DOI: 10.1002/cphc.200800469

IRMPD Spectroscopy of a Protonated, PhosphorylatedDipeptide**Catarina F. Correia,[a] Carine Clavaguera,[a] Undine Erlekam,[b] Debora Scuderi,[b] andGilles Ohanessian*[a]

1. Introduction

Reversible phosphorylation of the side-chain alcohol group ofserine (S), threonine (T), or tyrosine (Y) residues is among themost frequent post-translational modifications (PTM) of pro-teins. Several thousand sites of post-translational phosphoryla-tion are now known, and estimates of the fraction of proteinsthat are phosphorylated in vivo range as high as 30 %.[1] Pro-tein phosphorylation has a very strong impact on protein func-tion. It is a ubiquitous mechanism to control various aspects ofcell proliferation, differentiation, metabolism, survival, mobility,and gene transcription.[2] It is often considered that the impactof phosphorylation on protein function is related to the con-formational changes it induces. It is therefore of crucial impor-tance to develop techniques to detect, locate, and structurallycharacterize these modifications in peptides and proteins. Theaim of the present work is to contribute to this endeavor.

Mass-spectrometric techniques have proved to be powerfultools for the identification and analysis of phosphorylated pep-tides.[3] Tandem mass spectrometry provides sequence informa-tion for library-based protein identification and the determina-tion of post-translational modifications. Alcohol phosphoryla-tion leads to a mass increase of about 80 units, depending onthe charge state of the phosphate. Mass spectrometry is there-fore an efficient method to detect the presence of one ormore phosphorylations, even for relatively large proteins, pro-vided that high mass accuracy and resolution are available.Tandem mass spectrometry allows the detection of phosphory-lated sites based on the loss of characteristic neutral species[�80 Da (HPO3) and/or �98 Da (H3PO4) in positive-ion MS/MS].[4] It is usually carried out by using collision-induced disso-ciation (CID) to activate and fragment ions. Several studieshave shown that infrared multiple photon dissociation (IRMPD)

at fixed wavelength is also very effective at selectively frag-menting phosphorylated versus unphosphorylated peptides.[5]

Infrared irradiation from a CO2 laser in the 10 mm region is res-onant with one or more phosphate vibrational modes andleads to more efficient fragmentation of phosphorylated pep-tides than non-phosphorylated ones. This provides an easyway to characterize which peptides are phosphorylated withina complex mixture. This has been achieved in both negative-[5]

and positive-ion[6] modes. Sequencing a peptide should in prin-ciple provide the location of any PTM. However, the covalentbonds connecting PTMs to peptide or protein backbones areamong the most fragile, and therefore they are easily brokenunder collisional or IR irradiation conditions, so that sequenc-ing becomes inefficient. The recent development of electron-capture dissociation (ECD) was prompted in part by the obser-vation that PTMs are preserved in this activation mode.[7] Be-cause ECD sequencing is incomplete, a combination of CIDand ECD activation is a method of choice for sequencing andPTM localization in phosphopeptides.[8] The importance of

The protonated, phosphorylated dipeptide [GpY + H]+ is charac-terized by mid-infrared multiple-photon dissociation (IRMPD)spectroscopy and quantum-chemical calculations. The ions aregenerated in an external electrospray source and analyzed in aFourier transform ion cyclotron resonance mass spectrometer,and their fragmentation is induced by resonant absorption ofmultiple photons emitted by a tunable free-electron laser. TheIRMPD spectra are recorded in the 900–1730 cm�1 range andcompared to the absorption spectra computed for the lowestenergy structures. A detailed calibration of computational levels,including B3LYP-D and coupled cluster, is carried out to obtain re-

liable relative energies of the low-energy conformers. It turns outthat a single structure can be invoked to assign the IRMPD spec-trum. Protonation at the N terminus leads to the formation of astrong ionic hydrogen bond with the phosphate P=O group in alllow-energy structures. This leads to a P=O stretching frequencyfor [GpY + H]+ that is closer to that of [pS + H]+ than to that of[pY + H]+ and thus demonstrates the sensitivity of this mode tothe phosphate environment. The COP phosphate ester stretchingmode is confirmed to be an intrinsic diagnostic for identificationof which type of amino acid is phosphorylated.

[a] Dr. C. F. Correia, Dr. C. Clavaguera, Prof. Dr. G. OhanessianLaboratoire des M�canismes R�actionnelsD�partement de Chimie, Ecole Polytechnique, CNRS91128 Palaiseau Cedex (France)Fax: (+ 33) 1-6933-4803E-mail : gilles.ohanessian@polytechnique.fr

[b] Dr. U. Erlekam, Dr. D. ScuderiLaboratoire de Chimie Physique, Universit� Paris-Sud 11CNRS, 91405 Orsay Cedex (France)

[**] IRMPD : Infrared multiple-photon dissociation.

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cphc.200800469.

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phosphorylation is such that improvements to existing analyti-cal techniques are still rapidly appearing.[9] Until now, however,three-dimensional structural characterization in a mass spectro-metric context has remained impossible.

An important breakthrough is now changing this situation,namely, the development of tunable IRMPD within tandemmass spectrometers.[10, 11] Compared to commonly used IRMPDwith fixed-wavelength CO2 lasers, free-electron lasers (FEL) pro-vide access to a wide frequency range that enables vibrationalspectra to be recorded in the mid-infrared region. This has thepotential for distinguishing between isomers, and also be-tween conformers, although the latter is much more difficult.This new spectroscopic tool has already found many applica-tions.[10b] Peptide ions which have been structurally probed byIRMPD include protonated12 and metal-cationized13 oligopepti-des, and a small potassiated protein.[14]

Previously, we obtained the IRMPD signatures of three pro-tonated, phosphorylated amino acids: phosphoserine ([pS +

H]+), phosphothreonine ([pT + H]+), and phosphotyrosine([pY + H]+).[15] The results indicated that phosphate-specificbands exist, as expected, and that they are easily detectableunder IRMPD conditions. Detailed band assignment based onquantum chemical calculations established that some featureswere common to all three species, while others were specific.Among the latter, the C�OP stretch of the phosphate esterwas found to be very different in [pS + H]+ and [pT + H]+ onthe one hand, and in [pY + H]+ on the other. The differencelies in the aliphatic versus aromatic character of the carbonatom, and in additional coupling of C�OP stretching with aro-matic CCH bends for [pY + H]+ . The C�OP stretching modeleads to a band with a maximum at 1080 cm�1 for [pS + H]+

and [pT + H]+ , respectively, while it lies at 1275 cm�1 for [pY +

H]+ . Thus, this band can be used as an intrinsic signature of[pS + H]+ or [pT + H]+ versus [pY + H]+ . Furthermore, asecond, environmental distinction was established. In [pY +

H]+, the amino acid and phosphate moieties are held apartfrom each other by the benzyl group of the tyrosine side chain(see Scheme 1). As a result, the P=O bond has little nonbond-ing interaction. In [pS + H]+ and [pT + H]+ , hydrogen bondingcan be established between the ammonium and phosphategroups. This interaction results in a strong redshift of the P=Ostretching band from 1228 and 1216 cm�1 in [pS + H]+ and[pT + H]+ , respectively, to 1265 cm�1 in [pY + H]+ . This canthen be used as a probe of the environment of the P=O bond.

These results indicate that some of the phosphate vibration-al modes, as well as those of groups that interact with thephosphate group, may be used as sensitive structural probes.Before this can be applied to significantly larger systems, thatis, phosphorylated peptides, we seek to strengthen this tool. Ifthe above reasoning is correct, then extending the chainbeyond the N terminus of [pY + H]+ with another residueshould enable direct interaction of the terminal ammoniumand phosphate groups that leads to P=O stretching and am-monium umbrella frequencies that are similar to those in [pS +

H]+ , while the C�OP stretch of the phosphate ester shouldremain at the same frequency as in [pY + H]+ . Such a situationcan be achieved by transforming [pY + H]+ into [GpY + H]+ . Asshown in Scheme 1, assuming that the most stable isomer in-volves protonation at the N terminus (see below), the longermain chain provided by the glycine (G) residue is enough toensure a strong hydrogen bond between the ammonium ter-minus and the phosphate group. In addition, the longer mainchain should lead to additional features in the IRMPD spec-trum, which we seek to characterize below.

Experimental Section

IRMPD Spectroscopy and CLIO-FEL and MS Operating Parameters:IRMPD spectra were recorded at the Centre Laser Infrarouged’Orsay (CLIO), where an IR-FEL is coupled to a modified BrukerAPEX-Qe Fourier transform ion cyclotron resonance (FT-ICR) massspectrometer.[16] The CLIO IR-FEL is based on a 10–50 MeV linearelectron accelerator.[11] At a given electron energy, the photonenergy is tuned by adjusting the gap of the undulator which isplaced in the optical cavity. The IR-FEL output consists of 8 ms-longmacropulses fired at a repetition rate of 25 Hz. Each macropulse iscomposed of 500 micropulses, each a few picoseconds long andseparated by 16 ns. For a typical IR average power of 500 mW, thecorresponding micropulse and macropulse energies are 40 mJ and20 mJ, respectively. The laser wavelength profile was monitored ateach reading with a monochromator associated with a pyroelectricdetector array (spiricon). The IR-FEL spectral width can be adjustedby tuning of the optical cavity length; the laser spectral width(FWHM) was less than 0.5 % of the central wavelength. The associ-ated output trigger was used to control the optical shutter, whichwas then opened for a controlled number of IR-FEL macropulses.Two different electron energies (42 and 45 MeV) were used inorder to optimize laser power while recording the spectra in the900–1730 cm�1 energy range. The maximum IR power was about900 and 750 mW for electron energies of 42 and 45 MeV, respec-tively. While scanning the undulator gap, the power dropped line-arly by a factor of two at the most.

Peptide MS Operating Parameters: Seventy four micromolar solu-tions of C-terminus-amidated GpY were prepared by mixing 40 mLof peptide stock solution (1 mm), 0.5 mL of H2O/MeOH (50/50), and10 mL of 98 % formic acid. The experiments were performed on amodified FT-ICR mass spectrometer (Bruker APEX-Qe). The FT-ICR isequipped with an Apollo II ESI ion source, a quadrupole mass filter,a collision cell (hexapole), and a 7 T magnet. The ions were gener-ated by electrospray, and the ESI conditions used were as follows:flow rate of 180 mL h,�1 spray voltage of 4600 V, drying-gas flow of4.5 L s,�1 nebulizer gas pressure of 1.5 bar, and drying-gas tempera-ture of 200 8C. The peptide ions were first mass-selected in thequadrupole mass filter and then accumulated in the collision cell

Scheme 1. Schematic 2D structures for [pS + H]+ , [pY + H]+ and [GpY + H]+ .The dotted lines stand for hydrogen-bonding interactions.

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IRMPD Spectroscopy of a Phosphorylated Peptide

filled with Ar over 150 and 250 ms for scans performed at 42 and45 MeV, respectively. Irradiation times of 50 and 100 ms were usedrespectively for the above scans. Subsequently the ions were accel-erated along the axis of the magnetic field, decelerated, and thentrapped in the ICR cell at a background pressure of about 1.5 �10�9 mbar. IRMPD spectroscopy was performed in the ICR cell byfocusing the IR-FEL laser beam with a two-meter focal mirror.

Computational Details : The potential-energy surface (PES) of[GpY + H+] was explored extensively, under the assumption thatprotonation occurs at the N terminus (other protonation sites arediscussed in the Section 2). The system is large enough that explo-ration was initially carried out at the molecular mechanics level,before obtaining reliable energetics and linear absorption spectrafor the most stable structures at the quantum mechanical level.

Conformational Searches: The AMBER99 force field was usedthroughout. Although it may be less accurate than force fields op-timized for organic molecules, its treatment of hydrogen bondingwas deemed useful in the present case. NBO atomic charges wereused, as obtained from the B3LYP/6-31G(d) wavefunction of an ini-tial conformer at its optimized geometry. Trial structures were gen-erated by simultaneous variation of one to six dihedral angles ran-domly selected out of a set of seven. This set was chosen to allowfor full conformational freedom within the main chain and phos-phate fragments, while keeping both on the same side of thephenyl plane (see Figure S1 in Supporting Information). The magni-tude of each variation ranged from 15 to 1808. The successivestructures to which these variations were applied were selected ac-cording to the usage directed method.[17] Geometry optimizationswere then carried out to yield energy minima, with a gradientthreshold of 0.02 kJ mol�1 and a maximum number of one thou-sand steps. After optimization, duplication tests based on geomet-ric and energy criteria were used to eliminate identical conformers.Those with RMS non-hydrogen atom coordinates, dihedral angles,and energy differences below 2.2 �, 58, and 0.02 kJ mol�1, respec-tively, were considered to be duplicates.

An initial search was performed by using the parameters describedabove and 1000 geometry optimizations. The lowest and highestenergy structures thus found were then used as starting points fortwo longer searches using the same parameters but with approxi-mately 10 000 geometry optimizations. The 150 lowest energy con-formers found were kept from each search. These 150 structuresare in a range of 46 kJ mol�1. The results from the two independentconformational searches were then compared, and all structureswith identical AMBER energies (within 0.0004 kJ mol�1) were con-sidered to be duplicates. From this comparison 105 structureswere considered to be unique. All searches were carried out withthe Hyperchem 7.5 package.[18]

Quantum Chemical Calculations: Because of the unreliability ofAMBER energies, all 105 conformers found above were subjectedto geometry re-optimization at the B3LYP/6–31G(d) level, fromwhich 22 unique conformers were selected in a 50 kJ mol�1

window. These were then re-optimized at the B3LYP/6-31 + G(d)level and classified into four different families (A–D) according totheir hydrogen-bonding patterns (see Figures 2 and 3 below). Vi-brational frequency calculations were carried out at the same level.Refined relative energies of these 22 structures were obtained atthe B3LYP/6-311 ++ GACHTUNGTRENNUNG(2d,2p) and MP2/6-311 ++ GACHTUNGTRENNUNG(2d,2p) levels.These calculations were performed with the Gaussian 03[19] pack-age. Because of the lack of dispersion interactions, standard DFTmethods are expected to be less accurate in predicting relative en-ergies of conformers than are MP2 and other post-Hartree–Fock

methods when used with sufficiently extended basis sets.[20] Forpeptides with aromatic residues such as GpY, this may lead to non-negligible errors.

To calibrate the results further, extended calculations were per-formed for structures A_1 and B_1 with original coupled-clusterand DFT methods as implemented in the Turbomole 5.10 programpackage.[21] The CC2 equations are an approximation to the cou-pled cluster singles and doubles (CCSD) equations in which the sin-gles equations are retained in the original form and the doublesequations are truncated to first order in the fluctuation poten-tial.[22, 23] A general empirical dispersion correction has been pro-posed by Grimme for density functional calculations.[24] We used amodified version termed DFT-D below, as recently implemented inTurbomole.[25] For all methods, the resolution-of-the-identity (RI)approximation was employed for molecular orbital two-particle in-tegrals. Provided that optimized auxiliary basis sets are used, theerrors due to this approximation are in general negligible as com-pared to those owing to incompleteness of the one-electron basisset. For DFT analytical frequency calculations, the RI approximationwas not used. Structures A_1 and B_1 were subjected to geometryoptimization and frequency calculation at the B3LYP-D and RI-MP2levels with the SVP basis set, which is similar in size to 6-31 + G(d).Final energies at these geometries were computed at the B3LYP-D/TZVPP, RI-MP2/TZVPP, and RI-CC2/TZVPP levels, where again theTZVPP basis set is similar in size to 6-311 ++ GACHTUNGTRENNUNG(2d,2p). The coupledcluster CC2 level is expected to provide an accurate referenceagainst which all other methods can be compared.

Calculated frequencies are in general blueshifted when comparedto the experimental ones. This is due in part to the harmonic ap-proximation, where anharmonic effects due to coupling of vibra-tional modes are not taken into consideration. This is expected tocontribute to the discrepancies that are often observed betweenexperimental and computed frequencies.[26] Provided an appropri-ate scaling factor is used, hybrid DFT methods have been shownto be very efficient in reproducing the observed fundamental IRfrequencies, with B3LYP average absolute differences of about30 cm�1 (and of 20–25 km mol�1 in IR intensities).[27, 28] A uniformscaling factor of 0.9614 has been proposed to correct IR frequen-cies computed at the B3LYP/6-31G(d) level for small organic mole-cules.[28, 29] Several scaling factors specific to phosphorus-containingmolecules have also been derived. Among these, corrections vary-ing between 0.98 and 1.05 have been proposed for the P=O andP�O stretching modes in compounds such as phosphine oxides,phosphites, and phosphorus oxides.[30] Analogous studies for phos-phate-containing molecules do not appear to exist to date. Basedon our previous work on phosphorylated amino acids, we decidedto leave our computed frequencies unscaled below 1300 cm�1

(where phosphate contributions are largely dominant) and scalethose above 1300 cm�1 by 0.96 (where phosphate contributionsare negligible). There is currently no experience accumulated onusing B3LYP-D calculations for deriving IR spectra. This work maybe considered as a benchmark in this respect. In the absence ofprevious calibration studies, we compared the B3LYP and B3LYP-Dfrequencies, and decided to apply the same scaling factors toboth: 1 for the phosphate modes below 1300 cm�1, and 0.96 forthe non-phosphate modes above 1300 cm�1. The computed IRspectra of all rotamers of structural family A (see below) are shownin Figure S5 of the Supporting Information. Their relative Gibbsfree energies at all computational levels used are gathered inTable S2 of the Supporting Information.

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2. Results

2.1. IRMPD Spectrum of [GpY + H]+

Irradiation of [GpY + H]+ at wavelengths in the 900–1730 cm�1

range leads to similar fragmentation patterns (see Figure S2 ofSupporting Information), although differences in absorptioncross sections lead to differing fragmentation ratios at thewavelengths of the main bands of the IRMPD spectrum. Thefragmentation ratio is defined as �ln ACHTUNGTRENNUNG(P/F+P), where P is theabundance of intact precursor ions, and F the sum of theabundances of fragment ions produced by IRMPD at a givenphoton energy. The main fragment ion at m/z 301 results fromloss of NH3 from the N-terminus ammonium or C-terminusamide group. Both pathways lead to the same fragment-ionstructure after formation of a six-membered ring between thetwo termini (see Figure S3 of Supporting Information). Thisstable structure helps to explain the relatively high intensity ofthis first fragment ion. When enough internal energy is avail-able, ring opening and successive eliminations of CO and ofCH2NH plus CO result in the formation of two additional frag-ment ions at m/z 273 and 216, respectively, the latter of whichis more intense. This is the well-documented phosphotyrosineimmonium ion that is often observed in MS/MS spectra of pY-containing peptides,[31] especially when pY is close to the N ter-minus.[32]

The IRMPD spectrum of [GpY + H]+ is shown in the lowerpart of Figure 1. It displays broad features at 900–1000 and1200–1300 cm�1, a series of four well-resolved narrower peaksin the 1450–1600 cm�1 range, and a slightly broader band at1650–1700 cm�1. While some of these features can be assignedon the basis of our previous work,[15] full assignment requiredextensive calculations to ensure that the lowest energy struc-tures were identified and their vibrational properties deter-mined.

2.2. Computed Structures

In phosphorylated amino acids, protonation at the aminogroup is more favorable than at the other possible sites (acid,phosphate P=O, and phosphate ester oxygen atoms) by 50–200 kJ mol�1.[15] We expected the same trend to hold here, andthus focused first on amine protonation. Other protonationsites were also considered (see below).

The extensive search for amino-protonated structures de-scribed in the Experimental Section led to a number of con-formers, all with a common motif : ionic hydrogen bonding be-tween one of the three ammonium N�H bonds and the P=Ooxygen atom. This is the most polar bond with the most basicof the four phosphate oxygen atoms. The lowest energy struc-tures A_1 and B_1 are shown in Figure 2, which also gives rela-tive free energies are indicated in Figure 2. Structure A_1 has asingle interaction between the main chain and the phosphategroup. Its stability is completed by a weaker ionic interactionbetween another ammonium N�H bond and the peptidic C=Ogroup. Structure A_1 was the most stable found, but anotherlow-energy structure located was B_1, which has two interac-

Figure 1. Experimental IRMPD spectrum for [GpY + H]+ (bottom) and theo-retical IR spectra for the lowest energy conformer of family A, computed atthe B3LYP/6-31 + G* (middle ) and B3LYP-D/SVP (top) levels. The experimen-tal spectrum shows the fragmentation ratio as a function of photon energy.

Figure 2. Most stable conformers of the four low-energy structural families(A, AI, B, and BI) for [GpY + H]+ . Relative Gibbs free energies computed atthe MP2/6-311 ++ (2d,2p)//B3LYP/6-31 + G(d) level are reported for eachconformer.

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IRMPD Spectroscopy of a Phosphorylated Peptide

tions between the main chain and the phosphate group. Be-sides the N�H···O=P interaction, as in family A, the main chainis oriented in such a way as to allow for a PO�H···O=C hydro-gen bond between one of the phosphate hydroxyl groups andthe peptidic C=O group. This structure is only 1 kJ mol�1

higher in enthalpy than A_1, while it lies 6 kJ mol�1 higher infree energy, probably because the two hydrogen bonds makeit more constrained than the single hydrogen bond in A_1.

Somewhat higher in enthalpy and free energy are structuralfamilies AI and BI, which have similar hydrogen-bonding pat-terns to A and B, respectively. The main difference is that in AIand BI, the peptide bond is in its cis configuration, while it istrans in A and B. While cis peptides are not commonly takeninto account in natural peptides and proteins, they cannot beruled out a priori. However, their higher energies make themless likely to be formed in the present case.

Other types of structures were found in which the N�H···O=

P motif is present. The lowest energy conformer that wasfound for each type is shown in Figure 3. Structures of type C

have an additional ionic hydrogen bond between another NHgroup of the N-terminus ammonium and C-terminus C=Ogroups. Structures of type D again have the same two hydro-gen-bonding interactions of the ammonium group as in A, butin addition the phosphate group is involved in a PO�H···O=Cinteraction with the C terminus. These combinations of hydro-gen bonds are significantly less favorable than those in A_1(D 1 is + 26 kJ mol�1 higher in energy than A_1, see Figure 3),probably because they lead to more structural constraints thatprevent each individual interaction from being optimal.

All other structures were found to be of significantly higherenergy. These include structures in which the hydrogen bondbetween the ammonium and the phosphate groups is of the

N�H···O(H)P type, and those in which these groups do not in-teract at all. In the former case, MP2 calculations provide Gibbsfree energies that are typically 50 kJ mol�1 higher than that ofA_1, while structures in which the ammonium charge solvationis only with the main chain are even less stable, typically80 kJ mol�1 above A_1.

Protonation at any phosphate oxygen atom or at the C ter-minus is expected to lead to much less stable structures of[GpY + H]+ , as was found for phosphorylated amino acids.[15]

For instance, MP2 calculations on protonation at the C-terminaloxygen atom yield Gibbs free energies typically 80 kJ mol�1

higher than that of A_1.Protonation at the peptidic oxygen atom must also be con-

sidered. It has been shown repeatedly that protons are mobilealong peptide main chains,[33] and recent IRMPD and theoreti-cal studies have shown that there is not much energetic differ-ence between protonation at the N terminus and at the nextpeptidic oxygen atom in di- and tripeptides.[12c–f] We thereforesearched for structures in which the peptidic oxygen atom isprotonated. It turns out that none of these structures is of lowenergy. The reason is that the charge may be partially stabi-lized by folding of the main chain, but this is much less favora-ble than interaction of the charge with the highly polar phos-phate group, which can only be achieved if the terminal aminogroup is protonated. As a result, protonation at the peptidicoxygen atom gives structures that are about 50 kJ mol�1 lessstable than the most stable amine-protonated structure.

To complete the picture of the low-energy structures of[GpY + H]+ , the rotamers of the phosphate group must betaken into account. Work on phosphorylated amino acids[15]

has shown that several rotamers exist with very similar ener-gies and very low interconversion barriers, so that the phos-phate group must be treated as a fluxional motif without astrictly defined conformation at room temperature. Thus, wecarefully considered all possible rotamers in structures oftypes A and B. As a general rule, each O�H bond has a stableposition when it is oriented trans to one of the P�O or P=Obonds. When two O�H bonds are free of hydrogen bonds asin A, this leads to six different rotamers. In B, the single avail-able hydroxyl group restricts this number to three. Table 1 liststhe relative energies for A and B rotamers, together with thepercentage of population estimated by assuming a Maxwell–

Figure 3. Most stable conformers of two high-energy structural families (Cand D) for [GpY + H]+ . Gibbs free energies relative to A_1, computed at theMP2/6-311 ++ (2d,2p)//B3LYP/6-31 + G(d) level are reported for each confor-mer.

Table 1. Relative Gibbs free energies [kJ mol�1] and Maxwell–Boltzmannpopulations at 298 K for the phosphate rotamers of [GpY + H]+ families Aand B, computed at the MP2/6-311 ++ G ACHTUNGTRENNUNG(2d,2p)//B3LYP/6-31 + G(d) level.

MP2 % A and B based on DG

A_1 0.0 28.7A 2 0.4 24.2A 3 1.9 13.0A 4 2.2 11.6A 5 2.8 8.9A 6 3.7 6.2B_1 6.0 2.5B 2 6.1 2.5B 3 6.4 2.2

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Boltzmann distribution at 298 K. Energies are reported relativeto A_1.

2.3. Refinement of Computed Energetics and Spectra

As shown in Table 1, the relative free energies of phosphate ro-tamers of families A and B are such that the populations arenearly negligible for the B family. This would mean that the as-signment of the IRMPD spectrum must be based on the com-puted spectra for the A family only. The computed free-energydifferences are small enough, however, that an additionalmethod calibration appears to be needed. As recalled above,DFT methods lack a description of dispersion interactions. Thismay have a significant influence on both relative energies andvibrational spectra of [GpY + H]+ conformers, since mainchain/aromatic ring interactions are expected to occur. Wetherefore re-optimized the structures of A_1 and B_1 and com-puted vibrational spectra at the B3LYP-D level, which includesan empirical 1/r6 term to account for dispersion interactions,and at the RI-MP2 level. The B3LYP and B3LYP-D geometriesare very similar, except for a few nonbonding distances whichare significantly reduced (by 0.1 to 0.2 �) at the B3LYP-D level,as expected due to the inclusion of dispersion interactions.The main decrease in distance occurs between the peptidicoxygen atom and the nearest aromatic carbon atom. Smallereffects are seen in the distances between the peptidic oxygenatom and the nearest ammonium hydrogen atom, and be-tween another ammonium hydrogen atom and the phosphateoxygen atom, with which it is involved in hydrogen bonding(see Table S1 and Figure S4 in Supporting Information). Thevery good agreement between B3LYP-D and RI-MP2 geome-tries indicates that the correction in B3LYP-D is able to capturedispersion interactions in a satisfactory manner. The geometrydifferences between B3LYP and B3LYP-D are expected toinduce some frequency displacements in the computed spec-tra. The full description of frequencies and vibrational modescomputed at the B3LYP/6–31 + G(d) and B3LYP-D/SVP levels isgiven in Table 2 and are be discussed below to assign theIRMPD spectrum.

Finally, we improved the level used for final energy calcula-tions, in order to derive the appropriate level for comparingthe relative energies of families A and B: B3LYP, B3LYP-D andMP2 geometries were obtained. At these geometries, B3LYPand B3LYP-D final energetics can be compared. The accuracyof these values can be assessed by comparison to coupledcluster CC2 results. Calculations at the MP2 level were also car-ried to complete this benchmark calibration. Table 3 lists theenthalpy and Gibbs free energy of B_1 relative to A_1 (relativeGibbs free energies of all rotamers of family A are given inTable S2, Supporting Information). When the B3LYP/6–31 + G(d)geometries are used, all methods except B3LYP are in excellentagreement, predicting B_1 to have a slightly more favorableenthalpy, but A_1 to have a more favorable free energy. Thistrend is reproduced by all methods including B3LYP-D whenthe B3LYP-D geometries are used. Very similar results are alsoobtained at the MP2 geometries. These results provide an ac-curate picture of the low-energy structures of [GpY + H]+ . It

Table 2. Measured and calculated vibrational frequencies [cm�1] for themost stable rotamer A_1 of [GpY + H]+ . Theoretical spectra were comput-ed at B3LYP/6-31 + G(d) and B3LYP-D/SVP levels. Intensities [km mol�1] arereported in parentheses.

Wavenumber Vibrational mode[a]

IRMPD B3LYP (a) B3LYP-D (b)

937 922 918 (a) n P�OH, d POH, d CCH*ACHTUNGTRENNUNG(267) ACHTUNGTRENNUNG(262) (b) d POH967 947 959 (a) n P�OH, n P-OC, d POH, d CCH*ACHTUNGTRENNUNG(523) ACHTUNGTRENNUNG(493) (b) d POH, n P�OC, d CCH*1005 1011 991 (a) d POHACHTUNGTRENNUNG(134) ACHTUNGTRENNUNG(174) (b) d POH, n P�OC, n P�OH, d CCH*

1034,1039

1028,1033

(a) d POH, d CCH*, n P�OHACHTUNGTRENNUNG(60, 90) ACHTUNGTRENNUNG(26, 99) (b) d POH, d CCH*1139 1134,

1148(a) d CCH*, d r NH3

+ , d w CH2 (G)

(30) ACHTUNGTRENNUNG(33, 33) (b1) d r NH3+ , d CCH*; (b2) d r NH3

+ ,n CCH (G,Y)

1193 (a) d CCH*, d PO�C(68)

1219 1214,1219

1220,1239

(a) d t CH2 (Y), d CCH (Y), d CCH*ACHTUNGTRENNUNG(23, 58) ACHTUNGTRENNUNG(48, 83) (b1) n P-OC, d CCH*, n P=O; (b2) n P=

O, n PO�C, d CCH (Y)1248 1257,

12601261,1279

(a) n P=O, d POH, d CNH (Y), d CCH(Y), d CCH*, d r NH3

+ACHTUNGTRENNUNG(98, 343) ACHTUNGTRENNUNG(445, 29) (b1) n P=O, d PO�C, d CCH*; (b2) d

CNH (Y), d CCH (G,Y)1289 1297

(23) (b) d r NH3+ , d t CH2 (G)

1336 1311 (a) d w CH2 (G), d CNH (Y), d CCH (Y),d CNH (C term)

(79) (34) (b) d w CH2 (G)1372 1366 (a) amide II (Y), d CCH (Y), d w CH2 (G)ACHTUNGTRENNUNG(162) ACHTUNGTRENNUNG(163) (b) d CCH (Y), d CNH (C term)

1389,1407ACHTUNGTRENNUNG(50,20) (b1) d t CH2 (G); (b2) d t CH2 (Y)1433 1420ACHTUNGTRENNUNG(210) (b) d u NH3

+

1470 1485,1489 1479 (a) d u NH3+ , d CCH*ACHTUNGTRENNUNG(94,288) (85) (b) d CCH*

1503 1509 1503 (a) amide II (Y)ACHTUNGTRENNUNG(193) ACHTUNGTRENNUNG(212) (b) amide II (Y)1529 (a) d u + d s NH3

+

(23)1540 1536ACHTUNGTRENNUNG(168) (b) d s NH2 (C term)1590 1587 1585 (a) d s NH2 (C term)ACHTUNGTRENNUNG(116) (48) (b) d s NH3

+

1616 (a) d u + d s NH3+

(34)1668 1666 (a) d s NH3

+

(46)1683 (a) n C=O (G)ACHTUNGTRENNUNG(244)1703 1713 (a) n C=O (C term), n C=O (G), d CNH

(C term)ACHTUNGTRENNUNG(249) ACHTUNGTRENNUNG(206) (b) n C=O (G)1747ACHTUNGTRENNUNG(251) (b) n C=O (Cter)

[a] *, r, s, t, u, and w, denote aromatic, rocking, scissoring, twisting, um-brella, and wagging motions, respectively.

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IRMPD Spectroscopy of a Phosphorylated Peptide

appears that B3LYP cannot be used to obtain final energetics,and that B3LYP-D is the best compromise between tractabilityand accuracy. The structural differences between B3LYP andB3LYP-D are relatively small and have little impact on the ener-getics. Based on these results, a clear picture emerges: theMaxwell–Boltzmann distribution, based on relative free ener-gies, indicates that only structures belonging to family A aresignificantly populated at 298 K (see Table 1).

2.4. Interconversion of Structures A and B.

Given the above results, the populations of structures B are in-significant at room temperature if A and B are in equilibrium.It may, however, be the case that a large activation barriermust be surpassed to interconvert them. In such a case, struc-tures A and B could be formed concurrently in the ESI process,and then coexist as independent populations in the gas phase.To settle this issue, we computed an interconversion pathwaybetween A_1 and B_1. There are seven torsions around singlecovalent bonds that are significantly modified between A_1and B_1. Several attempts were made to determine a concert-ed or a stepwise mechanism. A concerted process was theonly one obtained, involving a single transition state betweenA and B, with a very small imaginary frequency of 29 i cm�1.The associated transition motion involves reorientation of thePO�H bond to establish a hydrogen-bonding interaction withthe peptidic carbonyl group and of the backbone torsions tofavor this interaction. The free energy of activation for this in-terconversion is computed to be 13 kJ mol�1 relative to A_1 atthe MP2/6-311 ++ GACHTUNGTRENNUNG(2d,2p)//B3LYP/6-31 + G(d) level. This lowactivation barrier is only slightly larger than those for the inter-conversion of phosphate rotamers. It appears then that A andB are in equilibrium at 298 K. Therefore, taking a Maxwell–Boltzmann population average at 298 K appears to be legiti-mate, regardless of which structures were formed by the elec-trospray process. As a consequence, the assignment of theIRMPD spectrum described in the next section is based on Aonly. As already discussed for phosphorylated amino acids,[15]

the frequency of a given vibrational mode is found to varyonly slightly among the rotamers. Thus, the assignment can besafely based on that of lowest energy, A_1. The other rotamersdo have a strong influence on band width whenever the vibra-

tional mode has a dominant contribution from the phosphategroup, as will be seen below.

2.5. Assignment of the IRMPD spectrum

The IRMPD and computed spectra for structure A_1 at theB3LYP and B3LYP-D levels are summarized in Table 2. The as-signments are very similar at these two levels, with only minordifferences between the mode components in most cases.There are only two significant differences, which are discussedbelow. Bands with computed intensities smaller than20 km mol�1 are not described. Based on our previous work,[15]

frequencies of phosphate modes (all below 1300 cm�1) werenot scaled, while those of all other modes (all above1300 cm�1) were scaled by 0.96.

The first strong absorption feature (located between 920and 1020 cm�1) results from at least three partially overlappingbands. The first two, with intensity maxima at 937 and967 cm�1, can be assigned to the antisymmetric coupling ofboth P�OH stretches, or of one P�OH and the P�OC stretch(symmetric mixtures of P�OH and P�OC stretches are predict-ed to appear in the 850 and 900 cm�1 range and are out ofthe scope of this study). There is often strong coupling withone of the POH bends. The third band, located at 1005 cm�1,can be attributed to POH bending modes. Bands dominatedby POH bending are computed to appear in the 1000–1040 cm�1 range. Their couplings and therefore their precisefrequencies depend on the rotamer. There are some contribu-tions of aromatic in-plane CCH bending modes here, sincetheir intrinsic frequencies are in this range. The second strongabsorption feature appears in the 1180–1300 cm�1 range. Itsshape and width suggests the presence of two to four overlap-ping bands. There may be a shoulder near 1200 cm�1, and thefirst clear band peaks at 1219 cm�1. The shoulder would corre-spond to in-phase and out-of-phase coupling of the PO�Cstretch and an in-plane CCH aromatic bend. The peak at1219 cm�1 is assigned to mixing of twisting motion of CH2 inpY with the previous modes. The second band, at 1248 cm�1,has a dominant contribution from P=O stretching.

Above 1300 cm�1, all bands are computed to be free ofphosphate contributions. In the region between 1300 and1370 cm�1, several bands of small to strong intensity are pre-dicted by the calculations. They correspond to coupling ofamide III modes with a strong contribution from the waggingmotion of the CH2 group of G or of the CH group of pY. Carefuloptimization of laser power and wavelength scanning in thisregion revealed absorption, but it was weak and unstructured.The weak absorption band at 1433 cm�1 may be assigned tothe umbrella motion of the ammonium group.

The region of strong absorption between 1450 and1600 cm�1 displays four well-resolved bands. The band at1470 cm�1 is attributed to a combination of ammonium um-brella motion and in-plane bending of the aromatic CCHangles. The B3LYP-D calculations also place the aromatic CCHbend at this position, but the ammonium umbrella motion ispredicted to be more to the red, in less good agreement withexperiment. The most intense band, at 1503 cm�1, corresponds

Table 3. Enthalpy and Gibbs free energy [kJ mol�1] of B_1 relative to A_1,computed with different methods and basis sets.

DH DG

B3LYP/6-311 ++ G ACHTUNGTRENNUNG(2d,2p)//B3LYP/631 + G(d) 11.5 19.0MP2/6-311 ++ G ACHTUNGTRENNUNG(2d,2p)//B3LYP/6-31 + G(d) �1.5 6.1RI-MP2/TZVPP//B3LYP/6-31 + G(d) �0.8 5.4RI-CC2/TZVPP//B3LYP/6-31 + G(d) �2.3 5.2B3LYP-D/TZVPP//B3LYP-D/SVP �4.2 5.4RI-MP2/TZVPP//B3LYP-D/SVP �2.5 7.2RI-CC2/TZVPP//B3LYP-D/SVP �4.2 5.4RI-MP2/TZVPP//ri-MP2/SVP �1.2 8.8RI-CC2/TZVPP//ri-MP2/SVP �2.8 7.2

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G. Ohanessian et al.

to the amide II mode of the peptide bond. The less intenseband at 1540 cm�1 is one for which some disagreement be-tween B3LYP and B3LYP-D predictions is found. In the B3LYP-Dspectrum this band is assigned to the scissoring motion of theC-terminus NH2 group, while it has no equivalent in the B3LYPspectrum. In the latter, this mode would be assigned to theband peaking at 1590 cm�1, whereas B3LYP-D calculations attri-bute it to scissoring of the ammonium group involving thetwo hydrogen-bonded hydrogen atoms.

The experimental band peaking at 1670 cm�1 is assigned tothe amide I modes. That corresponding to the peptidic COgroup is predicted to occur at 1680 and 1713 cm�1 at theB3LYP and B3LYP-D levels, respectively. Because of strongthrough-space dipole coupling with ammonium N�H bonds, asignificant contribution from ammonium deformation is pres-ent in this mode. The amide I frequency of the C terminus ispredicted to be 1702 or 1747 cm�1 (B3LYP and B3LYP-D, re-spectively). This is in less good agreement with experiment. Ithas been repeatedly reported in the literature that amide Imodes are sometimes not responsive in IRMPD experi-ments.[12d] This may be the case here for the C-terminus amidegroup, which computations predict to lie somewhat “outside”of the molecule, without any significant interaction with theother functional groups.

3. Discussion

The IRMPD bands have been assigned on the basis of the com-puted IR spectra of the lowest-energy conformers of family A.In this section, we compare these results to those obtainedpreviously for phosphorylated amino acids. This is useful fordeciding whether some of the phosphate bands can be re-garded as specific to certain residues and how they are influ-enced by their environment.

Figure 4 compares the IRMPD spectra of [GpY + H]+ and[pS + H]+ (top) and of [GpY + H]+ and [pY + H]+ (bottom).Band assignments for [pS + H]+ and [pY + H]+ IRMPD spectrahave been reported previously.[15] The first experimental bandfor [GpY + H]+ , around 950 cm�1, was assigned to the couplingof POH stretches and bends together with a contribution froman aromatic CCH bend. This band is equally present in [pY +

H]+ . In [pS + H]+ , due to the absence of an aromatic ring, thisband is slightly redshifted. A second band around 1000 cm�1

in [GpY + H]+ is assigned mainly to POH bending. In thisregion a weak band in the spectrum of [pY + H]+ peaks at1026 cm�1, and the spectrum of [pS + H]+ shows a weak ab-sorption shoulder just below 1000 cm�1. Both are mainly dueto POH bends. It appears that the combination of strong hy-drogen bonding of the P=O group to the ammonium moiety(as in [pS + H]+ but not in [pY + H]+) and coupling with aro-matic CCH bends (as in [pY + H]+ but not in [pS + H]+) leadsto increased intensity for this mode in [GpY + H]+ .

The region between 1050 and 1120 cm�1 is absorption-freefor [GpY + H]+ and [pY + H]+ , while [pS + H]+ has an intenseband around 1080 cm�1, assigned to the PO�C stretch. The dif-ference between [GpY + H]+ and [pY + H]+ on the one handand [pS + H]+ on the other is due to the carbon atoms in-

volved in the PO�C stretch, which are aromatic in [GpY + H]+

and [pY + H]+ but aliphatic in [pS + H]+ . In addition, thisstretch is coupled to the aromatic CCH bend in [GpY + H]+

and [pY + H]+ , and therefore it is significantly blueshifted (seebelow). The region around 1190 cm�1 is also absorption-freefor [GpY + H]+ , while there is a well defined band around1185 cm�1 for [pY + H]+ and a partially overlapped band at1190 cm�1 for [pS + H]+ . These bands were assigned to theacidic COH bend in the phosphorylated amino acids, with noequivalent in the [GpY + H]+ spectrum because the C terminusis amidated. The [GpY + H]+ spectrum shows an intense andbroad feature between 1200 and 1300 cm�1, assigned to twooverlapping bands corresponding to the PO�C and P=Ostretches, and computed at 1219 and 1260 cm�1, respectively.As discussed above, the PO�C stretching frequency is stronglyblueshifted compared to that of [pS + H]+ , by about 140 cm�1.In the case of [pY + H]+ , a further blueshift of 50 cm�1 occursbecause coupling with the nearby P=O stretch is weaker inthis case due to the lack of hydrogen bonding to the ammoni-um group. Overall the PO�C stretch is a good sensor of the ali-phatic versus aromatic side chain to which the phosphategroup is attached, but well-resolved spectra could also makethis band environment-sensitive. The P=O bond is expected tohave similar environments in [GpY + H]+ and [pS + H]+ , whereit establishes a strong hydrogen bond with the ammoniumgroup that is lacking in [pY + H]+ . Theory predicts intensebands around 1260 and 1240 cm�1 for [GpY + H]+ and [pS +

H]+ , respectively, and a much less intense band at 1295 cm�1

Figure 4. Comparison of the IRMPD spectra of [GpY + H]+ and [pS + H]+

(top) and of [GpY + H]+ and [pY + H]+ (bottom). The experimental spectrashow the fragmentation ratios as functions of photon energy.

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IRMPD Spectroscopy of a Phosphorylated Peptide

for [pY + H]+ . As can be seen in Figure 4, the superposition ofthe bands between 1220 and 1290 cm�1 shows very clearly theinfluence of the phosphate environment on the spectra of[GpY + H]+ and of the phosphorylated amino acids.

The region between 1300 and 1400 cm�1 is absorption-freefor [GpY + H]+ and [pS + H]+ , while [pY + H]+ has a band peak-ing at 1350 cm�1, assigned to the C�C stretch and COH bendat the C terminus, coupled with CCH wagging at the a-carbonatom and some rocking motion of the ammonium group. Thisis the analogue of the amide III band for a carboxylic acid. Asdiscussed above and shown in Figure 1, a relatively intenseamide III band is computationally predicted to appear in thesame region for [GpY + H]+ . However, careful scanning andlaser-power optimization in this region only revealed weak andunstructured absorption. It is unclear why analogous acid andamide III bands appear not to be equally responsive underIRMPD conditions.

The spectrum of [GpY + H]+ shows a band at 1470 cm�1 as-signed to the NH3

+ umbrella motion mixed with aromatic CCHbending. In [pY + H]+ this band is redshifted to about1450 cm�1, since the ammonium group is not constrained tothe P=O group by a hydrogen bond. In [pS + H]+ , on the con-trary, this hydrogen bond exists, but the absence of couplingwith aromatic modes leads to a blueshift of this band to 1510–1530 cm�1. As a result, this band has varying composition andappears at rather different frequencies in all three cases. Theintense band with a maximum at 1503 cm�1 for [GpY + H]+

arises from the peptidic amide II motion, and therefore it hasno equivalent in either [pS + H]+ or [pY + H]+ . For the samereason, the band of [GpY + H]+ at 1540 cm�1 that is assignedto the scissoring mode of the C-terminus NH2 group has noequivalent in phosphorylated amino acids in which the C ter-mini are not amidated. In addition, in the [pY + H]+ spectrumthe band around 1520 cm�1 arises from an aromatic CCHbending, blueshifted relative to that of the dipeptide men-tioned above because there is no NH3

+ umbrella componentin this case. The last weak band in this region, around1590 cm�1, is assigned to an HNH scissoring motion of NH3

+ . Itis common to [GpY + H]+ and [pY + H]+ , and is not seen for[pS + H]+ , presumably due to experimental loss of laser powerwithin this region.[15]

Finally, [GpY + H]+ has one intense feature around1670 cm�1 that corresponds to the amide I motion, a convolu-tion of two bands arising from the peptidic C=O stretch (tothe red) and to the C-terminus C=O stretch (to the blue). Hereagain no equivalent can exist for [pS + H]+ or [pY + H]+ , sinceneither has an amide group. The acidic C=O stretch is signifi-cantly blueshifted as expected, as seen in the spectrum of[pY + H]+ . That of [pS + H]+ was not recorded in this region; itwould be expected to show a C=O stretching band very similarto that of [pY + H]+ .

4. Conclusions

IRMPD experiments were successfully carried out on the pho-phorylated peptide [GpY + H]+ . Extensive DFT and ab initio cal-culations allowed assignment of all experimental bands within

the 900–1730 cm�1 fingerprint region. Two low-energy amine-protonated conformers A_1 and B_1 were identified. Theirfree-energy barrier of interconversion was computed to be assmall as 13 kJ mol�1 relative to the most stable A_1, and thusthe two conformers are in equilibrium at room temperature.Based on a careful calibration of computational levels, we con-cluded that the free energy of B_1 is high enough that thisconformer is not significantly populated at room temperature.Several low-energy rotamers of A_1 were identified and are ex-pected to contribute to broadening of the experimentalIRMPD absorption bands. From the comparison of the IRMPDspectrum of [GpY + H]+ with those of [pS + H]+ and [pY + H]+ ,previous speculations regarding the environmental depend-ence of the vibrational signatures of phosphorylation wereconfirmed. Extending the chain beyond the N terminus of[pY + H]+ with a glycine residue enabled direct interaction ofthe terminal ammonium and phosphate groups and led to aP=O stretching frequency similar to that of [pS + H]+ . On theother hand, the C�OP stretching mode of the phosphate esterPO�C bond was confirmed to be intrinsic to the phosphorylat-ed residue, and therefore similar in [GpY + H]+ and in [pY +

H]+ but very different in [pS + H]+ . The environmental sensitiv-ity will be probed as well in the future, and the results shouldprove useful for interpretation of the IRMPD spectra of largerphosphorylated peptides. Such studies are currently underway.

Acknowledgements

Dr. O. P. Balaj is thanked for his help with the preliminary experi-ments. Acquiring the FT-ICR mass spectrometer used in this workwas made possible by the support of the European Community,through the NEST program EPITOPES project 15637. Supportfrom the ANR Blanc PROBIO 2006-BLAN-0002 project is alsogratefully acknowledged, in particular for the post-doctoral fel-lowship of U.E. C.F.C thanks Fundażo para a CiÞncia e Tecnolo-gia (SFRH/BPD/22482/2005) and Ecole Polytechnique for postdoc-toral grants. We thank Dr. J. M. Ortega and the CLIO team fortechnical assistance and T. Besson and C. Boisseau for their helpwith the operation of the FT-ICR mass spectrometer.

Keywords: ab initio calculations · density functionalcalculations · IR spectroscopy · mass spectrometry · peptides

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IRMPD Spectroscopy of a Phosphorylated Peptide