Infrared Multiple Photon Dissociation Spectroscopy and

Article pubs.acs.org/ac

Infrared Multiple Photon Dissociation Spectroscopy and Computational Studies of O-Glycosylated Peptides Kazuhiko Fukui*,† and Katsutoshi Takahashi‡ †

Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology, 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan ‡ Super-Spectroscopy System Research Group, Research Institute of Instrumentation Frontier, National Institute of Advanced Industrial Science and Technology, 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8566, Japan S Supporting Information *

ABSTRACT: The infrared multiple photon dissociation (IRMPD) spectra of O-glycosylated peptides in the gas phase were studied in the IR scanning range of 5.7−9.5 μm. Fragmentation of protonated and sodiated Oglycopeptides was investigated using electrospray ionization (ESI) Fouriertransform ion cyclotron resonance (FTICR) mass spectrometry (MS) with a free electron laser (FEL). FEL is used in the IRMPD technique as a tunable IR light source. In the IRMPD spectroscopic analysis of the protonated Oglycopeptide, fragment ions of the b/y and B/Y types were observed in the range of 5.7−9.5 μm, corresponding to the cleavage of the backbone in the parent amino acid sequence and glycosyl bonds, whereas the spectra of the sodiated glycopeptide showed major peaks of photoproducts of the B/Y type in the range of 8.4−9.5 μm. The IRMPD spectra of the Oglycopeptides were compared with simulated IR spectra for the structures obtained from the molecular dynamics.

P

connects the peptide to the glycan, is more labile than the peptide bond during collision-induced dissociation (CID) in the mass spectrometer. A novel strategy for selective cleavage of peptide bonds from O-glycoside bonds is therefore desired for the direct analysis of O-linked glycopeptides by a mass spectrometer. Recently, the utility of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) mass spectrometry (MS) has been demonstrated for the sequence analysis of PTMs. In terms of diversity of cleavages, the fragmentation efficiency of ECD and ETD is superior to that of other activation/dissociation procedures. The method for cleaving the chemical bonds in ECD and ETD appears to have the greatest usefulness for de novo sequencing of small proteins10−12 and for determining post-translational modification sites.13−15 Large proteins, however, are still intractable by this method.12 Moreover, precursor ions with a low abundance are not amenable to ECD, because the large fragmentation diversity, i.e., the large number of possible cleavage sites in ECD, may result in a weakness of intensity of individual fragment peaks. In this study, we investigate O-linked glycopeptides using infrared multiple photon dissociation (IRMPD) with a tunable IR free electron laser (FEL)16−19 in conjunction with MS,

roteomics has contributed to large-scale identification of proteins and determination of their expression levels in the living body. The next important subject in protein science is functional elucidation, which requires analysis of post-translational modifications (PTMs) tuning the functions of proteins. Glycosylation is a relatively common PTM that is associated with protein folding, stability, cellular localization, recognition, and immune reactions, all of which are necessary for normal biological processes.1,2 Glycosylation analysis of proteins has three approaches: structural analysis of glycan moieties released from glycoproteins,3,4 determination of glycosyl sites using the resulting peptide isotope-tagged or chemical-tagged from the glycan-releasing reaction,5−7 and direct analysis of glycopeptides to determine both the glycosyl sites and the structure of the glycan moiety.8,9 However, once glycans are released from proteins, information as to which glycans are attached to which positions of the peptides is completely lost. Direct analysis of glycopeptides is therefore the best approach. Currently, mass spectrometry is the most promising tool for these three approaches. In N-linked glycopeptides, the number of glycan moieties is normally one in a single peptide. Additionally, the consensus sequence for N-linked glycosylation often helps to determine the glycosyl site. These facts make the direct analysis of N-linked glycopeptides relatively easy. On the other hand, analysis of O-linked glycopeptides is quite difficult, despite their biological importance. They often have plural glycan attachment sites and do not have a consensus sequence, unlike Nlinked glycopeptides. Additionally, the O-glycoside bond, which © 2012 American Chemical Society

Received: September 16, 2011 Accepted: January 26, 2012 Published: January 26, 2012 2188

dx.doi.org/10.1021/ac202379v | Anal. Chem. 2012, 84, 2188−2194

Analytical Chemistry

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Figure 1. Peptide sequence (Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln: PSDTPILPQ) and glycan structure of the investigated O-glycosylated peptide of GP2. The abbreviations used are as follows: Gal, galactose; GalNAc, N-acetylgalactosamine. The fragmentation was compared between GP2 and the synthesized peptide (PSDTPILPQ).

provide detailed information about the fragmentation efficiency at a certain wavelength on the electrical ground state. To our knowledge, this is the first time that IR fingerprint spectroscopy of O-glycosylated peptides has been achieved in the gas phase. We also performed theoretical calculations of normal-mode analysis of glycopeptides with Hartree−Fock (HF)/3-21G* level and molecular dynamics (MD) simulations in order to investigate the normal modes activated by the IR excitation energy. In this study, MD simulations were used as a tool in studying structural and dynamic details at the atomic/molecular level and in linking these details to the experimentally observable properties of the molecules.

leading to a better understanding of the photochemistry of biopolymer systems. A high-efficiency protein and oligosaccharide cleavage method employing a powerful wavelengthtunable pulsed laser, highly specific with respect to the absorption bands of amino acid and saccharide residues, has been established using laser-induced chemical reactions in proteins and oligosaccharides.20,21 The development of soft ionization methods such as matrixassisted laser desorption/ionization (MALDI)22,23 and electrospray ionization (ESI)24,25 combined with MS has made it possible to investigate many biologically relevant molecules in the gas phase. Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) employing a soft ionization method is useful for highly sensitive analysis to identify small amounts of peptides/proteins, oligosaccharides, and glycopeptides/ proteins and to determine which amide bonds and glycosyl bonds are cleaved in the charged state.26,27 For the photochemical analysis of biopolymers, FTICR MS can be considered a suitable tool due to its ability to store selected mass ions in a collision-free environment for a period of up to several minutes and its adaptability with respect to introducing the laser beam into the cell, while yielding accurate fragmented molecular weights with a high resolution.28 In general, a CO2 laser at an IR wavelength of 10.6 μm is used to cleave the peptide and glycosyl bonds in the fragmentation analysis of biopolymers.21,29 The key process in IRMPD is an increase in internal energy resulting from the absorption of multiple IR photons, which is transferred into the internal vibrational and rotational states of the ions via intramolecular vibrational redistribution (IVR). FTICR MS in combination with the IRMPD technique, employing an intense mid-IR pulse with a tunable wavelength, has been used to investigate laser-induced chemical reactions in metal ions and peptides,26,30−36 making it possible to determine the binding sites for complexes of a transition metal ion with aromatic molecules.34 IR spectroscopy for molecular analysis employing IRMPD with a tunable infrared FEL in conjunction with ESI FTICR MS can divulge important information about molecular structures and dynamics. Our previous studies of peptides and oligosaccharides probed the effective IR range for cleavage of peptide and glycosyl bonds in the fingerprint IR region.37 Fragmentation from positively ionized peptide and oligosaccharide molecules took place mainly in peptide bonds (b/y type) and glycosyl bonds at the nonreducing end side called B/ Y-type fragmentation (nomenclature by Domon and Costello38). In the present study, we extended the findings thus obtained to investigate O-glycopeptides. FEL can be tuned over different vibrational absorption lines for the glycopeptide to probe the efficiency of IRMPD by monitoring the fragment ions. This study addresses fundamental questions concerning the photochemical dynamics of glycopeptides that will ultimately lead to a better understanding of photochemistry in biopolymer systems. The IR spectroscopic observations

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EXPERIMENTAL AND COMPUTATIONAL SECTION Experiments. Figure 1 shows the structures of the Oglycosylated peptide and peptide. The glycopeptide, GP2, was chemically synthesized using a method described elsewhere and prepared as a 100 pmol/μL solution in water.39,40 The peptide (amino acid sequence: Pro-Ser-Asp-Thr-Pro-Ile-Leu-Pro-Gln) was purchased from Peptide Institute, Inc. (Osaka, Japan) and used without additional purification. A 50 nmol portion of the peptide was dissolved in 1 mL of HPLC-grade 50:50 methanol/ water mixture to give a concentration of 50 μM. Each aliquot of this solution was diluted to a final concentration of 20 pmol/ μL. For the protonated and sodium adduct solutions of GP2, the stock solution was diluted with 50:50 methanol/water with 2% acetic acid for [GP2 + 2H]2+ and mixed with 5 mM of Na solution in methanol/water with 2% acetic acid for [GP2 + H + Na]2+. A final concentration of 20 pmol/μL for GP2 was used. A 7 μL aliquot of the sample was pipetted into a metal-coated fused-silica needle (New Objective, Woburn, MA). The protonated and sodiated sample ions were delivered to the ICR cell by means of a nanoelectrospray ionization source. The FEL system was coupled to a BioAPEX III FTICR mass spectrometer equipped with a 4.7 T superconducting magnet (Bruker Daltonics, Billerica, MA). The FEL experiments were carried out using the facilities at the IR FEL Research Center, Research Institutes for Science and Technology, Tokyo University of Science (FEL-TUS). Details on the experimental conditions for the FEL beam and FTICR MS are described in the Supporting Information S-1. Calculations. Theoretical calculations were performed in order to obtain IR spectral information. A large amount of time is required to calculate the proton paths in the charge-directed fragmentation of GP2, which has 195 atoms and 579 vibrational degrees of freedom. The difficulties involved in quantum studies of large molecules make classical MD calculations the most feasible method for studying the reaction mechanisms and interpreting experimental data.41 For the empirical potential functions of the MD simulations, the potential parameters were obtained from GLYCAM.42 In this study, the geometry of GP2 was first optimized using molecular mechanics (MM3), then constant-temperature MD were propagated at a temperature of 2189



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Figure 2. (a) MS spectra at intervals of 0.2 μm in the range of 5.7−9.5 μm. (b) Relative intensity of the fragment ions of [GP2 + 2H]2+ as a function of the IR wavelength.

identified by resolving the isotopic peak spacing) were isolated in the FTICR ion-trap cell using a correlated sweep excitation method. Figure 2a shows the IRMPD MS spectra of [GP2 + 2H]2+ as a function of the FEL laser wavelength in the range of 5.7−9.5 μm. Figure 2b shows the relative intensity of the fragment ions, which are normalized to the sum of all ions. The fragment ions were observed from the cleavage of the peptide bonds in the parent peptide [M + 2H]2+ at Leu-Pro and Ile-Leu assigned as y2 (244 Da) and y3 (357 Da). The ions form the cleavage of the side chain and carbohydrate at Ser-GalNAc and of the glycosyl bond GalNAc-Gal were assigned [(GalNAc + Gal) − H2O + H]+ (367 Da), [GP2 − Gal + 2H]2+ (585 Da), and [anhydro-GalNAc + H]+ (204 Da). In order to investigate the dependence of fragment ions on the attached saccharides (GalNAc-Gal) of GP2, the synthesized peptide (amino acid sequence: PSDTPILPQ) was obtained (Peptide Institute). The greatest abundance of fragment ions was observed around the wavelength of 6.0 μm (1667 cm−1) (see Supporting Information S-2), corresponding to the amide I band, which absorbs around 6.1 μm (1655 cm−1) and is predominately due to contributions from the CO stretching vibration of the peptide backbone (local mode).43 The major fragment ions were observed from the cleavage in the parent

298 K for 10 ns. At 10 ps intervals during the trajectory, the nuclear coordinates were saved to obtain the average structure as an initial conformation for ab initio calculations of IR spectra analysis and for MD simulations of the energy flow of the thermally excited molecule. Quantum chemical calculations were carried out using the HF/3-21G* basis set. A geometric optimization was performed for the O-glycosylated peptide, and all of the optimized geometries were confirmed to have positive vibrational frequencies by calculation of the Hessian. These calculations were carried out using the AMBER MD program run on a PC cluster and the Gaussian03 program on the IBM Regatta computer system at the Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology, Japan.

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RESULTS AND DISCUSSION Protonated GP2. The O-glycopeptide, GP2, in Figure 1 was selected as a model glycopeptide for investigation of the IRMPD spectra as a function of FEL laser wavelength. The mass spectrum of GP2 shows that the most abundant ion produced by the nano-ESI is a doubly charged peptide ion at m/z = 666.83 Da, corresponding to [GP2 + 2H]2+. The most abundant parent ions (the whole group of GP2 at charge +2 2190



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peptide [M + 2H]2+ at Leu-Pro, Leu-Ile, and Thr-Pro assigned as b7/y2, b6/y3, and b4/y5, showing a proline effect, that is, a rich abundance of the proline-directed fragmentation process in CID and IRMPD. In this study, the results of our investigation into which bonds can be more effectively and easily cleaved at a specific wavelength indicate that cleavage at the peptide bond of Xaa-Pro is a preferential reaction path. The effective IR wavelength for cleavage of the peptide bonds was in the range of 5.9−7.8 μm. This region is consistent with the results of our previous experiments on protonated peptide ions of substance P.37 In the analysis of the synthesized peptide, fragment ions were not observed in the 8.5−9.5 μm range, indicating no photoabsorption of the peptide ions in this region (Supporting Information Figure S-2). In contrast, the fragment ions produced from [GP2 + 2H]2+ were observed at the range of over 8.5 μm (see Figure 2b). The major product ion over the entire examined wavelengths is the y2 ion from cleavage at LeuPro. At wavelengths of 5.7 and 6.5 μm, the dominant product ion of [(GP2 − Gal) + 2H]2+ was observed. Interestingly, the population of the parent ion at those wavelengths is higher than that at other wavelengths, suggesting less photoabsorption to energize and decompose the molecule. Our previous experiment shows that the effective irradiation wavelength for cleavage of the glycosyl bonds of saccharides is in the range of 8.6−9.5 μm.37 For the wavelength range higher than 8.5 μm, the unit of the saccharide (Gal-GalNAc) in the GP2 ion mainly absorbed multiple photons, showing that the effective wavelength of glycopeptide has a broader photoabsorption range than that of peptides and oligosaccharide ions in the IR fingerprint region. The effective wavelength in the range of 8.5−9.5 μm for cleavage of the glycosyl bonds is attributed to the highly coupled modes (global modes). Figure 2 indicates that the lowest competing two dissociation channels of all the bonds in GP2 ion are the peptide bond of Leu-Pro and the glycosyl bond of Gal-GalNAc. Sodiated GP2. It is known that the activation energy of protonated oligosaccharides leading to the cleavage of the glycosyl bond that produces B/Y- and C/Z-type fragmentation is smaller than that of alkali-cationized samples. In the dissociation mechanism, a proton may be localized at the glycosyl oxygen atom, leading to a charge-induced reaction. A similar mechanism was suggested by Hofmeister et al. to explain the bond cleavages in lithium-cationized gentiobiose.44 It has also been theorized that metal ions interact with several oxygen atoms in oligosaccharides to stabilize the glycosidic bond, leading to a remote-charge reaction. The multiple binding between the cation and the oxygen atoms leads to a highly stable form of the complex ion.45,46 The GP2 ions that interacted with the alkali metal ions produced by the nano-ESI were mainly doubly charged sodium adduct ions at m/z = 677.8 Da corresponding to [GP2 + H + Na]2+. [GP2 + H + Na]2+ was therefore selected as a precursor ion to investigate the dependence of fragment ions on the adduct ion of GP2. The observed major product ions were due to cleavages at the peptide bond of Leu-Pro and the glycosyl bond of GalNAc-Gal (Supporting Information S-3). The population of fragment ions of the sodiated [GP2 + H + Na]2+ is much smaller than that of the doubly protonated [GP2 + 2H]2+, indicating that the energy required to cleave the bonds of sodium-interacted glycopeptide ion is higher than that of protonated glycopeptide ion. This result is consistent with that in our previous study in which fragment ions were not observed

for the stable complex ion of [Ley + Na]+ at the FEL wavelength of 8.9 μm, whereas fragment ions formed by cleavages at the glycosyl bonds of Gal-Fuc and GalNAc-Fuc were observed for [Ley + H]+.28 The effective irradiation wavelength for cleavage of the bonds of the sodiated ion in Figure 3 was in the higher wavelength range, mainly

Figure 3. Relative intensity of the fragment ions of [GP2 + H + Na]2+ as a function of the IR wavelength.

corresponding to the photoabsorption of global modes of saccharides. Interestingly, the observed major fragment ions were doubly charged ions, suggesting a strong interaction between the slightly negatively charged saccharide (GalNAc) and positively charged Na ion. IRMPD Spectroscopic Analysis. The most pronounced spectral feature differentiating oligosaccharides from peptides was found in the IR region around 9 μm. The region corresponds to the highly mixed global modes of oligosaccharides involving the stretching and bending modes of C1−O−C1 in glycosyl bonds and ring vibrations. It is noted that the monosaccharide units of GalNAc and Gal have the absorption bands of amide I and II due to the presence of NHCOCH3 in the C-2 position. The O-glycopeptides have characteristic spectral features associated with the absorption bands of the amide group (amide bands I, II, and III) and the highly coupled modes (around 9 μm) of the two monosaccharide units of Gal and GalNAc. The infrared absorption of GP2, the peptide (PSDTPILPQ), and the saccharide unit (Gal-GalNAc) were calculated to analyze the vibrational modes in the gas phase. Calculations of the IR vibrational frequencies allow the assignment of IR bands. In the calculations, the alkali metal complex ion, [GP2 + Na + H]2+, was optimized by placing Na+ at the appropriate position and obtaining the geometry of the preferential binding position of Na+46,47 (Supporting Information S-4). Quantum chemical calculations were performed at the HF/3-21G* level on the IBM Regatta computer system at CBRC. Parts a−c of Figure 4 show the IR spectra of the peptide and GP2 calculated from the vibrational frequency analysis at HF/ 3-21G*. The calculated stick spectra were convoluted with a 40 cm−1 wide full width at half-maximum (fwhm) Lorentzian profile to compare the experimental IRMPD spectra. The IRMPD spectra were obtained by monitoring the sum of relative intensities of all fragment ions as a function of the FEL wavelength. It is noted that the IRMPD spectra were corrected 2191



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Figure 4c, the discrepancy between the IRMPD and calculated spectra is due to stability of the sodium adduct complex ion having multiple binding between the Na cation and anion atoms. This result suggests that it may be difficult to obtain the experimental IRMPD spectrum (red line in Figure 4c), because of the lack of considering the interaction via photodissociation dynamics in [GP2 + H + Na]2+ which have multiple strong binding interaction with the Na cation. Using the MD method, we investigated the intramolecular vibrational energy redistribution of GP2. The internal energy increases through the absorption of multiple IR photons in the process of IRMPD, when the tuned wavelength from FEL coincides with that of the fundamental frequency, ν, of the vibrational mode i. The resonantly absorbed energy (hνi) is redistributed among other modes due to coupling of the vibrational modes and then transferred into the internal vibrational and rotational states of the ion. The single-photon energy at a wavelength of 6.1 μm is 3.26 × 10−20 J, and the number of photons in a micropulse (2.22 μJ) emitted from the FEL laser is approximately 6.81 × 1013 photons/s with a laser power of 30 mW. Assuming the beam diameter to be 1 mm, the photon density per pulse becomes 2.17 × 1019 photons/m2. For an ion having an absorption cross section of 1 × 10−9 m, the number of absorbed photons is roughly estimated to be 68 photons. If the photons absorbed by the GP2 molecule (196 atoms) are converted into thermal energy, the temperature of the molecule is about 550 K. To simulate the GP2 molecule vibrationally excited by the resonant absorption of FEL photons, the photon energy is placed in the kinetic energy by scaling the atomic velocities of the molecule.48 MD simulations of GP2 were carried out. These simulations computed the momenta and coordinates of each atom in the system as a function of time by numerically integrating Hamilton’s equations of motion written in Cartesian coordinates. To obtain the initial conditions of the IVR trajectories, the constant-temperature dynamics were propagated at a temperature of 298 K for 100 ps. The averaged structure was fully optimized at the HF/3-21G* level of theory, and normal-mode analysis was performed. The fundamental frequencies and eigenvectors corresponding to the irradiated wavelengths (6.1 and 8.8 μm) within the fwhm of ±0.08 were selected from the calculated normal modes. By scaling the velocities of the normal modes along with the eigenvectors, the GP2 molecule was thermally excited. Parts a and b of Figure 5 show the kinetic energy of the amino acids in the GP2 molecule as a function of time. In the simulations, the excited energy at the wavelength of 6.1 μm at time = zero is mainly localized to the CO stretching bond of the Thr and Pro residues. Figure 5a shows that the localized energy is distributed into the system at around 30 ps. This indicates that the absorbed energy is redistributed into each peptide before the absorption of the next FEL micropulse. On the other hand, the photon energy at the wavelength of 8.9 μm is mainly absorbed by the saccharide units of Gal and GalNAc. A duration of around 600 ps is required to distribute the excited energy to the global modes of the stretching and bending modes of C1−O−C1 and the ring vibrations of GalNAc-Gal. In this process, the GP2 molecule absorbs photons of the next FEL micropulse before IVR since the time of initiation of IVR in the system is earlier than the interval of 350 ps between micropulses. This may lead to the effective fragmentation of the glycosyl bonds in the glycopeptide. It is noted that, although this fragmentation pathway may appear to be the most favorable at certain

Figure 4. Comparison between the calculated IR spectra and experimental IRMPD spectra for the peptide (PSDTPILPQ) and GP2. The experimental spectra are corrected as follows: intensity at the wavelength = (sum of photoproduct ions) × (FEL power/average of FEL power). The average FEL power was 62.3 mW.

linearly by the FEL intensity (Supporting Information Figure S1) and the vibrational frequencies produced by ab initio calculations were scaled (multiplied by 0.918 for the peptide and 0.90 for GP2) to fit experimental data. In the calculated IR spectrum of the peptide, three dominant absorption bands, amide I, II, and III, are observed. The amide I band around 6.1 μm (1655 cm−1) due to contributions from the CO stretching vibration of the peptide backbone (local mode) is the main absorption band. The amide II band around 6.45 μm (1550 cm−1) arises from an out-of-plane combination of N−H bending and C−N stretching of the backbone. The amide III band is an in-phase combination of C−N stretching and N−H bending having a broad absorbance around 7.1 μm (1400 cm−1). The IRMPD spectra of the peptide and GP2 in Figure 4, parts a and b, show good agreement with the calculated IR spectra, with the presence of the strong photoabsorption of the amide bands. In a comparison of the spectra of the peptide and GP2, clear differences are observed at wavelengths of less than 1300 cm−1 owing to the photoabsorption of saccharide units. In 2192



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study confirm the applicability of a combination of FTICR MS and FEL as a spectroscopic molecular characterization technique for glycopeptides.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the New Energy and Industrial Technology Development Organization (NEDO) as a part of the Structural Glycomics Project in Japan. K.F. acknowledges the valuable discussions with and advice received from Professor M. Suzuki at RIKEN and Professor H. Narimatsu at RCMG, AIST. Thanks are also due to Professor K. Nakai, Professor M. Ikekita, Professor K. Tsukiyama, and Dr. T. Imai at FEL-TUS and A. Yano for their support.

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Figure 5. MD IVR simulations of kinetic energy of amino acids in the GP2 molecule at an excitation of (a) 6.1 and (b) 8.9 μm.

wavelengths, it is not necessarily the lowest energy pathway for dissociation. The local heating effect caused by poor IVR may make higher energy dissociation pathways more accessible than lower energy pathways.

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CONCLUSIONS The coupled instrument of a nano-ESI FTICR MS and a tunable FEL allows the study of IRMPD spectroscopy of probe molecules absorbed from the gas phase. This technique was used to study the fragment ions of protonated and sodiated Oglycosylated peptide ions as a function of the wavelength of incident IR laser irradiation. For the glycopeptide, GP2, containing the sugar units of GalNAc and Gal, the effective wavelength for cleavage of the peptide and glycosyl bonds was found to be broader than the molecules of the peptide and oligosaccharide. The rich abundance of product ions is due to a wide range of photoabsorption in the IR region. The major product ions in the protonated GP2 were observed from the cleavage at the peptide bond of Xaa-Pro, the glycosyl bond of GalNAc and Gal, and the bond of the peptide and saccharide of Ser-GalNAc. The objective of the present study was to demonstrate the unique possibilities offered by the combination of FTICR MS and FEL for the analysis of photoproduct ions and acquisition of IRMPD spectra for O-glycosylated peptides. The IRMPD spectrum of the protonated GP2 showed good agreement with the calculated IR spectrum. The results of this 2193



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Infrared Multiple Photon Dissociation Spectroscopy and

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