Ion Mobility Spectrometry in Food Analysis: Principles, Current Applications and Future Trends

In the last decade, ion mobility spectrometry (IMS) has reemerged as an analytical separation technique, especially due to the commercialization of ion mobility mass spectrometers. Its applicability has been extended beyond classical applications such as the determination of chemical warfare agents and nowadays it is widely used for the characterization of biomolecules (e.g., proteins, glycans, lipids, etc.) and, more recently, of small molecules (e.g., metabolites, xenobiotics, etc.). Following this trend, the interest in this technique is growing among researchers from different fields including food science. Several advantages are attributed to IMS when integrated in traditional liquid chromatography (LC) and gas chromatography (GC) mass spectrometry (MS) workflows: (1) it improves method selectivity by providing an additional separation dimension that allows the separation of isobaric and isomeric compounds; (2) it increases method sensitivity by isolating the compounds of interest from background noise; (3) and it provides complementary information to mass spectra and retention time, the so-called collision cross section (CCS), so compounds can be identified with more confidence, either in targeted or non-targeted approaches. In this context, the number of applications focused on food analysis has increased exponentially in the last few years. This review provides an overview of the current status of IMS technology and its applicability in different areas of food analysis (i.e., food composition, process control, authentication, adulteration and safety).M.H.-M. was granted a postdoctoral fellowship (University Research Plan, Program “Perfeccionamiento de doctores en el extranjero 2017”) by the University of Granada (Spain)

Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina

International audienceBackground: The understanding of enzymatic polysaccharide degradation has progressed intensely in the past few years with the identification of a new class of fungal-secreted enzymes, the lytic polysaccharide monooxygenases (LPMOs) that enhance cellulose conversion. In the fungal kingdom, saprotrophic fungi display a high number of genes encoding LPMOs from family AA9 but the functional relevance of this redundancy is not fully understood. Results: In this study, we investigated a set of AA9 LPMOs identified in the secretomes of the coprophilous ascomycete Podospora anserina, a biomass degrader of recalcitrant substrates. Their activity was assayed on cellulose in synergy with the cellobiose dehydrogenase from the same organism. We showed that the total release of oxidized oligosaccharides from cellulose was higher for PaLPMO9A, PaLPMO9E, and PaLPMO9H that harbored a carbohydrate-binding module from the family CBM1. Investigation of their regioselective mode of action revealed that PaLPMO9A and PaLPMO9H oxidatively cleaved at both C1 and C4 positions while PaLPMO9E released only C1-oxidized products. Rapid cleavage of cellulose was observed using PaLPMO9H that was the most versatile in terms of substrate specificity as it also displayed activity on cello-oligosaccharides and beta-(1,4)-linked hemicellulose polysaccharides (e.g., xyloglucan, glucomannan). Conclusions: This study provides insights into the mode of cleavage and substrate specificities of fungal AA9 LPMOs that will facilitate their application for the development of future biorefineries

A fungal family of lytic polysaccharide monooxygenase-like copper proteins

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-ef-fective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans—a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxida-tive cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications

Gangliosides characterization by high resolution mass spectrometry and ion mobility

Gangliosides (GG) are sialic acid-containing glycosphingolipids particularly abundant in the nervous system. They exhibit a wide variety of structures depending on both the oligosaccharide chain and the ceramide moiety. About 200 GG species have been described so far, but their diversity and biological roles are far from being completely elucidated. An efficient method of identification and quantification is crucial to apprehend this huge heterogeneity. In this study, we focused on the characterization of the ceramide portion of GG, identifying the long chain base (LCB) and the fatty acid. A commercial standard of GD3 was analysed with both a Thermo LTQ-Orbitrap XLTM and a Waters Synapt G2-Si equipped with ion mobility.Whatever the equipment used, fragmentation of GD3 40:1 molecular species revealed the presence of four LCBs (d16:1, d17:1, d17:0 and d18:1) for one ceramide type, suggesting the presence of four GG isomers. This observation was confirmed by the MS3 analyses conducted with the Orbitrap, which allowed isolation and fragmentation of the ion corresponding to the ceramide. The technique of ion mobility offered the possibility to separate the four detected LCBs, after MS/MS fragmentation of GD3 40:1. We were thus able to detect and separate the LCBs, according to their differences of steric hindrance, for each isomer of GG. In this way we deduced the combination LCB/fatty acid (ex: GD3 d16:1/24:0) for each GD3 molecular species. Moreover, this separation presents the advantage to individualize each molecular isobaric species of GG for their ulterior quantification.Ion mobility applied to GG analysis allowed separation and further characterization and quantification of the various ceramide isomers

Distribution of cell wall hemicelluloses in the wheat grain endosperm: a 3D perspective

International audienceMain conclusionUneven distribution of AX and BG in lateral and longitudinal dimensions of a wheat grain was observed by three-dimensional MS imaging, presumably related to specific physicochemical properties of cell walls.Arabinoxylans (AX) and -glucans (BG) are the main hemicelluloses that comprise the primary walls of starchy endosperm. These components are not evenly distributed in the endosperm, and the impact of their distribution on cell wall properties is not yet fully understood. Combined with on-tissue enzymatic degradation of the cell walls, mass spectrometry imaging (MSI) was used to monitor the molecular structure of AX and BG in thirty consecutive cross-sections of a mature wheat grain. A 3D image was built from the planar images, showing the distribution of these polymers at the full-grain level, both in lateral and longitudinal dimensions. BGs were more abundant at the vicinity of the germ and in the central cells of the endosperm, while AX, and especially highly substituted AX, were more abundant close to the brush and in the cells surrounding the crease (i.e., the transfer cells). Compared with the previously reported protocol, significant improvements were made in the tissue preparation to better preserve the shape of the fragile sections. This allowed to us achieve a good-quality 3D reconstruction from the consecutive 2D images. By providing a continuous view of the molecular distribution of the cell wall components across and along the grain, the three-dimensional images obtained by MSI may help understand the structure-function relationships of cell walls. The method should be readily extendable to other parietal polymers by selecting the appropriate enzymes

Acid-detoxified Inaba lipopolysaccharide (pmLPS) is a superior cholera conjugate vaccine immunogen than hydrazine-detoxified lipopolysaccharide and induces vibriocidal and protective antibodies

International audienceWorldwide, in endemic areas of cholera, the group most burdened with cholera is children. This is especially vexing as young children (2-5 years of age) do not respond as well, or for as long as adults do, to the current killed oral cholera vaccines (OCV). Conjugate vaccines based on the hapten-carrier paradigm have been developed for several bacterial pathogens that cause widespread and severe diseases in young children. We and others have studied different formulations of Vibrio cholerae (Vc) O1 lipopolysaccharide (LPS, a T-independent antigen) conjugates. Detoxified LPS is a central component of a LPS-based conjugate vaccine. pmLPS, which is detoxified by acid treatment, is a superior immunogen compared with hydrazine-detoxified LPS (DetAcLPS) that has altered lipid A acyl chains. The other feature of pmLPS is the ability to link carrier proteins to a core region of sugar. pmLPS readily induced vibriocidal antibodies following one intraperitoneal dose in a MPL-type adjuvant One dose of the pmLPS conjugate was suggestive of being protective; a booster resulted in protective antibodies for infant mice challenged with virulent cholera

New insight into the mode of action of a GH74 xyloglucanase on tamarind seed xyloglucan: Action pattern and cleavage site

International audienceStructural elucidation of plant cell wall xyloglucan through the analysis of enzymatically produced fragmentsrequires detailed knowledge of enzymes hydrolytic mechanism. In this note, the mode of action and cleavage siteof commercial recombinant xyloglucanases (GH74, Paenibacillus sp.) was studied on native and fluorescent-tagged tamarind xyloglucan. In complement to information provided by the manufacturer, GH74 hydrolysiswas shown dual endo/exo- and exo-processive with low affinity towards labelled reducing-ends. GH74 accom-modated X/G in its subsite --1 and X/L in its subsite +1. Moreover, hydrolysis kinetic indicated a GH74 activityinhibition by excess products. These results will help for application of this enzyme in xyloglucans structuralanalysis or for processing cell walls