Letter pubs.acs.org/JPCL
Isomer-Specific IR−IR Double Resonance Spectroscopy of D2-Tagged Protonated Dipeptides Prepared in a Cryogenic Ion Trap Christopher M. Leavitt,† Arron B. Wolk,† Joseph A. Fournier,† Michael Z. Kamrath,† Etienne Garand,† Michael J. Van Stipdonk,‡ and Mark A. Johnson*,† †
Sterling Chemistry Laboratory, Yale University, P.O. Box 208107, New Haven, Connecticut 06520, United States Department of Chemistry, Lawrence University, 711 East Boldt Way, Appleton, Wisconsin 54911, United States
‡
S Supporting Information *
ABSTRACT: Isomer-specific vibrational predissociation spectra are reported for the gasphase GlySarH+ and SarSarH+ [Gly = glycine; Sar = sarcosine] ions prepared by electrospray ionization and tagged with weakly bound D2 adducts using a cryogenic ion trap. The contributions of individual isomers to the overlapping vibrational band patterns are completely isolated using a pump−probe photochemical hole-burning scheme involving two tunable infrared lasers and two stages of mass selection (hence IR2MS2). These patterns are then assigned by comparison with harmonic (MP2/6-311+G(d,p)) spectra for various possible conformers. Both systems occur in two conformations based on cis and trans configurations with respect to the amide bond. In addition to the usual single intramolecular hydrogen bond motif between the protonated amine and the nearby amide oxygen atom, cis-SarSarH+ adopts a previous unreported conformation in which both amino NH's act as H-bond donors. The correlated red shifts in the NH donor and CO acceptor components of the NH···OC linkage to the acid group are unambiguously assigned in the double H-bonded conformer. SECTION: Spectroscopy, Photochemistry, and Excited States
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cryogenic ion trap, where D2 adducts are prepared prior to spectroscopic interrogation. We specifically focus on the GlySarH+ and SarSarH+ ions because our earlier report of their vibrational spectra, obtained by D2 tagging, suggested that at least two conformers were present in each case.42 Figure 1 presents selected regions of the GlySarH+ (b) and SarSarH+ (c) spectra. Both of these ions display extra peaks and splittings pointing to contributions from at least two conformers, thus providing ideal cases with which to demonstrate the power of the IR2MS2 technique, as well as to explore the possible formation of new intramolecular H-bonding motifs. Here, we use the method to empirically determine the number of conformers present and to isolate the spectrum of each. Ab initio calculations are then used to identify the structures of the different conformers. The vibrational predissociation spectrum of GlySarH+·(D2)2, presented in Figure 1b, was previously analyzed in the context of a dominant structural element, in which one of the protons on the NH3+ group strongly interacts with the nearby amide carbonyl in an intramolecular ionic H-bond.42 Mullikan analysis of the calculated minimum-energy structure indicates that ≥70% of the excess positive charge is localized on the amino group, and consequently, intramolecular H-bonds involving this
here has recently been great progress in the structural characterization of simple peptide ions in the gas phase using vibrational spectroscopy.1 Most of this work has involved mass selection of ambient ions and detection of vibrational resonances in the fingerprint region using IR multiple photon dissociation (IRMPD).2−18 A very promising development in this regard has been the introduction of cryogenic techniques in which either ionic or neutral target molecules are cooled close to 10 K prior to laser interaction, and the absorptions are detected either by population transfer in an IR/UV double resonance scheme19−29 or by predissociation of a weakly bound “messenger” species like H2.30−32 Either way, it is often the case that several conformers are present in the size-selected ion packet, and methods for isolating the spectra of a single conformer include differential ion mobility33−40 and, when available, detection through the sharp bands in the electronic spectrum.19−21 We have recently demonstrated a more general isomer-specific scheme, carried out entirely within the vibrational manifold, that is based on infrared photochemical holeburning of tagged species.41 This approach requires two independently tuned infrared lasers and at least two stages of mass selection (hence denoted IR2MS2). Furthermore, this technique has the advantage that it can be used on any tagged ion, unlike the IR/UV experiments that require a chromophore such as a tyrosine or phenylalanine residue. In this Letter, we adapt the hole-burning approach, initially carried out on Artagged ions created in a supersonic jet,41 for application to ions generated by electrospray ionization and cooled in a ∼10 K © 2012 American Chemical Society
Received: March 14, 2012 Accepted: April 9, 2012 Published: April 9, 2012 1099
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Figure 1. Vibrational predissociation of (b) GlySarH+·(D2)2 and (c) SarSarH+·(D2)2 obtained by monitoring the photoevaporation of both D2 messenger molecules as a function of laser wavelength. The harmonic frequency calculations of the minimum-energy structures of bare GlySarH+ and SarSarH+, calculated at the MP2/6-311+G(d,p) level of theory, are presented in (a) and (d), respectively. The 2500− 3800 cm−1 range was scaled by a factor of 0.944 for GlySarH+ and 0.945 for SarSarH+ to bring the OH stretches into agreement with the experimentally observed positions, while the 1600−1900 cm−1 range was scaled by 0.977 for both protonated peptides (a value that brought the calculated acid CO stretch into agreement with the experimental GlyGlyH+·(D2)2 spectrum).42
Figure 2. IR2MS2 spectra of GlySarH+·(D2)2 collected by probing at (b) 3571 (α) and (c) 3561 cm−1 (β) while scanning the pump laser from 3200 to 3700 cm−1. The total ion depletion spectrum of GlySarH+·(D2)2 is presented in panel (d), which was collected by monitoring the GlySarH+·(D2)2 signal as a function of the pump laser wavelength. The α-band is assigned to the trans conformer, while the β-band is assigned to a cis conformer, with both structures presented in the right inset. The corresponding scaled (see Figure 1 caption) harmonic frequency calculations (MP2/6-311+G(d,p)) are presented in (a) and colored in blue and red, respectively.
group are analogous to intermolecular proton bonds discussed at length by Roscioli et al.43 The bonded NH stretch appears very strongly red-shifted (by >2000 cm−1),42 while the remaining NH2 group yields symmetric and asymmetric collective NH stretching modes split by about 50 cm−1 and centered at 3350 cm−1. That work also established that the hydrogen tag molecules preferentially attach to these “free” NH's.30 The band highest in energy at 3575 cm−1 is due to the nonbonded OH stretch, but is also split into a close doublet separated by ∼9 cm−1 (see the inset in Figure 1b). The calculated harmonic frequencies of the minimum-energy structure (Figure 1a) are generally in good agreement with the experimental positions of the NH2 and OH stretches, but the splitting of the OH mode cannot be explained solely with this species. In our previous study, we calculated many lowlying minimum-energy structures that could account for the observed splitting of the OH stretch.42 To experimentally determine the existence as well as the spectral contributions of individual isomers, we obtained IR2MS2 spectra of the GlySarH+·(D2)2 ion, with the results displayed in Figure 2. In this approach, a probe laser is fixed on a particular transition to monitor the population of the isomer responsible for that band, while a powerful pump laser intersects the same ion packet upstream and is scanned through the entire spectrum. The spectrum of the isomer selected by the probe laser transition is then revealed as dips in the probe signal, which is described in detail in the Experimental Methods section. Figure 2b and c presents the depletion of the probe signal when the probe laser is fixed on the right (α) and left (β) members of the OH doublet at 3571 and 3561 cm−1, respectively. The absence of the doublet in each of the dip spectra immediately establishes the presence of two separate species that display remarkably similar overall patterns, each
consisting of a sharp OH band above a pair of somewhat broader features in the NH stretching region. Closer inspection reveals that all three peaks in Figure 2c (red) occur at slightly (about 5 cm−1) lower energies than the corresponding bands in Figure 2b (blue). The likely structural motifs of the two conformers were explored by comparing the isomer-specific spectra with those anticipated for the predicted isomers at the harmonic level. Two of the calculated low-lying conformers indeed yield spectra that accurately account for the observed bands. Their structures are indicated to the right of Figure 2, while the (scaled) harmonic fundamentals are color-coded in Figure 2a. On the basis of the slight red shift anticipated by these calculations (MP2/6-311+G(d,p)), we assign the red spectrum to the higher-energy cis form, calculated to lie 290 cm−1 above the global minimum that was recovered in our previous report. Interestingly, there are actually three other conformers calculated to lie below this cis species (Figure 2c), but two of these are O-protonated, which we discard based on poor agreement of their calculated NH2 stretches (Figure S1, Supporting Information). The other lower-lying local minimum is N-protonated and has a cis configuration but contains two intramolecular H-bonds. That structure would result in a single, strong free NH stretch at 3347 cm−1, which again is not observed in the experimental spectrum.42 The two observed GlySarH+ structures basically differ by 180° rotation about the amide bond. Both adopt very similar intramolecular H-bonding motifs at the N-terminal residue, accounting for the similarities in the symmetric and asymmetric NH2 stretching fundamentals observed experimentally. There is more variation at the C-terminal end, however, where the dihedral angle defined by the N−Cα−C(O)−OH arrangement 1100
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differs by ∼20° in going from the trans to the cis conformer. This rotation brings the OH group into a nearly eclipsed configuration with respect to a hydrogen atom on the adjacent CH2 group, possibly accounting for the slight red shift in its OH stretching fundamental. We note that cis/trans isomerization about the amide bond is commonly observed (e.g., by NMR and ion mobility MS) for peptides containing tertiary amides,35,44,45 and the observation of similar motifs in the cold ions thus provides a powerful means with which to quantify these structures. It is interesting that both conformers are efficiently prepared in the cryogenic ion source despite the fact that they are calculated to be quite different in energy (∼290 cm−1). We note that both NMR spectroscopy and theoretical analysis of similar systems have shown that cis/trans conformers are separated by large barriers,15,16,44,46 which would allow them to be trapped as metastable species. It is not presently clear whether these structures are extracted directly from solution or quenched into local minima after injection into the 10 K ion trap. The SarSarH+ system presents a more interesting and complicated case. In our previous analysis of its spectrum (Figure 1c),42 we suggested that in addition to the OH doublet, two very broad features (labeled by †) also appeared that were most consistent with a qualitatively different arrangement in which both NH groups on the amine form intramolecular Hbonds. That arrangement also explained the red-shifted shoulder (again labeled †) observed in the amide I region (Figure 1c) by its assignment to an H-bond-accepting acid carbonyl. Note that this double H-bonding motif requires the strong free NH stretch at 3331 cm−1 to be completely absent in the spectrum of this putative conformer, providing an unambiguous target for double resonance sorting of the various contributions to the spectrum. We remark that an analogous double H-bonded structure was calculated to be low-lying in GlySarH+ but was not evident in its IR2MS2 spectra (Figure S1, Supporting Information). To unravel the distinct spectral contributions to the SarSarH+ spectrum, we first focused on the amide I region highlighted in Figure 3e, where the candidate for the H-bonded acid carbonyl stretch at 1754 cm−1 (denoted β) appears 24 cm−1 below the 1778 cm−1 transition assigned to the nonbonded acid carbonyl stretches (denoted α). The left panel of Figure 3 presents the IR2MS2 spectra of the SarSarH+·(D2)2 ion obtained when the probe laser is fixed on either the α or β feature while the pump laser was scanned through the region of the NH and OH stretching fundamentals. Note that, unlike the GlySarH+ system, the two IR2MS2 spectra obtained for SarSarH+ are qualitatively different, thus establishing that the latter peptide adopts dramatically different folded structures. Figure 3b, obtained by probing at the α-CO transition, confirms that this is derived from a distinct species with a free NH band at 3331 cm−1 but does not display the broader features at 3120 and 3210 cm−1. The carrier of the free NH bands does, however, contribute to the doublet in the OH stretching region. The middle (green) trace (Figure 3c), obtained by probing at the β-CO band, displays a sharp, single feature at the free OH stretching position in addition to the two broad features at 3120 and 3210 cm−1. Most importantly, the fact that the two acid carbonyl probe transitions (α and β) yield distinct IR2MS2 patterns confirms that these CO bands are in fact due to different structures and do not, for example, arise from intramolecular couplings leading to extra bands.
Figure 3. The D2 predissociation spectrum of SarSarH+·(D2)2 in the 1650−1825 cm−1 range is presented in trace (e). The acid CO stretch is split into a doublet with bands at 1778 (α) and 1754 cm−1 (β). IR2MS2 spectra obtained by scanning the pump laser from 3050 to 3700 cm−1 while probing on the α and β transitions are presented in traces (b) and (c), respectively. The inverted total ion depletion obtained by monitoring the SarSarH+·(D2)2 parent as a function of the pump laser frequency is presented in trace (d). The scaled (see Figure 1 caption) harmonic frequency stick spectra (MP2/6-311+G(d,p)) of the trans, cis(1), and cis(2) conformers, indicated in blue, red, and green, respectively, are presented over the 3050−3700 cm−1 range (trace a) and over the 1650−1825 cm−1 range (trace e). The ion-dip spectrum obtained by probing on the α transition (purple, trace b), is assigned to a mixture of the trans and cis(1) conformers, with their structures presented in (f) and (g), while the spectrum obtained by probing on the β transition (green, trace c) is assigned to the cis(2) conformer, with its structure presented in (h).
The isomer-selective spectra in Figure 3 confirm the presence of a conformer that accounts for both the red-shifted CO stretching band of the acid group (β) as well as the two broad transitions centered at ∼3160 cm−1. This behavior is consistent with the double H-bonded structure presented in Figure 3h, which is a variation on the cis framework where the acid functionality folds back onto the amino group. The two Hbonded NH's thus account for the two broad bands at 3120 and 3210 cm−1, where the asymmetrical H-bond lengths (calculated at 2.112 and 2.078 Å, respectively) slightly increase the typical splitting (50−60 cm−1)42 expected for the collective normalmode behavior of the symmetric and asymmetric stretches of the embedded NH2 group. The 24 cm−1 red shift in the CO acceptor is also recovered at the harmonic level, as indicated by the green stick spectrum in Figure 3e. This double H-bonded conformer is calculated to lie 120 cm−1 above the global minimum and is likely separated from it by the typical barrier (5600−7700 cm−1) for cis/trans isomerization of a tertiary amide.44 The structural motif that accounts for the SarSarH+ species with free NHs is similar to that identified in GlySarH+, where one of the NH groups is H-bonded to the nearby amide carbonyl. It is of interest that probing the α-CO band yields a doublet in the OH stretching region (Figure 3b), again suggesting the presence of cis and trans conformers formed by rotation about the amide bond. This was verified to be the case using IR2MS2, and the individual spectral contributions are included in Figure S2 (Supporting Information). The structures 1101
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differing by 180° rotation about the amide bond are again found to be local minima (MP2/6-311+G(d,p)) and are included in Figure 3 (insets of (f) and (g)). The observed OH stretch splitting is accurately recovered, and the global minimum corresponding to the trans configuration yields the highest-energy OH stretching band as before. The lower-energy OH transition is then assigned to the cis configuration, calculated to lie 240 cm−1 above the global minimum. We note, however, that the lower-energy member of the OH doublet is actually more complicated because the IR2MS2 spectrum of the double H-bonded structure (obtained by probing the β-CO stretch in Figure 3e) also displayed a single OH stretch fundamental in almost the same location, as indicated by the hole-burning scans in Figure S2c and S2d (Supporting Information). The calculated OH fundamentals associated with the three minimum-energy structures indeed explain this OH stretching pattern, specifically with the double H-bond structure yielding the lowest-energy OH stretch, as is observed. Finally, we remark on the extreme variation on the NH stretching frequencies associated with the partially charged, intramolecular H-bonds. The most difficult spectroscopic signature to assign involves the single H-bond between the protonated amine and the amide oxygen in GlySarH+, which we tentatively attributed to a broad feature at 1340 cm−1 in our earlier report.42 Further studies exploiting isotopic substitution (e.g., 15N) are currently underway to challenge this assignment in both GlyGlyH+ and GlySarH+. Introduction of the methyl group at the N-terminus acts to increase the basicity of the amino group,47 which results in dramatically blue-shifted NH stretches in SarSarH+ that are readily assigned. Interestingly, these H-bonded NH stretches are quite dependent on whether one or two of the NH groups are involved in intramolecular Hbonding. We previously assigned the very broad NH feature near 2700 cm−1 to the single H-bonded conformer, and this conjecture was verified in the course of this work using IR2MS2. The resulting spectra are shown in Figure 4, where (b) presents the pump scan obtained when the probe was fixed on the strong free NH stretch (3331 cm−1, labeled ε). The broad 2700 cm−1 band is clearly present, while the features (green, Figure 4c) near 3200 cm−1, traced to the double H-bonded conformer, are conspicuously absent. Although the calculated harmonic Hbonded NH values, displayed in Figure 4a, clearly underestimate the observed red shifts of the cis and trans single Hbonds (HB−NH), they do indicate that the single H-bonded conformers should exhibit much more red-shifted NH stretches than those associated with the double H-bonded conformation (HB−NH2). In particular, the experimental band patterns establish that when the second NH engages the acid group to form the cis(2) conformer, the NH bonded to the amide dramatically blue shifts, while free NH red shifts by approximately 150 cm−1. This type of anticooperativity among ionic H-bonds donated by the same center are well documented in the analogous case where multiple ligands are added to H3O+.48,49 As a result, we anticipate that the ability to follow the evolution of the charged H-bonds in a series of folded peptides, now available through isomer-selective IR2MS2 and IR2MS3 spectroscopies,41 will yield considerable insight into the trade-offs between single and double linkages that control the secondary and tertiary structures of the isolated systems.
Figure 4. (a) Calculated harmonic frequency stick spectra (MP2/6311+G(d,p), with scaling details described in Figure 1 caption) of the trans, cis(1), and cis(2) conformers, colored in blue, red, and green, respectively. (b) IR2MS2 spectrum of SarSarH+·(D2)2, collected by scanning the pump laser from 2600 to 3700 cm−1 while probing at the 3331 cm−1 feature (ε). (c) Total ion depletion spectrum, collected by monitoring the SarSarH+·(D2)2 parent ion intensity during the pump laser scan. The probed transition (ε) is common to both the trans and cis(1) conformers, which are shown at the right inset of the figure.
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EXPERIMENTAL METHODS Both single-photon predissociation and IR2MS2 vibrational spectra of D2-tagged protonated peptide ions were recorded using the Yale time-of-flight mass spectrometer50,51 in the configuration diagrammed schematically in Figure S3 (Supporting Information). Ions were generated and tagged with D2 using a custom ESI source coupled to a cryogenic quadrupole ion trap cooled to ∼10 K, as described in detail in our earlier studies of di- and tripeptides.30,31,42,51 The isomer-selective scheme is an IR−IR pump−probe approach using two independently tunable IR lasers (LaserVision) that intercept an ion packet at two different locations along the time-of-flight path. This technique relies on removal of the population by saturating predissociative transitions with a scanning pump laser while monitoring the population of a particular isomer by fixing a probe laser at one of its characteristic transitions. When the pump excites a transition arising from the same isomer monitored by the probe, the removal of population by the pump is registered by a dip in the probe signal; these dips then record the entire spectrum corresponding to that isomer. The key is to intercept the ion packet with the pump laser and then isolate undissociated parent ions before they are interrogated by the probe. Although our group has applied the isomer-specific IR−IR double resonance technique to a variety of Ar-tagged systems created in free jet ions sources,41,52,53 the smaller mass loss inherent in the D2-tagged ions places much higher demands on the intermediate mass selection stage essential for separating the action of the pump and probe lasers. Here, we exploited the fact that ion packet of tagged peptides prepared in a quadrupole ion trap is quite small and, as such, can be intercepted by the pump laser immediately upon ejection from the trap en route to the TOF photofragmentation spectrometer.54 The layout of the scheme used in this study is depicted in Figure S3 (Supporting Information), where the ensemble of tagged and untagged ions is extracted from the trap and intersected with 1102
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the pump laser in a cylindrical multipass arrangement, modeled after the design of Riedel et al.,55 which resulted in the depletion of about 50% of the tagged ions upon resonant absorption of a 10−15 mJ/laser pulse. Because these experiments were carried out in the evaporative regime, source conditions were optimized for formation of the [peptideH+·(D2)2] complex while minimizing the abundance of the larger D2 adducts in order to prevent an increase in the population of the 2D2 complex from photodepletion of the larger adducts. Using standard time-of-flight techniques,50,56 the tagged ions were mass-separated, and the [peptideH+·(D2)2] adduct was isolated and intersected with the probe laser tuned to a resonant transition. A reflectron was used to separate the initial parent ion, which was depleted by the pump laser, from the probe photoproduct. We note that when probing weaker transitions, isolation of the population modulated by the pump laser required background subtraction (Figure S4, Supporting Information) to remove the weak (
