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.

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/β 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 (MS). These findings allow for integration of MS with ion mobility spectrometry (IM-MS) and lead to a strategy to distinguish α- and β-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.

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.

IR spectroscopy of gas phase ions is proposed to resolve positional isomers of sulfated carbohydrates. Mass spectrometric fingerprints and gas phase vibrational spectra in the near and mid IR regions were obtained for sulfated monosaccharides, yielding unambiguous signatures of sulfated isomers. We report the first systematic exploration of the biologically relevant but notoriously challenging deprotonated state in the near IR region. Remarkably, anions displayed very atypical vibrational profiles, which challenge the well--established DFT (Density Functionnal Theory) modeling. The proposed approach was used to elucidate the sulfate patterns in glycosaminoglycans - a ubiquitous class of mammalian carbohydrates - which is regarded as a major challenge in carbohydrate structural analysis. Isomeric glycosaminoglycan disaccharide from heparin and chondroitin sources where resolved, highlighting the potential of InfraRed Multiple Pho--ton Dissociation spectroscopy as a novel structural tool for carbohydrates.

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.

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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.