Mass spectrometry is a vital tool for molecular characterization, and the allied technique of ion mobility is enhancing many areas of (bio)chemical analysis. Strong synergy arises between these two techniques because of their ability to ascertain complementary information about gas-phase ions. Ion mobility separates ions (from small molecules up to megadalton protein complexes) based on their differential mobility through a buffer gas. Ion mobility-mass spectrometry (IM-MS) can thus act as a tool to separate complex mixtures, to resolve ions that may be indistinguishable by mass spectrometry alone, or to determine structural information (for example rotationally averaged cross-sectional area), complementary to more traditional structural approaches. Finally, IM-MS can be used to gain insights into the conformational dynamics of a system, offering a unique means of characterizing flexibility and folding mechanisms. This Review critically describes how IM-MS has been used to enhance various areas of chemical and biophysical analysis.

Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

Carbohydrates possess a variety of distinct features with stereochemistry playing a particularly important role in distinguishing their structure and function. Monosaccharide building blocks are defined by a high density of chiral centers. Additionally, the anomericity and regio-chemistry of the glycosidic linkages carry important biological information. Any carbohydrate-sequencing method needs to be precise in determining all aspects of this stereo-diversity. Recently, several advances have been made in developing fast and precise analytical techniques that have the potential to address the stereochemical complexity of carbohydrates. This perspective seeks to provide an overview of some of these emerging techniques, focusing on those that are based on NMR and MS-hybridized technologies including ion mobility spectrometry and IR spectroscopy. Associated Content Supporting Information. Two tables containing software and databases that facilitate glycan analysis can be found in the supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

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.

N. (2015). A recycling pathway for cyanogenic glycosides evidenced by the comparative metabolic profiling in three cyanogenic plant species. Biochemical Journal, 469(3), 375-389. DOI: 10.1042/BJ20150390 1 A RECYCLING PATHWAY FOR CYANOGENIC GLYCOSIDES EVIDENCED BY THE COMPARATIVE METABOLIC PROFILING IN THREE CYANOGENIC PLANT SPECIES ABSTRACTCyanogenic glycosides are phytoanticipins involved in plant defence against herbivores by virtue of their ability to release toxic HCN upon tissue disruption. In addition, endogenous turnover of cyanogenic glycosides without the liberation of HCN may offer plants an important source of reduced nitrogen at specific developmental stages. To investigate the presence of putative turnover products of cyanogenic glycosides, comparative metabolic profiling using LC-MS/MS and HR-MS complemented by ion-mobility mass spectrometry was carried out in three cyanogenic plant species: cassava, almond and sorghum. In total, the endogenous formation of 36 different chemical structures related to the cyanogenic glucosides linamarin, lotaustralin, prunasin, amygdalin and dhurrin was discovered, including di-and triglycosides derived from these compounds. The relative abundance of the compounds was assessed in different tissues and developmental stages. Based on results common to the three phylogenetically unrelated species, a potential recycling endogenous turnover pathway for cyanogenic glycosides is described in which reduced nitrogen and carbon are recovered for primary metabolism without the liberation of free HCN. Glycosides of amides, carboxylic acids and anitriles derived from cyanogenic glycosides appear as common intermediates in this pathway and may also have individual functions in the plant. The recycling of cyanogenic glycosides and the biological significance of the presence of the turnover products in cyanogenic plants open entirely new insights into the multiplicity of biological roles cyanogenic glycosides may play in plants.Abbreviations: HR-MS, high-resolution mass spectrometry; EIC, extracted ion chromatogram; IM-MS, ionmobility mass spectrometry; ATD, arrival time distribution; CID, collision-induced dissociation 2 SUMMARY STATEMENTA potential recycling pathway for cyanogenic glycosides is presented wherein reduced nitrogen and carbon are recovered for primary metabolism without HCN liberation. Common types of glycosylated pathway intermediates were found in three cyanogenic plant species: cassava, almond and sorghum.

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.

IM-MS is a promising technique that fills an important gap within the Glycomics toolbox, namely identifying and differentiating the three-dimensional structure of chemically similar carbohydrates and glycoconjugates. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

We have constructed a two-dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low-copy-number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope-bound protein fraction using either immobilized pH gradient strips or a two-detergent system with a cationic detergent in the first and an anionic detergent in the second-dimensional separation. Different proteins (502) were identified by matrix-assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two-dimensional proteome databases.