Philippe Maitre

Infrared Spectroscopy of Fragments of Protonated Peptides: Direct Evidence for Macrocyclic Structures of b5 Ions

b ions are of fundamental importance in peptide sequencing using tandem mass spectrometry. These ions have generally been assumed to exist as protonated oxazolone derivatives. Recent work indicates that medium-sized b ions can rearrange by head-to-tail cyclization of the oxazolone structures generating macrocyclic protonated peptides as intermediates. Here, we show using infrared spectroscopy and density functional theory calculations that the b(5) ion of protonated G(5)R exists in the mass spectrometer as an amide oxygen protonated cyclic peptide rather than fleetingly as a transient intermediate. This assignment is supported by our DFT calculations which show this macrocyclic isomer to be energetically preferred over the open oxazolone form despite the entropic constraints the cyclic form introduces.

Vibrational signatures of protonated, phosphorylated amino acids in the gas phase.

Structural characterization of protonated phosphorylated serine, threonine, and tyrosine was performed using mid-infrared multiple photon dissociation (IRMPD) spectroscopy and density functional theory (DFT) calculations. The ions were generated and analyzed by an external electrospray source coupled to a Paul ion-trap type mass spectrometer. Their fragmentation was induced by the resonant absorption of multiple photons from a tunable free electron laser (FEL) beam. IRMPD spectra were recorded in the 900-1850 cm(-1) energy range and compared to the corresponding computed IR spectra. On the basis of the frequency and intensity of two independent bands in the 900-1400 cm(-1) energy range, it is possible to identify the phosphorylated residue. IRMPD spectra for a 12-residue fragment of stathmin in its phosphorylated and nonphosphorylated forms were also recorded in the 800-1400 cm(-1) energy range. The lack of spectral congestion in the 900-1300 cm(-1) region makes their distinction facile. Our results show that IRMPD spectroscopy may became a valuable tool for structural characterization of small phosphorylated peptides.

Tautomerism of Uracil Probed via Infrared Spectroscopy of Singly Hydrated Protonated Uracil

Tautomerism of the nucleobase uracil is characterized in the gas phase through IR photodissociation spectroscopy of singly hydrated protonated uracil created with tandem mass spectrometric methods in a commercially available Fourier transform ion cyclotron resonance mass spectrometer. Protonated uracil ions generated by electrospray ionization are re-solvated in a low-pressure collision cell filled with a mixture of water vapor seeded in argon. Their structure is investigated by IR photodissociation spectroscopy in the NH and OH stretching region (2500-3800 cm(-1)) with a tabletop IR laser source and in the 1000-2000 cm(-1) range with a free-electron laser. In both regions the IR photodissociation spectrum exhibits well-resolved spectral signatures that point to the presence of two different types of structure for monohydrated protonated uracil, which result from the two lowest-energy tautomers of uracil. Ab initio calculations confirm that no water-catalyzed tautomerization occurs during the re-solvation process, indicating that the two protonated forms of uracil directly originate from the electrospray process.

Investigation of the protonation site in the dialanine peptide by infrared multiphoton dissociation spectroscopy

Protonated dialanine cations have been isolated in a Fourier transform ion cyclotron resonance mass-spectrometer (FT-ICR-MS) and subjected to infrared multiphoton dissociation (IRMPD) at the free electron laser facility CLIO in Orsay (France). The spectral dependence of the IR induced fragmentation pattern in the mid-infrared region (800–2000 cm−1) is interpreted with the help of structure and vibrational spectrum calculations of the different protonated conformers. This comparison allows for the assignment of the proton on the terminal amino group, as the most favourable proton site, the neighbouring amide bond being in the trans conformation.

Infrared Spectroscopy of Fragments from Doubly Protonated Tryptic Peptides

Most proteins in proteomics are identified from tandem mass spectra of doubly protonated tryptic peptides. Statistical studies indicate that these spectra fall into two distinct classes. IR spectroscopy experiments and DFT calculations performed on model b(2) ions show that peptides producing Class I spectra form protonated oxazolone ions (see figure) and not protonated diketopiperazines as proposed elsewhere.

COVALENT, IONIC AND RESONATING SINGLE BONDS

Abstract Three bond types of electron-pair bonding emerge from multi-structure valence bond (VB) computations of 10 different single bonds. The first bond type is observed in HH, LiLi, CH and SiH. These are all covalent bond types whose major bonding comes from the covalent Heitler-London (HL) configuration, with a minor perturbation from the resonance interaction between the covalent and zwitterionic (Z) configurations. The second bond type is observed for NaF. This is an ionic bond type in which the major bonding is provided by the electrostatic stabilization of the ionic configuration, Na + F − , with a slight perturbation from the HLZ resonance interaction. The third bond type is observed for FF, HF, CF and SiF. These are the resonating bond types in which the major bonding event is the resonance energy stabilization due to the HLZ mixing. No special status should be attached to either the covalency or ionicity of these last bond types, even if they may appear purely “covalent”, such as FF, or “highly ionic”, as CF, by charge distribution criterion. The phenomenon of resonating bonding is shown to emerge from weakly bound or unbound covalent HL configurations which originate when the “preparation” for bonding of the fragments becomes energy demanding, as for fluorine. The mechanism of HL bond weakening is through costly promotion energy and overlap repulsion of a lone pair with a bond pair of the same symmetry. The essential requirements for a fragment A to qualify as a resonating binder are therefore: (a) to possess two AOs which maintain a very large energy gap between them, and which by virtue of overlap capability can both enter into bonding; and (b) to have three electrons in these two AOs which thereby mutually antagonize each others bonding. The propensity for resonating bonding is discussed, in the light of these qualifications, for the main elements across the Periodic Table. It is concluded that the elements with the highest propensity for resonating bonding are F, O and N. Any combination AB where either A or B or both are resonating binders is likely to lead to a resonating bond (e.g. OO, NF, CF, CO, and so on). The resonating bonds are shown to coincide with the group of “weakened” bonds in the classification of Sanderson, and with those bonds which exhibit negative or marginally positive deformation densities in electron density determinations. Negative or marginally positive deformation densities may serve as the experimental signature of the theoretical concept of resonating bonding. The LiH bond appears to possess a special status. While the computations tend to classify this bond among the covalent types, the results also show that the HL and ionic Li + H − configurations are nearly degenerate and maintain a very weak coupling. Therefore the LiH bond will have a metastable character, as far as ionicity-covalency, in the presence of medium perturbations which are at least of the magnitude of the coupling between the ionic and covalent structures.

Structure of Electron-Capture Dissociation Fragments from Charge-Tagged Peptides Probed by Tunable Infrared Multiple Photon Dissociation

In this study, we propose the first spectroscopic structural characterization of c-type ions produced by ECD of a peptide. The structure of c-type ions formed by electron capture dissociation and the overall mechanism leading to their formation are still a question of debate. Depending on the mechanism, c-type ions have been proposed to have either an enol-imine structure (-C(OH)NH) or an amide one (-C(O)-NH2). Since these ions are isomeric, mass spectrometry only cannot discriminate between them, but infrared spectroscopy can bring experimental evidence and help determine which scheme is operative. Using the coupling between a tunable free electron laser and a FT-ICR mass spectrometer, we show that c-type ions have an amide structure, characterized by an IR signature of the C=O stretch at 1731 cm(-1). This result is particularly interesting from the perspective of the elucidation of the ECD mechanism.

Probing Mobility-Selected Saccharide Isomers: Selective Ion-Molecule Reactions and Wavelength-Specific IR Activation.

Differential Ion Mobility Spectrometry (DIMS) provides orthogonal separation to mass spectrometry, and DIMS combined with the high sensitivity of a quadrupole ion-trap is shown to be useful for the separation and identification of saccharides. A comprehensive analysis of the separation of anomers (α- and β-methylated glucose) and epimers (α-methylated glucose and mannose) ionized with Li(+), Na(+), and K(+) is performed. DIMS separation is found to be better for saccharides cationized with the two latter species. The corresponding resolving power for the two glucose anomers with Na(+) is found to be very close to the corresponding drift-tube IMS value. The lithiated complexes are investigated further using a combination of infrared spectroscopy integrated to ion-trap mass spectrometry and quantum chemical calculations. Together with DIMS, consistent results are obtained. It is found that two competing structural motifs might be at play, depending on the subtle balance between the maximization of the coordination of the metal cation and the intrinsic conformational energetics of the saccharide, which is for a large part driven by hydrogen bonding. The comparison of simulated and observed spectra clearly shows that a band at ∼3400 cm(-1) is specific to a structural motif found in the lithiated glucose complexes, which could explain the trends observed in the DIMS spectra of the saccharide complexes. It is shown that DIMS-MS/MS using wavelength specific IR activation would provide a new orthogonal dimension to mass spectrometry.

Molecular complexes of simple anions with electron-deficient arenes: spectroscopic evidence for two types of structural motifs for anion-arene interactions.

Anion-pi interactions between a pi-acidic aromatic system and an anion are gaining increasing recognition in chemistry and biology. Herein, the binding features of an electron-deficient aromatic system (1,3,5-trinitrobenzene (TNB)) and selected anions (OH-, Br-, and I-) are examined in the gas phase by using the combined information derived from collision-induced dissociation experiments at variable energy, infrared multiple-photon dissociation spectroscopy, and quantum chemical calculations. We provide spectroscopic evidence for two different structural motifs of anion-arene complexes depending on the nature of the anion. The TNB-OR- complexes (R=H, or alkyl groups which were studied earlier) adopt an anionic sigma-complex structure whereby RO- attacks the aromatic ring with covalent bond formation, and develops a tetrahedral ring carbon bound to H and OR. The halide complexes rather conform to a structure in which the TNB moiety is hardly altered, and the halogen is placed on an unsubstituted carbon atom over the periphery of the ring at a C-X distance that is appreciably longer than a typical covalent bond length. The ensuing structural motif, previously characterized in the solid state and named weak sigma interaction, is now confirmed by an IR spectroscopic assay in the gas phase, in which the sampled species are unperturbed by crystal packing or solvation effects.

Gas phase infrared multiple-photon dissociation spectra of methanol, ethanol and propanol proton-bound dimers, protonated propanol and the propanol/water proton-bound dimer

The infrared multiphoton dissociation (IRMPD) spectra of three homogenous proton-bound dimers are presented and the major features are assigned based on comparisons with the neutral alcohol and with density functional theory calculations. As well, the IRMPD spectra of protonated propanol and the propanol/water proton-bound dimer (or singly hydrated protonated propanol) are presented and analysed. Two primary IRMPD photoproducts were observed for each of the alcohol proton bound dimers and were found to vary with the frequency of the radiation impinging upon the ions. For example, when the proton-bound dimer absorbs weakly a larger amount of S(N)2 product, protonated ether and water, are observed. When the proton-bound dimer absorbs more strongly, an increase in the simple dissociation product, protonated alcohol and neutral alcohol, is observed. With the aid of RRKM calculations this frequency dependence of the branching ratio is explained by assuming that photon absorption is faster than dissociation for these species and that only a few photons extra are necessary to make the higher-energy dissociation channel (simple cleavage) competitive with the lower energy (S(N)2) reaction channel.