residues while the other displayed less access to these residues (see discussion below) as is suggested to be required by the “relay” mechanism for HDX [76]. Hydrogen accessibility was computed as described previously [77] where each carbonyl site was scored based on its relative access to charge sites on the hypothetical peptide ions. Then the distances between exchange sites and these scored carbonyls were used to obtain relative theoretical reactivities of each exchange site which were summed for individual residues to provide a residue score. Finally, the residue scores were scaled based on relative values of surface accessibility using the solvent accessibility surface area (SASA) approach in the VMD software suite. Using these residue scores, the deuterium incorporation was estimated for [M+3H]3+ ions using an HDX kinetics model described previously [57]. Briefly, a population of ions (1000) was stepped (1 μs) through a mobility separation (~ 10 ms) and the exchange nature of each site was computed at each step based on the contribution of the residue to the overall rate of exchange for the ions. After stepping all ions through the mobility separation time, the HDX simulation algorithm determined the amount of deuterium incorporation for each residue; this theoretical uptake value is compared to the experimental results below.
Results and Discussion
Peptide Ion Collision Cross Sections
Summing the spectral counts for the [M+3H]3+ peptide ions at each mobility selection allows the generation of the tD distribution shown in Fig. 1 (bottom trace). The distribution is dominated by a feature having a tD of ~ 8.5 ms. A shoulder, indicating the presence of larger ion conformer types is also observed. With the addition of ~ 0.02 Torr of D2O, the entire distribution shifts to longer times. The dominant peak is observed to have a tD of ~ 9.3 ms representing a shift of ~ 0.8 ms. Again, a shoulder of similar intensity is observed at longer tD values. The shift in the shoulder feature is also observed to be of the same magnitude. That the distribution is similar in the presence and absence of D2O suggests that the HDX process does not result in structural transformations and it is therefore possible to monitor the reactivities of select ion conformer types. This is similar to results that have been presented for protein ions [61].
From the ion tD distribution, it is possible to determine the collision cross section of the [M+3H]3+ ions. Collision cross sections are determined using Eq. 1 [78]:
In Eq. 1, ze and kB are the charge of the ion and Boltzmann’s constant, respectively. The variables mI and mB are the mass of the ion and the mass of the buffer gas, respectively. E, L, T, and P represent the electric field, the length of the drift tube, and the temperature and pressure of the buffer gas, respectively. Finally, N is the neutral number density at standard temperature and pressure (STP) conditions. Using Eq. 1, the HeΩDT (DT = linear drift tube) is determined to be 424 Å2 and 464 Å2 for the prominent feature and shoulder, respectively. These values are larger but similar to those determined for more compact and partially unfolded A-containing peptide conformers (417 Å2 and 438 Å2) [34, 77].
A goal in examining the model peptide ions is to begin to determine the degree of similarity in solution- and gas-phase peptide structure in order to better understand ion conformer establishment. A number of studies suggest that a high degree of structure preservation is observed for relatively small proteins [79, 80]. The fate of even smaller peptide ions is less clear. As mentioned above, comparisons of the data reported here for the S-containing peptide are made with those obtained from previous studies of the A-containing peptide because of expected differences in predominant solution conformers. Indeed, CD spectroscopy of the A-containing peptide suggests a high degree of helical character as indicated by the negative bands at ~ 207 and 223 nm as well as the positive band at ~ 190 nm shown in Fig. S1 in the Supplementary Information section. The CD spectrum for the S-containing peptide (Fig. S1 in the Supplementary Information section) shows a very clear difference in predominant solution structure for this peptide where the features indicating α-helix are clearly missing and a broad band at lower wavelengths is indicative of more random coil character.
Because of these differences in solution structure, it is of interest to consider the compact ion conformers for both the S- and A-containing peptides. Notably, the collision cross section of the S-containing peptide is only ~ 1.7% larger than that of the A-containing peptide ion (see above). This occurs even though the molecular weight of the S-containing peptide is ~14.1% larger than that of the comparative peptide. Thus, the overall gas-phase “packing” of the S-containing peptide is more extensive than that of the A-containing peptide. This increased atom density for the S-containing peptide ions offers the opportunity to determine whether or not distinctive HDX behavior can be observed that would be consistent with such ion structures. Thus for the S-containing peptide, the residue specific deuterium content was examined in detail; the deuterium content was then compared to that of the A-containing peptide and the results are discussed below. Here, it is noted that it was not possible to compare the more elongated conformer of the [M+3H]3+ ions as the signal level for the mobility-selected ions was insufficient to obtain ion fragmentation spectra.
Per-residue Deuterium Uptake
Upon introduction of D2O to the drift tube, the m/z of the [M+3H]3+ ions shifted to higher values (m/z~ 695.4) indicating the incorporation of 20.1 deuteriums (19.9 ± 0.9, on average). Overall there are 48 exchangeable hydrogens (including the three protonation sites). Thus, ~ 41% of the exchangeable hydrogens are observed to undergo exchange. This is similar to the level of deuterium incorporation observed for the A-containing peptide [77]. Therefore, it is useful to consider the amount of deuterium incorporation occurring at the neutral S and A residues. Notably, because the ETD approach used here does not differentiate between side-chain and backbone sites, the question of relative accessibility of different site types for the S residues cannot currently be answered. However, a semi-quantitative comparison of residue accessibility can still be achieved based on the per-residue deuterium content.
To determine the S-residue deuterium content for the gas-phase HDX studies, the more compact [M+3H]3+ precursor ions were mobility selected and subjected to ETD both before and after introduction of the D2O reagent gas. Figure 2a shows the fragmentation spectrum produced for the unlabeled ions. Two strong features are observed at m/z~ 688.7 and m/z~ 1032.5 and correspond to the [M+3H]3+ precursor ions and the charge-reduced, doubly charged ions. Overall, a general lack of low-m/z ions is observed in the fragmentation spectrum; dataset features for the z4 through z20 and the c6 through c20 fragment ions are observed. Observed ions used in deuterium content calculations with their associated m/z values are provided in Table S1 in the Supplementary Information section. Because many lower m/z fragment ions are not observed, the deuterium uptake values for individual amino acid residues have been determined using a combination of c and z ions. There are two advantages in using both c and z ions to obtain the uptake values. The first is that nearly complete sequence coverage can be obtained and the second is that a double check of deuterium uptake is provided especially for dataset features of lower intensity.
After undergoing gas-phase HDX, the fragment ions produced by ETD broaden due to an increased number of isotopologues and shift to higher m/z values as shown in Fig. 2b. In the case of the z ions, by subtracting the zi − 1 ion from the zi, the deuterium uptake value for the residue numbered N − i − 1 can be determined for which N is the number of total peptide residues (21 in this case). For the c ions, by measuring the difference between the ci − 1 and the ci ions, the deuterium content of the residue number i can be calculated. For example, the average m/z for the peak corresponding to z15 ions is 1432.68 and the same peak when D2O is introduced is m/z 1445.40. Thus the deuterium content is calculated to be 12.72. Another example in Fig. 2 is the c6 ions. In this case, the average m/z is 633.70 and the corresponding average m/z in the presence of D2O is 638.78. Here, the deuterium content is 5.08. Table S1 in the Supplementary Information section provides the deuterium uptake values for the amino acid residues obtained from the z and c ion fragments.
To provide further insight into the determination of deuterium uptake, it is instructive to examine expanded regions of the mass spectra shown in Fig. 2. Fig. S2 in the Supplementary Information section shows expanded regions for three fragment ions. In general, as the fragment ion mass increases, an increased shift to higher m/z is observed and a wider isotopic distribution is encountered for ions produced after HDX (Fig. S2 in the Supplementary Information section). For fragment ions produced after HDX, the average m/z value is computed by locating the dataset feature edges (typically ≤ 20% peak height) and then computing the weighted average of the isotope peaks between the edges. The average m/z of the fragment ion produced prior to introduction of D2O is then subtracted from this value to obtain the overall m/z shift resulting from deuterium incorporation.
S- and A-Residue Deuterium Content Comparison
To investigate whether the per-residue deuterium content reveals differences in relative residue accessibility between the S- and A-containing peptides, the per-residue deuterium content is here compared. As mentioned above, one comparison that is readily available is that of overall HDX efficiency for these resideus. For the S-containing peptide, on average, the number of deuteriums incorporated for all S residues is ~ 9.4 (~ 28.5% of the 33 total available). For the A-containing peptide, a value of ~ 19.5% efficiency (~ 3.3 of 17 available sites), on average, is obtained. The higher efficiency could be reflective of the more compact nature of the S-containing peptide as more exchange sites are in closer proximity to charge sites and deuterium incorporation sites as required by the reaction mechanism [76].
For the reactivity comparisons at the per-residue level, a question arises as to how the evaluation should be performed. For example, the S-containing peptide contains 16 more exchangeable hydrogens due to the side-chain site on the S residues when compared with the A-containing peptide ions. Therefore, a direct comparison of per-residue deuterium content may not provide the simplest representation of the relative accessibility. Thus the S and A deuterium content values are scaled according to their fractional values of the total deuterium incorporated into the neutral residues. K residues are not included in this analysis because, considering the HDX mechanism, different charge site configurations could contribute to the observed differences. Here, rather the interest is in comparing the average differences in accessibility to different regions of the peptide through the use of the S and A residues.
Figure 3 shows the comparison of the deuterium content of the S and A residues on the respective peptide ions. For the A-containing peptide, only four residues show significantly greater accessibility; these are the A4, A5, A19, and A20 residues. For the S-containing peptide, the residues showing significantly increased accessibility are the S2, S9, S10, S13, S15, and S17 residues. Overall, the results suggest a greater accessibility to middle residues for the S-containing peptide ion. This could result from increased charge solvation by the S residues as discussed below. Notably, the relative per-residue deuterium uptake values shown in Fig. 3 exhibit different levels of precision (also shown as independent values in Table S1). This appears to primarily result from variability in determining the peak centers of low-abundance fragment ions. For example, two required fragment ions used to determine the deuterium content for S3 and S18 residues are ~ 40% lower in intensity relative to those used for the S13 and S19 residues. That said, the error levels are consistent with those reported previously [67, 71, 77, 81].
Comparison to Candidate Structures from MD Simulations
The HDX behavior of the S-containing peptide was further considered using MD simulations. To find the best structural type (or types) exhibiting similar exchange site accessibility to the gas-phase conformer type, simulated annealing was conducted for [M + 3H]3+ peptide ions using two different charge configurations as well as two different simulation times (see “Experimental”). As shown in Fig. S3 in the Supplementary Information section, the collision cross sections obtained from MD simulations are, for the most part, significantly smaller than the experimental value. An examination of some of these structures, reveals extremely compact ion conformer types with significant charge solvation of the charge sites by the serine residues as shown in Fig. S4 (Supplementary Information section). Indeed, as an indicator of how the MD simulations captured such charge solvation, consider that only 71 of 8000 structures were observed to have collision cross sections within ± 2% of the experimental value for the compact [M+3H]3+ ions.
To consider which of the structural types with matching collision cross sections best represented the experimental data, HDX kinetics modeling was employed (see “Experimental” above). Notably, this work does not include the very extensive MD trajectory modeling that was performed previously for the A-containing peptide [34, 35, 77]. Rather, here the MD simulations are only performed to provide an idea of conformer types that could account for the observed HDX accessibility [73]. From HAS scoring and kinetics modeling of the 71 structures, several conformer types appeared to exhibit significant charge solvation by middle residues. Based on the best ability to match the experimental results (see below), one of these ion structures was selected for discussion. This ion structure is shown in Fig. 4C.
The agreement between experimental and theoretical (HDX kinetics modeling) relative deuterium content is shown as a bar graph in Fig. 4A. A number of trends are shown to agree between the experimental and theoretical values. Here, we note that the theory favors sites that are in relative proximity to backbone carbonyls that are in relative proximity to charge sites. A trend captured by the theory for the first ion conformer is the relatively low levels of uptake for S3 and S4 and an increase in uptake for S5 and S7. This is followed by a slight decrease for S8 and then increases for S9 and S10. The model slightly overestimates the deuterium content for S12 to S14. This is followed by relatively good agreement between S15 and S17. The worst agreement is observed at the C-terminal and N-terminal ends for the S2, S18, and S20 residues. Overall, the average percent difference between experiment and theory is observed to be ~ 27.5% across all S residues.
Figure 4D shows a separate ion conformer for which increased accessibility is observed for the terminal peptide portions relative to middle residues. For example, for this ion conformer there is an increase in estimated deuterium content for the N-terminal residues S3, S4, and S5 with an increase also observed for the C-terminal residues S18, S19, and S20. These increases are offset by decreases in theoretical deuterium content for residues S7 to S14 as well as S17. For this structure, the average percent difference between experiment and theory is ~ 41.4%.
The differences in the quality of HDX reactivity matching for the ion candidate structures can be described via the HAS scores and the structure images shown in Fig. 4. For the ion structure providing the best HDX reactivity matching, the charge site assignments are K6, K16, and K21 while the charge site assignments for the second ion structure are K6, K11, and K21. One noticeable difference in the structures is that the former ion conformer with its charge arrangement exhibits less charge density. Indeed, the relative Coulomb energy of the former ion conformer is only 69% that of the latter. Thus a more even dispersion of charge throughout the ion conformer is observed. For the first ion conformer (Fig. 4C), the relative increase in theoretical deuterium content would be elevated for the S17 residue due to its hydrogens proximity to the S13 (2.9 Å), S14 (2.1 Å), and S17 (3.6 Å) carbonyls. These carbonyls are highly scored because they are in close proximity to the K21 charge site (all ≤ 1 Å) and the S13 carbonyl is also within 2.5 Å of the K16 charge site. The S15 residue has a large theoretical deuterium uptake as well due to its hydrogens proximity to the K11 (1.9 Å), S12 (3.0 Å), and S14 (3.1 Å) carbonyls. The K11 and S12 carbonyls are scored highly because they are within 3 Å of the K16 and K21 charge sites. In comparison, only one scored carbonyl is positioned at ≤ 4 Å from the S19 hydrogens. The increased theoretical deuterium uptake for the S10 to the S14 sites results from the fact that these residues fall between the K6 and K16 charge sites and there is a significant compaction of this portion of the ion such that exchange sites are in close proximity to a greater number of carbonyl sites. For example, the S10 residue has hydrogens that are within 1.8 Å and 2.9 Å of scored carbonyls on the S4 and S7 residues. Additionally, labile hydrogens are ≤ 4 Å of scored carbonyls on the S8 and S9 residues. These scored carbonyls are in closer proximity to the K6 charge site. Finally, although the S2, S3, and S4 residues are in relative proximity to the K6 charge site, there is a decreased number of close carbonyl sites. For example, for hydrogens on the S2 residue, only the S2 carbonyl is within ≤ 4 Å.
For the ion conformation presenting decreased agreement in theoretical and experimental deuterium content (Fig. 4), the decrease at S2 results from the placement of this residue further away from charge sites. This is evident from the fact that the only carbonyl sites within 4 Å of any hydrogen on the S2 residue are the N-terminal acetyl group and the P1 residue and both of these carbonyls are not highly scored due to their distance to charge sites. Decreases in middle residues S9 and S10 can be seen to result from the placement of these residues further away from charge sites and scored carbonyl sites. For example, hydrogens on the S10 residue only encounter the S8 carbonyl at a distance that is ≤ 4 Å. Finally, the increased theoretical deuterium content at the C-terminal residues (S18, S19, and S20) results from the fact that these residues are in close proximity to the K6 and K21 charge sites as well as a number of carbonyl sites. For example, the hydrogens on the S19 residue are within 1.5 Å and 2.5 Å of the scored carbonyls on the S18 and S19 residues. These carbonyls are scored highly as they are 0.5 Å and 1.7 Å from the K21 charge site. Additionally, the latter carbonyl is also within 5.7 Å of the K6 and K11 residues. As shown in Fig. 4, the structure exhibiting the decreased HDX reactivity match appears to position the S4 and S5 residues such that they would not exhibit greater accessibility as determined by the HAS approach. However, upon rotation of the ion structure, it becomes apparent that the exchangeable hydrogens on the S4 and S5 residues are near a number of highly scored carbonyl sites as shown in Fig. S5 in the Supplementary Information section. Indeed, the hydrogens on these residues are ≤ 2 Å from the N-terminal acetyl group carbonyl and the P1 carbonyl.
It is instructive to consider the type of ion structure that would be required to result in increased exchange of the S2 and S20 residues as is observed experimentally (Fig. 4). One consideration is that more compact structures in which the N-terminal and C-terminal ends turn back toward the middle residues would provide increased HAS scores for these residues. Fig. S6 in the Supplementary Information section shows a compact ion structure (HeΩDT = 363.0 Å2) for which HAS scoring and HDX kinetics modeling was performed. The relative deuterium incorporation levels for several residues are also shown in Fig. S6. Notably, the value for the S20 residue is the largest of all residues and that of S2 is among the largest values as well. Additionally, the deuterium incorporation values for the neighboring S4 and S18 residues are among the lowest. These results are similar to what is observed experimentally for N- and C-terminal residues (Figs. 3 and 4). The high value for the S2 residue results from the fact that a N-terminal bend positions this residue such that the S2 backbone hydrogen is in relatively close proximity of the acetyl carbonyl (3.3 Å), the P1 carbonyl (3.1 Å), and the S2 carbonyl (2.1 Å). These carbonyl sites also have relatively high scores as they are located near the K6 charge site. The side-chain hydrogen of S2 is located near (1.7 Å) the S10 carbonyl which is one of the highest scoring carbonyl sites being near multiple charge sites. In contrast, the backbone hydrogen and the side-chain hydrogen of the S4 residue are not within 3 and 4 Å, respectively, of any scored carbonyl. For the S20 residue, hydrogens are in close proximity to the highly scored carbonyls on S19 (3.1 Å) and S8 (2.0 Å). The S18 residue hydrogens are not within 3 Å of any scored carbonyl.
It is necessary to present a note of caution regarding the comparisons between theoretical and experimental deuterium uptake here. Such comparisons are not intended to be definitive as they do not consider ion conformer dynamics or the possible contribution by multiple ion conformers with different charge site configurations. As mentioned above, the failings of the approach are especially obvious in the inability of either ion structure to account for the increased deuterium content of the S2 and S20 residues observed experimentally. The improved matching of HAS scores with experimental values for these residues when using a far too compact ion structure is intriguing. For example, such analysis may suggest that multiple ion conformer types comprise the mobility selection and a portion of these contain bends at either the N-terminal or the C-terminal end with the other end extended (in order to exhibit the correct cross section). That said, the comparisons are useful insofar as they demonstrate conformer types exhibiting specific charge solvation that could account for the observed differences in relative deuterium content of specific peptide regions. Additionally, such comparisons represent the foundational work that will be extended to exhaustive MD simulations that do account for conformer dynamics as well as the presence of different conformer types. In future works, such simulations will be conducted in the gas and solution phases as was performed for the A-containing peptide [34, 35] and the results will be compared with regard to the preferred types of structure formed for the different ions.
Conclusions
IMS-HDX-MS/MS measurements coupled with MD simulations have been used to gain insight into the behavior of [M+3H]3+ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK. From the IMS measurements a dominant, compact ion conformer is observed. In comparison to the same ions for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK, the S-containing peptide is observed to be relatively more compact. This may be somewhat expected as CD spectroscopy suggests that the former peptide exhibits more random coil nature while the A-containing peptide exhibits significantly more α-helical character. In addition to, and likely attributed to, the increased compactness, the S-containing peptide shows increased accessibility of interior residues to gas-phase HDX. MD simulations suggest that ion conformers demonstrating specific, relatively disperse charge solvation by interior residues could account for the observed HDX behavior of the [M+3H]3+ ions.
The disparity in hypothetical and experimental deuterium uptake suggests the need for highly extensive MD simulations to better understand the contributions of ion structure dynamics as well as contributions from multiple ion conformers. This limitation was also encountered for the A-containing peptide where exhaustive gas-phase and ESI droplet simulations have been conducted to better fit the experimental data [34, 35]. Thus, in order to gain insight into the ion structures resulting from very different solution structures, it will be necessary, in future studies, to conduct these same exhaustive analyses where a convergence between gas-phase ion structures and those emerging from ESI droplets is obtained. With these structures it will be possible to compare the major differences in structures arising from the S- and A-containing peptides. The comparison of such predominant structures could provide information about the degree of preservation of secondary structure for these smaller biomolecules and could thus help with our understanding of the process of gas-phase ion structure establishment.
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We are grateful for financial support from the National Institutes of Health (R01GM114494).
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Karanji, A.K., Khakinejad, M., Kondalaji, S.G. et al. Comparison of Peptide Ion Conformers Arising from Non-Helical and Helical Peptides Using Ion Mobility Spectrometry and Gas-Phase Hydrogen/Deuterium Exchange. J. Am. Soc. Mass Spectrom. 29, 2402–2412 (2018). https://doi.org/10.1007/s13361-018-2053-4
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DOI: https://doi.org/10.1007/s13361-018-2053-4