Very highly charged proteins, so-called “supercharged” ions, can lose (excess) protons to background gases like N2. It is remarkable that such extremely acidic species can be generated in electrospray ionization, in the presence of not just N2 but also much higher-basicity solvents. What mechanism(s) can explain such high charging, and what is the ultimate limit?

Sehr hoch geladene Proteine – „supercharged” – können überschüssige Protonen an Hintergrundgase wie N2 abgeben. Bemerkenswerterweise lassen sich derart saure Spezies mittels Elektrospray-Ionisierung nicht nur in Gegenwart von N2, sondern auch in Gegenwart von Lösungsmitteln mit viel höherer Basizität erzeugen. Durch welche Mechanismen lässt sich eine solch hohe Ladung erklären und wo ist die Grenze?

The effects of electrospray ionization (ESI) solvent and source temperature on the relative abundance of the preferred solution-phase (N-protonated; i.e. amine) versus preferred gas-phase (O-protonated; i.e., acid) isomers of p-aminobenzoic acid (PABA) were investigated. When PABA was electrosprayed from protic solvents (i.e., methanol/water), the infrared multiple photon dissociation (IRMPD) spectrum recorded was consistent with that for O-protonation, according to both calculations and previous studies. When aprotic solvent (i.e., acetonitrile) was used, a different spectrum was recorded and was assigned to the N-protonated isomer. As the amine is the preferred protonation site in solution, this suggests that an isomerization takes place under certain conditions. Photodissociation at the diagnostic band for the O-protonated isomer (NH2 stretching mode) was used to quantify the relative contributions of each isomer to ion signal as a function of ESI conditions. For mixtures of methanol and acetonitrile, the relative contribution of the O-protonated gas-phase structure increased as a function of methanol content. Yet, substituting methanol for water resulted in a marked decrease of isomerization to the O-protonated structure. The source temperature (i.e., temperature of a heated desolvation capillary) was found to play a key role in determining the extent of isomerization, with higher temperatures yielding increased presence of gas-phase structures. These results are consistent with a protic bridge mechanism, in which the ESI droplet desolvation, dependent on the solvent system and radiative heating from the capillary, may determine the isomerization yield.Download high-res image (131KB)Download full-size image

Three lithiated N-acetyl-D-hexosamine (HexNAc) isomers, N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), and N-acetyl-D-mannosamine (ManNAc) are investigated as model monosaccharide derivatives by gas-phase infrared multiple-photon dissociation (IRMPD) spectroscopy. The hydrogen stretching region, which is attributed to OH and NH stretching modes, reveals some distinguishing spectral features of the lithium-adducted complexes that are useful in terms of differentiating these isomers. In order to understand the effect of lithium coordination on saccharide structure, and therefore anomericity, chair configuration, and hydrogen bonding networks, the conformational preferences of lithiated GlcNAc, GalNAc, and ManNAc are studied by comparing the experimental measurements with density functional theory (DFT) calculations. The experimental results of lithiated GlcNAc and GalNAc show a good match to the theoretical spectra of low-energy structures adopting a 4C1 chair conformation, consistent with this motif being the dominant conformation in condensed-phase monosaccharides. The epimerization effect upon going to lithiated ManNAc is significant, as in this case the 1C4 chair conformers give a more compelling match with the experimental results, consistent with their lower calculated energies. A contrasting computational study of these monosaccharides in their neutral form suggests that the lithium cation coordination with Lewis base oxygens can play a key role in favoring particular structural motifs (e.g., a 4C1 versus 1C4) and disrupting hydrogen bond networks, thus exhibiting specific IR spectral features between these closely related lithium-chelated complexes.

The detailed chemical information contained in the vibrational spectrum of a cryogenically cooled analyte ion would, in principle, make infrared (IR) ion spectroscopy a gold standard technique for molecular identification in mass spectrometry. Despite this immense potential, there are considerable challenges in both instrumentation and methodology to overcome before the technique is analytically useful. Here, we discuss the promise of IR ion spectroscopy for small molecule analysis in the context of metabolite identification. Experimental strategies to address sensitivity constraints, poor overall duty cycle, and speed of the experiment are intimately tied to the development of a mass-selective cryogenic trap. Therefore, the most likely avenues for success, in the authors’ opinion, are presented here, alongside alternative approaches and some thoughts on data interpretation.

We report on the intermolecular transfer of sulfuric acid (H2SO4) and sulfur trioxide (SO3) from an acidic sulfopeptide (sSE) to a basic peptide (R3); this is achieved by subjecting a noncovalent complex of sSE + R3 to collisional activation in a quadrupole ion trap. The product ions resulting from the sulfo-group transfers were characterized by MS3 experiments. Peak assignments were additionally supported by isotope-labeling and energy-resolved collision induced dissiciation (CID) experiments. The observed reactions and their potential implications for proteomics and post-translational modification discovery experiments are discussed.

•The CID fragmentation pathway of protonated sulfoserine was investigated.•The two dominant fragment ions resulted from loss of SO3 and H2SO4.•The SO3-loss ion was found to be structurally analogous to protonated serine.•The H2SO4-loss ion was assigned an aziridine structure as its major constituent.The fragmentation chemistry of protonated sulfoserine was probed using a combination of collision-induced dissociation (CID) mass spectrometry, infrared multiple photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations. The IRMPD spectra of the dominant fragment ions at m/z 106 and 88 (i.e., loss of SO3 and H2SO4) were obtained and used to determine the corresponding structures. By comparison to a synthetic standard and calculations, it was determined that the m/z 106 ion is structurally identical to protonated serine. The m/z 88 fragment ion was assigned an aziridine structure based on a comparison to theory, analogous to the structure previously proposed by others for phosphoric acid loss from phosphoserine. This work provides the first spectroscopic insights into the dissociation pathways of a sulfated amino acid, laying the groundwork for future studies on related amino acids and peptides with this important, labile post-translational modification.

Carbohydrates and their derivatives play important roles in biological systems, but their isomeric heterogeneity also presents a considerable challenge for analytical techniques. Here, a stepwise approach using infrared multiple-photon dissociation (IRMPD) via a tunable CO2 laser (9.2–10.7 μm) was employed to characterize isomeric variants of glucose-based trisaccharides. After the deprotonated trisaccharides were trapped and fragmented to disaccharide C2 fragments in a Fourier transform ion cyclotron resonance (FTICR) cell, a further variable-wavelength infrared irradiation of the C2 ion produced wavelength-dependent dissociation patterns that are represented as heat maps. The photodissociation patterns of these C2 fragments are shown to be strikingly similar to the photodissociation patterns of disaccharides with identical glycosidic bonds. Conversely, the photodissociation patterns of different glycosidic linkages exhibit considerable differences. On the basis of these results, the linkage position and anomericity of glycosidic bonds of disaccharide units in trisaccharides can be systematically differentiated and identified, providing a promising approach to characterize the structures of isomeric oligosaccharides.

The post-translational modifications sulfation and phosphorylation pose special challenges to mass spectral analysis due to their isobaric nature and their lability in the gas phase, as both types of peptides dissociate through similar channels upon collisional activation. Here, we present resonant infrared photodissociation based on diagnostic sulfate and phosphate OH stretches, as a means to differentiate sulfated from phosphorylated peptides within the framework of a mass spectrometry platform. The approach is demonstrated for a number of tyrosine-containing peptides, ranging from dipeptides (YG, pYG, and sYG) over tripeptides (GYR, GpYR, and GsYR), to more biologically relevant enkephalin peptides (YGGFL, pYGGFL, and sYGGFL). In all cases, the diagnostic ranges for sulfate OH stretches are established as 3580–3600 cm–1 and can thus be distinguished from other characteristic hydrogen stretches, such as carboxylic acid OH, alcohol OH, and phosphate OH stretches.

We report infrared multiple photon dissociation (IRMPD) spectra for a series of crown-adducted, protonated amino acids, generated by electrospray ionization. The tight chelation of 18-crown-6 on the protonated NH3+ moiety results in a considerable red shift of the NH3+ stretch modes, notably the antisymmetric NH3+ stretch. This is rationalized by a distortion of the NH3+ normal mode potential energy surface, as verified by quantum chemical calculations. On the other hand, the local oscillator modes, such as the carboxylic acid OH stretch, indole NH stretch, and phenol OH stretches, remain well-resolved and are subject to minor and predictable blue shifts of 5–15 cm–1. Other chemically diagnostic modes, such as the guanidine NH stretch and alcohol OH stretches, also have discernible band positions. Crucially, some of these diagnostic band positions have little to no overlap with one another and can hence be readily distinguished. In addition, the complexes are often found to efficiently photodissociate by neutral loss of 18-crown-6, particularly for higher-basicity amino acids. This in principle opens the door on multiplexing the IRMPD experiment, where the IR spectra of multiple precursors are recorded simultaneously.

We present an infrared laser-based mass spectrometric strategy to differentiate peptides that are phosphorylated (i.e., containing pS, pT, or pY) from those that are nonphosphorylated (i.e., containing S, T, or Y), and those peptides that have none of these moieties (i.e., containing neither pS, pT, pY nor S, T, Y). This is demonstrated for a series of tripeptides and for two larger octapeptides, showing that the diagnostic phosphate OH stretch (indicative for pS, pT, or pY) can be distinguished from the alcohol OH stretch (indicative for S, T, or Y). In addition, the infrared multiple photon dissociation (IRMPD) spectra of multiple peptide analytes are recorded simultaneously in a multiplexed fashion. This is achieved by complexing each peptide precursor with a noncovalently bound 18-crown-6 ether, which is detached upon resonant infrared photon absorption.

The mechanism of peptide “b” fragment formation in collision-induced dissociation (CID) is generally understood as a nucleophilic attack from a carbonyl oxygen onto the electron deficient carbon of the dissociating amide bond forming a five-membered oxazolone ring structure. Nonetheless, other nucleophiles, such as the N-terminus and side-chain moieties (e.g., imidazole, guanidine), can in principle engage in a nucleophilic attack to induce amide backbone cleavage. Here, we apply a combination of infrared multiple photon dissociation (IRMPD) spectroscopy and computational chemistry to characterize the water loss, [M+H-H2O]+, product ions from protonated ArgGly and GlyArg. IRMPD spectra for [M+H-H2O]+ from ArgGly and GlyArg differ in the presence and absence of a characteristic band at 1885 cm−1, which is indicative of an oxazolone structure for ArgGly. The remaining parts of the vibrational spectra are consistent with the vibrational signatures of diketopiperazine structures. Conversely, there is no match between the experimental spectra and any of the putative structures arising from guanidine side-chain attack. These results show that the presence of a basic residue, such as arginine, facilitates the formation of diketopiperazine structures, and that residue order matters in the competition between diketopiperazine and oxazolone pathways.Graphical abstractHighlights► The water loss products from protonated ArgGly and GlyArg are investigated by infrared multiple photon dissociation spectroscopy and computational chemistry. ► While ArgGly exclusively yields diketopiperazine structures, a mixture of diketopiperazine and oxazolone structures are confirmed for GlyArg. ► Product structures due to nucleophilic attacks from the guanidine side chain can be excluded.

While recent studies have shown that for some peptides, such as oligoglycines and Leu-enkephalin, mid-sized b fragment ions exist as a mixture of oxazolone and macrocycle structures, other primary structure motifs, such as QWFGLM, are shown to exclusively give rise to macrocycle structures. The aim of this study was to determine if certain amino acid residues are capable of suppressing macrocycle formation in the corresponding b fragment. The residues proline and 4-aminomethylbenzoic acid (4AMBz) were chosen because of their intrinsic rigidity, in the expectation that limited torsional flexibility may impede “head-to-tail” macrocycle formation. The presence of oxazolone versus macrocycle b6 fragment structures was validated by infrared multiple photon dissociation (IRMPD) spectroscopy, using the free electron laser FELIX. It is confirmed that proline disfavors macrocycle formation in the cases of QPWFGLM b7 and in QPFGLM b6. The 4AMBz substitution experiments show that merely QWFG(4AMBz)M b6, with 4AMBz in the fifth position, exhibits a weak oxazolone band. This effect is likely ascribed to a stabilization of the oxazolone structure, due to an extended oxazolone ring-phenyl π-electron system, not due to the rigidity of the 4AMBz residue. These results show that some primary structures have an intrinsic propensity to form macrocycle structures, which is difficult to disrupt, even using residues with limited torsional flexibility.

This tutorial review presents the technique of infrared multiple-photon dissociation (IRMPD) spectroscopy of mass-selected trapped ions. This requires coupling of a tunable infrared laser with mass spectrometry instrumentation. IRMPD spectroscopy has recently blossomed due to the emergence of widely tunable free electron lasers, as well as on-going developments of benchtop lasers. The merits of different trapping approaches in mass spectrometry are discussed in the light of photodissociation experiments. This tutorial discusses current capabilities, as well as limitations of the technique.

We report trends in the theoretically derived number of compositionally distinct peptides (i.e., peptides made up of different amino acid residues) up to a nominal mass of 1000. A total of 21 amino acid residues commonly found in proteomics studies are included in this study, 19 natural, nonisomeric amino acid residues as well as oxidated methione and acetamidated cysteine. The number of possibilities is found to increase in an exponential fashion with increasing nominal mass, and the data show a periodic oscillation that starts at mass ∼200 and continues throughout to 1000. Note that similar effects are reported in the companion article on fragment ions from electron capture/transfer dissociation (ECD/ETD) (Mao et al. Anal. Chem.2011, DOI: 10.1021/ac201619t). The spacing of this oscillation is ∼15 mass units at lower masses and ∼14 mass units at higher nominal masses. This correlates with the most common mass differences between the amino acid building blocks. In other words, some mass differences are more common than others, thus determining the periodicity in this data. From an analytical point of view, nominal masses with a larger number of compositionally distinct peptides include a substantial number of isomers, which cannot be separated based on mass. Consequently, even ultrahigh mass accuracy (i.e., 0.5 ppm) does not lead to a substantially enhanced rate of identification. Conversely, for adjacent nominal masses with a lower number of isomers, moderately accurate mass (i.e., 10 ppm) gives a higher degree of certainty in identification. These effects are limited to the mass range between 200 and 500 Da. At higher masses, the percentage of uniquely identified peptides drops off to close to zero, independent of nominal mass, due the inherently high number of isomers. While the exact number of isobars/isomers at each nominal mass depends on the amino acid building blocks that are considered, the periodicity in the data is found to be remarkably robust; for instance, inclusion of phosphorylated residues barely affects the pattern at lower masses (i.e., <500 Da).

In collision-induced dissociation (CID) of peptides, it has been observed that rearrangement processes can take place that appear to permute/scramble the original primary structure, which may in principle adversely affect peptide identification. Here, an analysis of sequence permutation in tandem mass spectra is presented for a previously published proteomics study on P. aeruginosa (Scherl et al., J. Am. Soc. Mass Spectrom.2008, 19, 891) conducted using an LTQ-orbitrap. Overall, 4878 precursor ions are matched by considering the accurate mass (i.e., <5 ppm) of the precursor ion and at least one fragment ion that confirms the sequence. The peptides are then grouped into higher- and lower-confidence data sets, using five fragment ions as a cutoff for higher-confidence identification. It is shown that the propensity for sequence permutation increases with the length of the tryptic peptide in both data sets. A higher charge state (i.e., 3+ vs 2+) also appears to correlate with a higher appearance of permuted masses for larger peptides. The ratio of these permuted sequence ions, compared to all tandem mass spectral peaks, reaches ∼25% in the higher-confidence data set, compared to an estimated incidence of false positives for permuted masses (maximum ∼8%), based on a null-hypothesis decoy data set.

We report infrared multiple-photon dissociation (IRMPD) spectra of protonated tryptophan, TrpH+, as well as the dissociation products from NH3 loss, [Trp+H−NH3]+, and subsequent CH2CO loss, [Trp+H−NH3−CH2CO]+. These results were obtained using a custom-built mass spectrometer, where the mass-isolated ions were photodissociated by a tunable optical parametric oscillator laser in a reduced-pressure (i.e., 10−5 mbar) “Paul-type” 3D ion trap. A comparison to computed spectra of putative conformations for TrpH+ shows that the protonated amino group, NH3+, interacts with the carboxylic acid carbonyl oxygen and the aromatic indole side chain. Ammonia loss is confirmed to occur via nucleophilic attack from C3 on the indole side chain, as opposed to C2 or C4, resulting in a spirocyclopropane structure with the charge located on the indole side chain. For the consecutive [Trp+H−NH3−CH2CO]+ product, the agreement with theory is slightly less persuasive, even if the qualitative trends are in accordance with the proposed structure.Keywords: amino acid; collision-induced dissociation; density functional theory; IRMPD; spectroscopy; tryptophan;

We present infrared multiple photon dissociation (IRMPD) spectra in the hydrogen stretching region of the simplest b fragment, b2 from protonated triglycine, contrasted to that of protonated cyclo(Gly-Gly). Both spectra confirm the presence of intense, diagnostic vibrations linked to the site of proton attachment. Protonated cyclo(Gly-Gly) serves as a reference spectrum for the diketopiperazine structure, showing a diagnostic O-H+ stretch of the protonated carbonyl group at 3585 cm–1. Conversely, b2 from protonated triglycine exhibits a strong band at 3345 cm–1, associated with the N-H stretching mode of the protonated oxazolone ring structure. Other weaker N-H stretches can also be discerned, such as the amino NH2 and amide NH bands. These results demonstrate the usefulness of the hydrogen stretching region, and hence benchtop optical parametric oscillator/amplifier (OPO/A) set-ups, in making structural assignments of product ions in collision-induced dissociation (CID) of peptides.

Despite significant developments in mass spectrometry technology in recent years, no routine proteomics sequencing tool is currently available for peptide anions. The use of a molecular open-shell cation is presented here as a possible reaction partner to induce electron transfer dissociation with deprotonated peptide anions. In this negative electron transfer dissociation (NETD) scheme, an electron is abstracted from the peptide anion and transferred to the radical cation. This is demonstrated for the example of the fluoranthene cation, C16H10+•, which is reacted with deprotonated phosphorylated peptides in a 3-D ion trap mass spectrometer. Selective backbone cleavage at the Cα−C bond is observed to yield a and x fragments, similarly to electron detachment dissociation (EDD) of peptide anions. Crucially, the phosphorylation site is left intact in the dissociation process, allowing an identification and localization of the post-translational modification (PTM) site. In contrast, NETD using Xe+• as the reagent cation results in sequential neutral losses (CO2 and H3PO4) from a/x fragments, which complicate the interpretation of the mass spectra. This difference in dissociation behavior can be understood in the framework of the reduced recombination energy of the electron transfer process for fluoranthene, which is estimated at 2.5−4.5 eV, compared to 6.7−8.7 eV for xenon. Similarly to ETD, proton transfer is found to compete with electron transfer processes in NETD. Isotope fitting of the charge-reduced species shows that in the case of fluoranthene-mediated NETD, proton transfer only accounts for <20%, whereas this process highly abundant for Xe+• (43 and 82%). Since proton abstraction from Xe+• is not possible, this suggests that Xe+• ionizes other transient species in the ion trap, which then engage in proton transfer reactions with the peptide anions.

Gas-phase binding of the alkali metal Rb+ to two monosaccharide isomers, glucuronic acid (GlcA) and iduronic acid (IdoA), is investigated by infrared photodissociation spectroscopy. The infrared spectra display striking differences, exemplified by the clear absence of a band at 3625 cm−1 in the case of IdoA + Rb+. Comparison of the experimental spectra to computed spectra of DFT-optimized structures suggests that Rb+-tagged GlcA and IdoA each adopt their own distinctive complexation pattern. For GlcA, mainly the β-anomer 4C1 chair complex is observed, whereas for IdoA the data are consistent with the α-anomer 1C4 chair structure, as well as the corresponding β-anomer. The differences in the Rb+ binding motif rationalize the disparities in the infrared multiple-photon dissociation (IR-MPD) spectra. Whereas Rb+ binding to GlcA leaves the intramolecular hydrogen-bonding network between the OH groups intact, this network is disrupted for IdoA. The lack of stronger hydrogen-bonding for IdoA + Rb+ thus correlates well with the absence of the red-shifted OH stretch band at 3625 cm−1.

Infrared multiple-photon dissociation (IR-MPD) spectra in the N–H and O–H stretching region (3000–3700 cm−1) are reported for gas-phase monomeric M2+Trp and dimeric M2+Trp2 complexes (where M = Mg, Ca, Sr, and Ba). The spectra are obtained by irradiating the complexes in the Penning trap of a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, using a tunable continuous-wave (cw) optical parametric oscillator (OPO) laser in combination with a fixed wavelength CO2 laser (10.6 μm). These spectra are compared with previously recorded mid-IR-MPD spectra, using the free electron laser FELIX, and are interpreted based on harmonic frequency calculations performed with density-functional theory (DFT). The experimental spectra show that a simple assignment of bands can be made to distinguish zwitterionic (ZW) from charge solvation (CS) complexes. In particular, the carboxylic acid O–H stretch at ∼3550 cm−1 identifies the presence of a CS structure, whereas the NH3+ antisymmetric stretching mode in the 3150–3375 cm−1 range is diagnostic of a ZW structure. For the monomeric Ba2+Trp complex, exclusively the ZW structure is observed. Conversely, for the dimeric complexes of M2+Trp2 (M = Sr and Ba) merely CS/CS geometries are confirmed. Surprisingly, for smaller alkaline earth metal dications (M = Mg and Ca), the IR-MPD spectra are consistent with the presence of mixed CS/ZW dimers. This is contrary to most previous trends for alkali metals, where larger cations typically favor ZW stabilization.Graphical abstractDiagnostic vibrations for alkaline earth-tryptophan dimers, M2+Trp2, confirm mixed CS/ZW (charge solvation/Zwitterion) complexes for Mg and Ca, as opposed to CS/CS for Sr and Ba.Research highlights▶ IR-MPD spectra of alkaline earth-tryptophan dimers, M2+Trp2, in the gas phase. ▶ Diagnostic vibrations confirm charge solvation (CS) or zwitterionic (ZW) structures. ▶ Smaller cations (Mg, Ca) adopt mixed CS/ZW configuration. ▶ Larger cations (Sr,Ba) adopt CS/CS configuration.

The collision-induced dissociation (CID) products b2-b4 from Leu-enkephalin are examined with infrared multiple-photon dissociation (IR-MPD) spectroscopy and gas-phase hydrogen/deuterium exchange (HDX). Infrared spectroscopy reveals that b2 exclusively adopts oxazolone structures, protonated at the N-terminus and at the oxazolone ring N, based on the presence and absence of diagnostic infrared vibrations. This is correlated with the presence of a single HDX rate. For the larger b3 and b4, the IR-MPD measurements display diagnostic bands compatible with a mixture of oxazolone and macrocycle structures. This result is supported by HDX experiments, which show a bimodal distribution in the HDX spectra and two distinct rates in the HDX kinetic fitting. The kinetic fitting of the HDX data is employed to derive the relative abundances of macrocycle and oxazolone structures for b3 and b4, using a procedure recently implemented by our group for a series of oligoglycine b fragments (Chen et al. J. Am. Chem. Soc.2009, 131(51), 18272–18282. doi: 10.1021/ja9030837). In analogy to that study, the results suggest that the relative abundance of the macrocycle structure increases as a function of b fragment size, going from 0% for b2 to ∼6% for b3, and culminating in 31% for b4. Nonetheless, there are also surprising differences between both studies, both in the exchange kinetics and the propensity in forming macrocycle structures. This indicates that the chemistry of “head-to-tail” cyclization depends on subtle differences in the sequence as well as the size of the b fragment.

The hydration of glycine is investigated by comparing the structures of bare glycine to its hydrated complexes, glycine·H2O and glycine·(H2O)2. The Fourier transform infrared spectra of glycine and glycine·water complexes, embedded in Ar matrices at 12 K, have been recorded and the results were compared to density functional theory (DFT) calculations. An initial comparison of the experimental spectra was made to the harmonic infrared spectra of putative structures calculated at the MPW1PW91/6-311++G(d,p) level of theory. The results suggest that bare glycine adopts a Cs symmetry structure (G-1), where the hydrogens of the amino NH2 hydrogen-bond intramolecularly with the carboxylic acid C═O oxygen. Also observed as minor constituents are the next two lowest-energy structures, one in which the carboxylic acid (O−)H group hydrogen-bonds to the amino NH2 group (G-2), and the other where intramolecular hydrogen bonding occurs between the NH2 and the carboxylic acid O(−H) groups (G-3). The abundances of these structures are estimated at 84%, 9% and 8%, respectively. The least favored structure, G-3, can be eliminated by annealing the matrix to 35 K. Addition of the first water molecule to G-1 takes place at the carboxylic acid group, with simultaneous hydrogen bonding of the water molecule to the carboxylic acid (C=)O and (O−)H. The results are consistent with the predominance of this structure, although there is evidence for a small amount of a hydrated G-2 structure. Addition of the second water molecule is less definitive, as only a small number of intense infrared modes can be unambiguously assigned to glycine·(H2O)2. Anharmonic frequency calculations based on second-order vibrational perturbation theory have also been carried out. It is shown that such calculations can generate improved estimates (i.e., ∼2%) of the experimental frequencies for glycine and glycine·H2O, provided that the potential energy surfaces are modeled with high-level ab initio approaches (MP2/aug-cc-pVDZ).

The chemistry of peptide fragmentation by collision-induced dissociation (CID) is currently being reviewed, as a result of observations that the amino acid sequence of peptide fragments can change upon activation. This rearrangement mechanism is thought to be due to a head-to-tail cyclization reaction, where the N-terminal and C-terminal part of the fragment are fused into a macrocycle (= cyclic peptide) structure, thus “losing” the memory of the original sequence. We present a comprehensive study for a series of b fragment ions, from b2 to b8, based on the simplest amino acid residue glycine, to investigate the effect of peptide chain length on the appearance of macrocycle fragment structures. The CID product ions are structurally characterized with a range of gas-phase techniques, including isotope labeling, infrared photodissociation spectroscopy, gas-phase hydrogen/deuterium exchange (using CH3OD), and computational structure approaches. The combined insights from these results yield compelling evidence that smaller bn fragments (n = 2, 3) exclusively adopt oxazolone-type structures, whereas a mixture of oxazolone and macrocycle b fragment structures are formed for midsized bn fragments, where n = 4−7. As each of these chemical structures exchanges at different rates, it is possible to approximate the relative abundances using kinetic fits to the H/D exchange data. Under the conditions used here, the “slow”-exchanging macrocycle structure represents ∼30% of the b ion population for b6−b7, while the “fast”-exchanging oxazolone structure represents the remainder (70%). Intriguingly, for b8 only the macrocycle structure is identified, which is also consistent with the “slow” kinetic rate in the HDX results. In a control experiment, a protonated cyclic peptide with 6 amino acid residues, cyclo(Gln-Trp-Phe-Gly-Leu-Met), is confirmed not to adopt an oxazolone structure, even upon collisional activation. These results demonstrate that in some cases larger macrocycle structures are surprisingly stable. While more studies are required to establish the general propensity for cyclization in b fragments, the implications from this study are troubling in terms of faulty sequence identification.

The influence of the physical environment on the structures of biomolecules is considered here for the dipeptide model H+AlaPhe cation, by making use of infrared multiple-photon dissociation (IRMPD) spectroscopy complemented by DFT calculations. The gas-phase structures of this peptide are also compared to the related peptide cations H+PheAla and H+AlaAla. The gas-phase IRMPD spectra of the Phe-containing cations are compared to previous studies, including the X-ray-crystallographic crystal structure for the H+AlaPheCl−·2H2O salt, a recent IRMPD spectrum of H+AlaAla, and a recent determination of the IR absorption spectrum of the H+AlaPheCl− salt in a liquid-crystal host matrix, as well as recent cryogenic ion trap results for H+TyrAla and H+AlaTyr. Between the gas-phase H+AlaPhe ion and the H+AlaPheCl−·2H2O crystal a conformational switch is observed, induced by hydrogen bonding with a water of crystallization, involving a 180° rotation of the COOH group. The hoped-for comparison of the gas-phase IR spectra with the liquid-crystal matrix IR spectrum was frustrated, because the literature matrix spectrum seems most likely to be that of a protonated homodimer of the dipeptide rather than the protonated monomer. The IRMPD spectra of H+AlaPhe and H+PheAla are very similar, with only minor peak shifts suggesting small differences in local interactions within a similar overall architecture. The H+AlaAla spectrum was also similar, and no significant reorganization of the structure seems to result from the presence or position of the aromatic ring. The spectra give highly satisfactory matches to the predicted IR spectra computed for the most stable conformers of the protonated dipeptides. It is suggested that the NH3+ proton is shared through hydrogen bonding to the amide CO, giving a distinctive broadening of the associated H-bending mode.The gas-phase IRMPD spectrum of H+AlaPhe shows that the gas-phase conformation differs from the crystalline chloride salt with respect to orientation of the terminal carboxyl.

•Protonation for PABA is on the carboxylic acid, but it is the amine for MABA and OABA.•For PABA and MABA resonance stabilization rationalizes the relative basicities.•The exception to this rule is OABA, where the proton is on the amine.Infrared multiple photon dissociation (IRMPD) spectroscopy and computational chemistry are applied to the ortho-, meta-, and para- positional isomers of aminobenzoic acid to investigate whether the amine or the carboxylic acid are the favored sites of proton attachment in the gas phase. The NH and OH stretching modes yield distinct patterns that establish the carboxylic acid as the site of protonation in para-aminobenzoic acid, as opposed to the amine group in ortho- and meta-aminobenzoic acid, in agreement with computed thermochemistries. The trends for para- and meta-substitutions can be rationalized simplistically by inductive effects and resonant stabilization, and will be discussed in light of computed charge distributions based from electrostatic potentials. In ortho-aminobenzoic acid, the close proximity of the amine and acid groups allow a simultaneous interaction of the proton with both groups, thus stabilizing and delocalizing the charge more effectively, and compensating for some of the resonance stabilization effects.

The collision-induced dissociation (CID) products b2-b4 from Leu-enkephalin are examined with infrared multiple-photon dissociation (IR-MPD) spectroscopy and gas-phase hydrogen/deuterium exchange (HDX). Infrared spectroscopy reveals that b2 exclusively adopts oxazolone structures, protonated at the N-terminus and at the oxazolone ring N, based on the presence and absence of diagnostic infrared vibrations. This is correlated with the presence of a single HDX rate. For the larger b3 and b4, the IR-MPD measurements display diagnostic bands compatible with a mixture of oxazolone and macrocycle structures. This result is supported by HDX experiments, which show a bimodal distribution in the HDX spectra and two distinct rates in the HDX kinetic fitting. The kinetic fitting of the HDX data is employed to derive the relative abundances of macrocycle and oxazolone structures for b3 and b4, using a procedure recently implemented by our group for a series of oligoglycine b fragments (Chen et al. J. Am. Chem. Soc. 2009, 131(51), 18272–18282. doi: 10.1021/ja9030837). In analogy to that study, the results suggest that the relative abundance of the macrocycle structure increases as a function of b fragment size, going from 0% for b2 to ∼6% for b3, and culminating in 31% for b4. Nonetheless, there are also surprising differences between both studies, both in the exchange kinetics and the propensity in forming macrocycle structures. This indicates that the chemistry of “head-to-tail” cyclization depends on subtle differences in the sequence as well as the size of the b fragment.For the b2-b4 CID fragment series of Leu-enkephalin, the relative abundance of the macrocycle structure increases as a function of the b fragment size.Download high-res image (193KB)Download full-size image

This tutorial review presents the technique of infrared multiple-photon dissociation (IRMPD) spectroscopy of mass-selected trapped ions. This requires coupling of a tunable infrared laser with mass spectrometry instrumentation. IRMPD spectroscopy has recently blossomed due to the emergence of widely tunable free electron lasers, as well as on-going developments of benchtop lasers. The merits of different trapping approaches in mass spectrometry are discussed in the light of photodissociation experiments. This tutorial discusses current capabilities, as well as limitations of the technique.

Gas-phase binding of the alkali metal Rb+ to two monosaccharide isomers, glucuronic acid (GlcA) and iduronic acid (IdoA), is investigated by infrared photodissociation spectroscopy. The infrared spectra display striking differences, exemplified by the clear absence of a band at 3625 cm−1 in the case of IdoA + Rb+. Comparison of the experimental spectra to computed spectra of DFT-optimized structures suggests that Rb+-tagged GlcA and IdoA each adopt their own distinctive complexation pattern. For GlcA, mainly the β-anomer 4C1 chair complex is observed, whereas for IdoA the data are consistent with the α-anomer 1C4 chair structure, as well as the corresponding β-anomer. The differences in the Rb+ binding motif rationalize the disparities in the infrared multiple-photon dissociation (IR-MPD) spectra. Whereas Rb+ binding to GlcA leaves the intramolecular hydrogen-bonding network between the OH groups intact, this network is disrupted for IdoA. The lack of stronger hydrogen-bonding for IdoA + Rb+ thus correlates well with the absence of the red-shifted OH stretch band at 3625 cm−1.