Fucose Migration Pathways Identified Using Infrared Spectroscopy.

Fucose is a ubiquitous monosaccharide associated to major classes of glycans. A main obstacle to the sequencing of fucosylated glycans is the migration of fucose, which leads to misinterpretations in mass spectrometry analysis. Here, using ion vibrational spectroscopy, we resolve the structure of fucosylated fragments of Lewis and blood group H antigen trisaccharides and we unveil the position and linkage of the fucose after migration. Our findings demonstrate that the structure of fragment ions resulting from fucose migration can be characterized. Additionally, we report a new type of fucose migration, which does not feature any change of mass and therefore had not been previously reported: it consists of a local migration where the fucose changes its position remaining on the initial residue. Our approach allows the characterization of glycans, an essential step to interpret glycomics data, as well as to understand underlying processes at play in mass spectrometry.

GlAIcomics: a deep neural network classifier for spectroscopy-augmented mass spectrometric glycans data.

Carbohydrate sequencing is a formidable task identified as a strategic goal in modern biochemistry. It relies on identifying a large number of isomers and their connectivity with high accuracy. Recently, gas phase vibrational laser spectroscopy combined with mass spectrometry tools have been proposed as a very promising sequencing approach. However, its use as a generic analytical tool relies on the development of recognition techniques that can analyse complex vibrational fingerprints for a large number of monomers. In this study, we used a Bayesian deep neural network model to automatically identify and classify vibrational fingerprints of several monosaccharides. We report high performances of the obtained trained algorithm (GlAIcomics), that can be used to discriminate contamination and identify a molecule with a high degree of confidence. It opens the possibility to use artificial intelligence in combination with spectroscopy-augmented mass spectrometry for carbohydrates sequencing and glycomics applications.

Rapid IRMPD (InfraRed multiple photon dissociation) analysis for glycomics.

Infrared vibrational spectroscopy in the gas phase has emerged as a powerful tool to determine complex molecular structures with high precision. Among the different approaches IRMPD (InfraRed multiple photon dissociation), which requires the use of an intense pulsed tuneable laser in the InfraRed (IR) domain, has been broadly applied to the study of complex (bio)molecules. Recently, it also emerged as a highly relevant approach for analytical purposes especially in the field of glycomics in which structural analysis is still a tremendous challenge. This opens the perspective to develop new analytical tools allowing for the determination of molecular structures with atomic precision, and to address advanced questions in the field. However, IRMPD experiments require non commercial equipment or/and long acquisition time which limits the data output. Here we show that it is possible to improve the IRMPD performances by optimizing the combination between a linear ion trap mass spectrometer and a high repetition tuneable laser. Two orders of magnitude are gained with this approach compared to the usual experiments ultimately leading to a completely resolved spectrum acquired in less than one minute. These results open the way to many new applications in glycomics with the possibility to include IRMPD in complex analytical workflows.

O-Acetylated sugars in the gas phase: stability, migration, positional isomers and conformation.

O-Acetylations are functional modifications which can be found on different hydroxyl groups of glycans and which contribute to the fine tuning of their biological activity. Localizing the acetyl modifications is notoriously challenging in glycoanalysis, in particular because of their mobility: loss or migration of the acetyl group may occur through the analytical workflow. Whereas migration conditions in the condensed phase have been rationalized, little is known about the suitability of Mass Spectrometry to retain and resolve the structure of O-acetylated glycan isomers. Here we used the resolving power of infrared ion spectroscopy in combination with ab initio calculations to assess the structure of O-acetylated monosaccharide ions in the gaseous environment of a mass analyzer. N-Acetyl glucosamines were synthetized with an O-acetyl group in positions 3 or 6, respectively. The protonated ions produced by electrospray ionization were observed by mass spectrometry and their vibrational fingerprints were recorded in the 3 µm range by IRMPD spectroscopy (InfraRed Multiple Photon Dissociation). Experimentally, the isomers show distinctive IR fingerprints. Additionally, ab initio calculations confirm the position of the O-acetylation and resolve their gas phase conformation. These findings demonstrate that the position of O-acetyl groups is retained through the transfer from solution to the gas phase, and can be identified by IRMPD spectroscopy.

Distinguishing Galactoside Isomers with Mass Spectrometry and Gas-Phase Infrared Spectroscopy.

Sequencing glycans is demanding due to their structural diversity. Compared to mammalian glycans, bacterial glycans pose a steeper challenge because they are constructed from a larger pool of monosaccharide building blocks, including pyranose and furanose isomers. Though mammalian glycans incorporate only the pyranose form of galactose (Galp), many pathogens, including Mycobacterium tuberculosis and Klebsiella pneumoniae, contain galactofuranose (Galf) residues in their cell envelope. Thus, glycan sequencing would benefit from methods to distinguish between pyranose and furanose isomers of different anomeric configurations. We used infrared multiple photon dissociation (IRMPD) spectroscopy with mass spectrometry (MS-IR) to differentiate between pyranose- and furanose-linked galactose residues. These targets pose a challenge for MS-IR because the saccharides lack basic groups, and galactofuranose residues are highly flexible. We postulated cationic groups that could complex through hydrogen bonding would offer a solution. Here, we present the first MS-IR analysis of hexose ammonium adducts. We compared their IR fingerprints with those of lithium adducts. We determined the diagnostic MS-IR signatures of the α- and ß-anomers of galactose in furanose and pyranose forms. We also showed these signatures could be applied to disaccharides to assign galactose ring size. Our findings highlight the utility of MS-IR for analyzing the unique substructures that occur in bacterial glycans.

Spectroscopic diagnostic for the ring-size of carbohydrates in the gas phase: furanose and pyranose forms of GalNAc.

Hexoses are mainly found in nature in the pyranose form (6-membered ring). Yet, furanose forms (5-membered ring) are observed in some rare polysaccharides. Using IRMPD spectroscopy (InfraRed Multiple Photon Dissociation), we propose a straightforward diagnostic of the ring-size of N-acetyl galactosamine ions. The furanose form of N-acetyl galactosamine was synthesized and its protonated ion was isolated in an ion trap to measure its gas phase vibrational spectrum by IRMPD. Comparison with the IRMPD spectrum of its pyranose counterpart reveals that they have distinctive optical fingerprints. This new MS-based diagnostic opens the way to facile identification of the ring-size in oligosaccharides. Our experimental data also provide new insights to support the theoretical description of the conformational behavior of the furanose ring, which is notoriously more flexible than the pyranose form but remains difficult to assess.

Online Separation and Identification of Isomers Using Infrared Multiple Photon Dissociation Ion Spectroscopy Coupled to Liquid Chromatography: Application to the Analysis of Disaccharides Regio-Isomers and Monosaccharide Anomers.

The vast array of molecular isomerisms which form the complex molecular structure of carbohydrates is the foundation of their biological versatility but defies the analytical chemist. Hyphenations of mass spectrometry with orthogonal structural characterization, such as ion mobility or ion spectroscopy, have recently shown great promise for distinction between closely related molecular structures. Yet, the lack of analytical strategies for identification of isomers present in mixtures remains a major obstacle to routine carbohydrate sequencing. In this context, an ideal workflow for glycomics would combine isomer separation and individual characterization of the molecular structure with atomistic resolution. Here we report the implementation of such a multidimensional analytical strategy, which consists of the first online coupling of high-performance liquid chromatography (HPLC)-MS and infrared multiple photon dissociation (IRMPD) spectroscopy. The performance of this novel workflow is exemplified in the case of monosaccharides (anomers) and disaccharides (regioisomers) standards. We report that the LC-MS-IRMPD approach offers a robust advanced MS diagnostic of mixtures of isomers, including carbohydrate anomers, which is critical for carbohydrate sequencing. Our results also explain the bimodal character generally observed in LC chromatograms of carbohydrates. More generally, this multidimensional analytical strategy opens the gateway to rapid identification of molecular isoforms with potential application in the "omics" fields.

Bottom-Up Elucidation of Glycosidic Bond Stereochemistry.

The lack of robust, high-throughput, and sensitive analytical strategies that can conclusively map the structure of glycans has significantly hampered progress in fundamental and applied aspects of glycoscience. Resolution of the anomeric α/ß glycan linkage within oligosaccharides remains a particular challenge. Here, we show that "memory" of anomeric configuration is retained following gas-phase glycosidic bond fragmentation during tandem mass spectrometry (MS2). These findings allow for integration of MS2 with ion mobility spectrometry (IM-MS2) and lead to a strategy to distinguish α- and ß-linkages within natural underivatized carbohydrates. We have applied this fragment-based hyphenated MS technology to oligosaccharide standards and to de novo sequencing of purified plant metabolite glycoconjugates, showing that the anomeric signature is also observable in fragments derived from larger glycans. The discovery of the unexpected anomeric memory effect is further supported by IR-MS action spectroscopy and ab initio calculations. Quantum mechanical calculations provide candidate geometries for the distinct anomeric fragment ions, in turn shedding light on gas-phase dissociation mechanisms of glycosidic linkages.

MS/IR, a new MS-based hyphenated method for analysis of hexuronic acid epimers in glycosaminoglycans.

We report an original MS-based hyphenated method for the elucidation of the epimerization in GAG fragments. It consists of measuring simultaneously the MS/MS spectrum and the gas phase IR spectrum to gain direct structural information. This is possible using a customized MS instrument, modified to allow injection of a tunable IR laser inside of the instrument for in situ spectroscopy of trapped ions. The proof of principle of this approach is performed in the case of a hyaluronic acid tetrasaccharide standard. In addition, we provide the reference IR fingerprint of glucuronic and Iduronic monosaccharide standards. Remarkably, we show that the gas phase IR fingerprint of reference hexuronic acid monosaccharides proves to be transposable to oligosaccharides. Therefore, the method presented here is predictive and allows structural elucidation of unknown GAG fragments, even in the absence of referenced standards.

Anharmonic simulations of the vibrational spectrum of sulfated compounds: application to the glycosaminoglycan fragment glucosamine 6-sulfate.

Mid-infrared spectroscopy coupled with mass spectrometry is an appealing tool for the sequencing and structural elucidation of functional modifications in biopolymers, as it offers direct spectroscopic identification of the functionality where the traditional mass spectrometric approach is insufficient. Whereas the gas phase vibrational spectroscopy of peptides (and to a lesser extent saccharides) has been widely investigated, sulfation has attracted much less attention, despite its prevalence in natural polymers. The simulation of the vibrational spectra of such functionalized compounds is however notoriously challenging, which impairs the interpretation of spectroscopic data in terms of structure. Driven by a striking case of such a failure for a sulfated glycosaminoglycan fragment, we elaborate on an original hybrid GVPT2 anharmonic approach. This strategy offers a significantly improved accuracy in the description of the sulfate modes, without the recourse to empirical scaling factors, and with a greatly reduced computational cost which is otherwise prohibitive for molecules of this size. Alternatively, we propose a selection of reasonably accurate harmonic methods with adequate scaling factors optimized on a set of benchmark compounds.

Distinguishing isobaric phosphated and sulfated carbohydrates by coupling of mass spectrometry with gas phase vibrational spectroscopy.

An original application of the coupling of mass spectrometry with vibrational spectroscopy, used for the first time to discriminate isobaric bioactive saccharides with sulfate and phosphate functional modifications, is presented. Whereas their nominal masses and fragmentation patterns are undifferentiated by sole mass spectrometry, their distinctive OH stretching modes at 3595 cm(-1) and 3666 cm(-1), respectively, provide a reliable spectroscopic diagnostic for distinguishing their sulfate or phosphate functionalization. A detailed analysis of the 6-sulfated and 6-phosphated d-glucosamine conformations is presented, together with theoretical scaled harmonic spectra and anharmonic spectra (VPT2 and DFT-based molecular dynamics simulations). Strong anharmonic effects are observed in the case of the phosphated species, resulting in a dramatic enhancement of its phosphate diagnostic mode.

Off-line coupling of capillary isotachophoresis separation to IRMPD spectroscopy for glycosaminoglycans analysis: Application to the chondroitin sulfate disaccharides model solutes.

Glycans analysis is challenging due to their immense structural diversity. Isotachophoresis was investigated as separation method for the purification of isobaric sulfated disaccharides prior to their characterization by Mass Spectrometry (MS) and tunable IR multiple photon dissociation (IRMPD). This proof of feasibility study was applied to the separation and characterization of chondroitin sulfate (CS) disaccharides. ITP separation conditions were optimized. Separation starts using a 2.5 mM chloride ions and 10 mM glycine at pH 3.2 solution as leading electrolyte and a terminating electrolyte composed of formic acid 2.5 mM and glycine 10 mM at pH 3.5. The CS disaccharides sample were prepared in the terminating electrolyte. The length of injection was also investigated in order to create longer plateau-like bands of pure solutes. This strategy was helpful for collecting fraction at such microseparation scale. Indeed, capillary ITP affords the injection of few tens of nanoliter of sample. Fractionation of the CS disaccharides mixture in isolated ITP bands and collection of solutes were successfully done using a HPC coated fused silica capillary of 1m-length and 75 µm of internal diameter. Collected fractions in a final of volume 10 µL were analyzed by CZE, tandem MS and IRMPD spectroscopy. The purity of each fraction is higher than 90% and is well-adapted to IRMPD characterization.

Lasers and ion mobility: new additions to the glycosaminoglycanomics toolkit.

Glycosaminoglycans are biopolymers present in mammalian cells or in the extracellular matrix. To address their structure, the nature of the hexuronic acids and the position of sulfate groups must be determined. Tandem mass spectrometry using collision induced dissociation or electron-based fragmentation techniques, is a well-established approach for the identification of glycans but suffers from the frequent lack of diagnostic fragments in the case of glycosaminoglycans. This review presents alternative fragmentation techniques, namely photofragmentation in the IR and the UV ranges. Alternative approaches based on the direct analysis of the molecular structure, including ion mobility spectrometry and ion spectroscopies are reviewed. The potential of future multidimensional workflows for glycosaminoglycanomics is discussed.

Ultrafast Nonadiabatic Cascade and Subsequent Photofragmentation of Extreme Ultraviolet Excited Caffeine Molecule.

Ultrafast XUV chemistry is offering new opportunities to decipher the complex dynamics taking place in highly excited molecular states and thus better understand fundamental natural phenomena as molecule formation in interstellar media. We used ultrashort XUV light pulses to perform XUV pump-IR probe experiments in caffeine as a model of prebiotic molecule. We observed a 40 fs decay of excited cationic states. Guided by quantum calculations, this time scale is interpreted in terms of a nonadiabatic cascade through a large number of highly correlated states. This shows that the correlation driven nonadiabatic relaxation seems to be a general process for highly excited states, which might impact our understanding of molecular processing in interstellar media.

Anomeric memory of the glycosidic bond upon fragmentation and its consequences for carbohydrate sequencing.

Deciphering the carbohydrate alphabet is problematic due to its unique complexity among biomolecules. Strikingly, routine sequencing technologies-which are available for proteins and DNA and have revolutionised biology-do not exist for carbohydrates. This lack of structural tools is identified as a crucial bottleneck, limiting the full development of glycosciences and their considerable potential impact for the society. In this context, establishing generic carbohydrate sequencing methods is both a major scientific challenge and a strategic priority. Here we show that a hybrid analytical approach integrating molecular spectroscopy with mass spectrometry provides an adequate metric to resolve carbohydrate isomerisms, i.e the monosaccharide content, anomeric configuration, regiochemistry and stereochemistry of the glycosidic linkage. On the basis of the spectroscopic discrimination of MS fragments, we report the unexpected demonstration of the anomeric memory of the glycosidic bond upon fragmentation. This remarkable property is applied to de novo sequencing of underivatized oligosaccharides.Establishing generic carbohydrate sequencing methods is both a major scientific challenge and a strategic priority. Here the authors show a hybrid analytical approach integrating molecular spectroscopy and mass spectrometry to resolve carbohydrate isomerism, anomeric configuration, regiochemistry and stereochemistry.

Fast and accurate hybrid QM//MM approach for computing anharmonic corrections to vibrational frequencies.

We have developed and tested a new time-effective and accurate hybrid QM//MM generalized second-order vibrational perturbation theory (GVPT2) approach. In this approach, two different levels of theory were used, a high level one (DFT) for computing the harmonic spectrum and a lower fast one (Molecular Mechanic) for the anharmonic corrections. To validate our approach, we used B2PLYP/def2-TZVPP as the high-level method, and the MMFF94 method for the anharmonic corrections as the low-level method. The calculations were carried out on 28 molecules (containing from 2 to 47 atoms) covering a broad range of vibrational modes present in organic molecules. We find that this fast hybrid method reproduces the experimental frequencies with a very good accuracy for organic and bio-molecules. The root-mean-square deviation (RMSD) is about 27 cm -1 while the full B3LYP/SNSD simulation reproduces the experimental values with a RMSD of about 41 cm -1. Concerning the computational time, the hybrid B2PLYP//MMFF94 approach considerably outperforms the full B3LYP/SNSD: for the larger molecule of our set (a dipeptide containing 47 atoms), the anharmonic corrections are 2300 times faster using hybrid MMFF94 rather than full B3LYP, which represents an additional computation time to the harmonic calculation of merely 9 %, instead of 32100 % with the full B3LYP approach. This time-effective and accurate alternative to the traditional GVPT2 approach will allow the spectroscopy community to explore anharmonic effects in larger biomolecules, which are generally unaffordable.