Diagnosing the Protonation Site of b2 Peptide Fragment Ions using IRMPD in the X–H (X = O, N, and C) Stretching Region
Charge-directed fragmentation has been shown to be the prevalent dissociation step for protonated peptides under the low-energy activation (eV) regime. Thus, the determination of the ion structure and, in particular, the characterization of the protonation site(s) of peptides and their fragments is a key approach to substantiate and refine peptide fragmentation mechanisms. Here we report on the characterization of the protonation site of oxazolone b2 ions formed in collision-induced dissociation (CID) of the doubly protonated tryptic model-peptide YIGSR. In support of earlier work, here we provide complementary IR spectra in the 2800–3800 cm–1 range acquired on a table-top laser system. Combining this tunable laser with a high power CO2 laser to improve spectroscopic sensitivity, well resolved bands are observed, with an excellent correspondence to the IR absorption bands of the ring-protonated oxazolone isomer as predicted by quantum chemical calculations. In particular, it is shown that a band at 3445 cm–1, corresponding to the asymmetric N–H stretch of the (nonprotonated) N-terminal NH2 group, is a distinct vibrational signature of the ring-protonated oxazolone structure.
Key wordsIRMPD IR spectroscopy Peptide fragmentation Collision-induced dissociation Oxazolone Diketopiperazine
Proton-driven amide bond cleavage is the key chemical reaction in mass spectrometry based peptide sequencing in proteomics [1, 2, 3, 4, 5]. The ionizing proton(s) play(s) a 2-fold role in the dissociation process by (1) weakening the amide bond acting as a catalyst and (2) attaching to dissociation product(s) allowing detection in the mass spectrometer. Hence, the characterization of the energetics and dynamics of protonation and “proton traffic” [6, 7] in the resulting ions are important issues for understanding the dissociation chemistry that leads to the bn and yn fragments formed upon collisional activation of (multiply-) protonated peptides. Peptides are multi-functional compounds with a plethora of likely protonation sites. Theoretical and experimental evidence exists for a mixture of initial protonation sites in some gas-phase peptides. Protonation can not only occur on the amino-terminus or basic side chains, but also on amide carbonyl oxygens. Theoretical study of protonated triglycine [8, 9] predicted that the two lowest energy structures correspond to carbonyl oxygen protonation. Additional experimental evidence [10, 11] for protonation of carbonyl oxygens has been recently provided by Wu and McMahon. Furthermore, direct evidence for the transfer of the ionizing proton has been provided by IR spectroscopy . Some of the complexity of peptide fragmentation mass spectra is also due to the fact that upon collisional activation, bn/yn primary fragment ions can subsequently dissociate by losing CO, NH3, or H2O, for example. Complex rearrangement chemistries can also occur upon peptide fragmentation and the formation of macrocyclic ring(s) has been proposed [13, 14, 15, 16, 17]. These studies indicate that the ionizing proton also plays an important role in rearrangement pathways of protonated peptides. In this context, the localization of the protonated sites of peptides and also of their fragments is an important issue.
Infrared spectroscopy has recently been critical for the structural characterization of peptide fragments. Most of the attention concerning peptide fragments has been devoted to bn ions [12, 17, 18, 19, 20, 21, 22, 23, 24, 25]. Nonetheless, an CID fragments [26, 27, 28] as well as cn fragment ions  generated via electron capture dissociation have also been investigated by IR spectroscopy. The highly intense and tunable IR beam delivered by free electron lasers (FELIX , CLIO ) is particularly well suited since it provides access to the so-called IR fingerprint region. As a result, structures bearing different functional groups can be clearly distinguished. It has been shown in particular that an IRMPD band appearing in the 1800–2000 cm–1 region is strongly diagnostic. Comparison with IR absorption determined using quantum chemical calculation shows that this band can be assigned to the oxazolone C = O stretching . In the case of small bn (n = 2) ions, it was found that two strong IRMPD bands in the 1600–1800 cm–1 spectral range constitute IR signatures of diketopiperazine structures [18, 19, 24]. For larger bn (n ≥4) ions, formation of macrocyclic structures predicted by theory has been confirmed by IR spectroscopy [12, 17, 25]. Nonetheless, although it has been suggested that an IRMPD band appearing at ~1460 cm–1 could be an IR signature of a macrocycle , the spectral congestion in this IR region prevents clear conclusions. Alternatively, at higher photon energy, a broad band observed between 2500 and 2700 cm–1 could be the IR signature of a macrocycle structure .
YIGSR [American Peptide Company (Sunnyvale, CA, USA)] was dissolved in CH3OH:H2O = 1:1 with 2% acetic acid in a concentration range of 50–80 μmol.l–1 and was sprayed with conventional ESI conditions.
2.2 Mass Spectrometry and IRMPD Spectroscopy
IR spectra in the 2800–3800 cm–1 spectral range of the clusters ions were recorded using a 7 Tesla Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometer (Bruker Apex Qe) coupled with an optical parametric oscillator/amplifier (OPO/OPA from LaserVision) laser system [32, 33]. This laser system is pumped by an Innolas Spitlight 600 non-seeded Nd:YAG (1064 nm, 550 mJ/pulse, bandwidth ~1 cm–1) laser running at 25 Hz and delivering pulses of 4–6 ns duration. Typical output energy of the OPO/OPA was 12–13 mJ/pulse at 3600 cm–1 with a 3–4 cm–1 (FWHM) bandwidth.
The ESI-formed doubly protonated YIGSR ions were mass selected and then allowed to collide with argon within the pressurized hexapole accumulation trap of the quadrupole-hexapole (Qh) interface of the 7 T hybrid FT-ICR mass spectrometer. Ions were then pulse-driven into the ICR cell, where b2 ions were mass-selected and then subjected to IR irradiation. For strongly bound ions as in the present case, the output energy of the OPO/OPA system is not sufficient for efficiently inducing their fragmentation. With the OPO/OPA tuned on a vibrational transition of the mass-selected ion, a significant enhancement of the fragmentation signal can be observed by irradiating the ions using a few ms long CO2 pulse [10 watt continuous wave (CW), BFi OPTiLAS, France] following each OPO/OPA pulse, the delay being on the order of ~1 μs. The total irradiation time was 1 s. This combination of a tunable OPO/OPA laser source with a broadband CO2 laser has been successfully used recently for enhancing the sensitivity of IR spectroscopy of [Mn(ClO4)(H2O)2–5]+ and [Mn2(ClO4)3(H2O)2–5]+ cluster ions . It should be noted that combination of tunable IR laser with line tunable CO2 laser has been used previously [35, 36]. Finally an auxiliary CO2 laser can also be useful for enhancing the spectroscopic sensitivity using the IR FEL at long wavelength, for probing metal-ligand modes for example .
Upon resonant vibrational excitation, dissociation of b2 ions was monitored via the a2 and a1 peaks. The abundances of these photofragments and their corresponding b2 precursors were recorded as a function of the IR wavelength in order to derive the IR action spectra where the IRMPD efficiency is plotted against the photon energy.
The spectral assignment of the infrared spectrum of b2 YI fragment ions was done considering the same structures as those considered for the spectral assignment in the 1000–2000 cm–1 spectral range . These structures were optimized at the B3LYP/6-31 + G(d,p) level. The theoretical IR spectra were determined using harmonic frequencies scaled by a factor of 0.955. The calculated stick spectra were convoluted assuming a Gaussian profile with a 10 cm–1 full-width at half-maximum. The Gaussian set of programs  was used for all ab initio and DFT calculations.
3 Results and Discussion
Relative energies (kJ/mol) of the four types of isomers described here
Oxazolone Ring N prot.
Oxazolone Nter prot.
Diketopiperazine I oxygen prot.
Diketopiperazine Y oxygen prot.
Experimental and theoretical (scaled by 0.955) frequencies in cm–1, and calculated intensities (in parentheses) in km/mol
Oxazolone ring N-protonated
Oxazolone N-terminally protonated
Diketopiperazine, Y-oxygen, protonated
Diketopiperazine, I-oxygen, protonated
NH3+ NH st
NH3+ NH st
Y NH st
NH3+ NH st
NH2 s st
I NH st
Y NH st
I NH st
NH2 as st
Finally, it should be noted that a broad IRMPD band can be observed in the lower frequency part of the spectrum. This band is likely to correspond to the aliphatic and aromatic CH stretches which are predicted to be weakly IR active in the 2900–2990 and 3030–3070 cm–1 ranges, respectively. As observed in other cases with the same experimental setup , although the cross-section of each individual CH stretching mode is low, the near-degeneracy of these modes favors the multiple absorption process associated the IRMPD process. The width and the maximum (~2950 cm–1) of the IRMPD band is consistent with the calculated frequencies of the alphatic and aromatic CH stretches, which are predicted to be in the 2900–2990 and 3030–3070 cm–1 spectral range, respectively.
Infrared spectroscopy allows for a clear determination of the protonation site of oxazolone. The predicted spectra of the two oxazolone structures differing by the protonation site (Figure 1b and c) are very different. The oxazolone N-terminally protonated is characterized by three strongly red-shifted NH stretching modes predicted at 3090, 3175, and 3347 cm–1. While an IRMPD band is observed at 3343 cm–1, no IRMPD signal corresponding to the strongly IR active NH stretching mode predicted at 3175 cm–1 or to the one at 3090 cm–1 was observed. It could be argued that the output power of the OPO/OPA system is maximal at 3 μm but decreases when scanning towards the lower frequencies. Nevertheless, the fact that a broad IRMPD signal could be observed on resonance with the CH stretches suggests that the laser power is large enough for inducing the fragmentation of the oxazolone N-terminally protonated on resonance with the strongly IR active NH stretching mode predicted at 3175 cm–1. It thus seems that the only oxazolone structure present under our experimental conditions corresponds to the proton attached to the ring nitrogen.
The IRMPD spectrum of the b2 fragment of doubly protonated YIGSR ions in the IR fingerprint range reported by us  clearly showed that the diketopiperazine structures are not formed under similar experimental conditions. This was supported separately by transition structure calculations and gas-phase hydrogen/deuterium exchange experiments, which both arrived at this conclusion . The calculated spectra of the diketopiperazine isomers either protonated on the oxygen of the Y or I residues are given in Figure 1d and e, respectively. These two spectra are significantly different from the experimental spectrum. In particular, they differ in the position of the O+–H stretching mode. In the case of the I-oxygen protonated isomer, it is predicted at 3581 cm–1, while it is significantly red-shifted to 3167 cm–1 in the case of the Y-protonated isomer. This strong shift to the red is characteristic of the hydrogen bond interaction of the OH+ group with the aromatic ring of the Y side chain. Since no IRMPD band was observed near 3167 cm–1 or 3581 cm–1, one can safely conclude that the diketopiperazine structures were not formed under our experimental conditions.
The IR spectrum of the b2 ion of doubly protonated YIGSR peptide recorded in the 2800–3800 cm–1 region is strongly diagnostic for localizing the protonation site of this peptide fragment. Three main experimental bands centered at 3343 (with a shoulder at 3360 cm–1), 3445, and 3648 cm–1 were observed. Overall, the agreement of the experimental IRMPD spectrum with the predicted IR absorption spectrum of the ring-protonated oxazolone isomer is remarkable. The band observed at 3445 cm–1, corresponding to the asymmetric N–H stretch of the free N-terminal NH2 group, is a clear vibrational signature of this isomer.
This structural assignment is fully consistent with that based on the IR spectrum recorded in the 1000–2000 cm–1 spectral range using the IR FEL. While the IR fingerprint region is well adapted for differentiating between isomeric forms, such as protonated-oxazolone and diketopiperazine of b2 ions, the IR range provided by relatively low output power table top laser systems (2800–3800 cm–1) is well suited to characterization of the protonation site. It should be stressed that a well resolved spectrum in the NH and OH stretching region could only be recorded by combining the table-top tunable laser with a high-power CO2 laser. In contrast to the limited available access to IR FEL facilities worldwide, the present results combine two easily accessible lasers and provide a practical means of obtaining detailed structural information from products of tandem MS/MS experiments.
B.B. thanks the DKFZ for a guest scientist fellowship. B.P. is grateful to the Deutsche Forschungsgemeinschaft for a Heisenberg fellowship. Financial support by the European Commission EPITOPES project (NEST program, project no. 15367) is gratefully acknowledged. The authors are grateful to J. M. Ortega, J. P. Berthet, and E. Nicol for technical support.
- 9.Paizs, B., Suhai, S.: Combined quantum chemical and RRKM modeling of the main fragmentation pathways of protonated GGG. I. Cis-Trans Isomerization around Protonated Amide Bonds. Rapid Commun. Mass Spectrom. 15(23), 2307–2323 (2001)Google Scholar
- 10.Wu, R.H., McMahon, T.B.: Infrared multiple photon dissociation spectroscopy as structural confirmation for GlyGlyGlyH+ and AlaAlaAlaH+ in the gas phase. Evidence for Amide Oxygen as the Protonation Site. J. Am. Chem. Soc. 129(37), 11312–11313 (2007)Google Scholar
- 19.Bythell, B.J., Erlekam, U., Paizs, B., Maitre, P.: Infrared spectroscopy of fragments from doubly protonated tryptic peptides. Chem Phys Chem 10(6), 883–885 (2009)Google Scholar
- 21.Gucinski, A.C., Somogyi, A., Chamot-Rooke, J., Wysocki, V.H.: Separation and identification of structural isomers by quadrupole collision-induced dissociation-hydrogen/deuterium exchange-infrared multiphoton dissociation (QCID-HDX-IRMPD). J Am Soc Mass Spectrom 21(8), 1329–1338 (2010)CrossRefGoogle Scholar
- 29.Frison, G., van der Rest, G., Turecek, F., Besson, T., Lemaire, J., Maitre, P., Chamot-Rooke, J.: Structure of Electron-Capture Dissociation Fragments from Charge-Tagged Peptides Probed by Tunable Infrared Multiple Photon Dissociation. J Am Chem Soc 130(45), 14916–14917 (2008)Google Scholar
- 38.Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J., J. A.;; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian Program Suite; Gaussian, Inc.: Wallingford, CT, 2004.Google Scholar
- 40.Wang, D., Gulyuz, K., Stedwell, C. N., Polfer, N. C.: Diagnostic NH and OH Vibrations for Oxazolone and Diketopiperazine Structures: b 2 from Protonated Triglycine. J Am Soc Mass Spectrom (in press). doi:10.1007/s13361-011-0147-3