Carbohydrate isomers with identical atomic composition cannot be distinguished by mass spectrometry. By separating the ions according to their conformation in the gas phase, ion mobility (IM) coupled ...to mass spectrometry is an attractive approach to overcome this issue and extend the limits of mass spectrometry in structural glycosciences. Recent technological developments have significantly increased the resolving power of ion mobility separators. One such instrument features a cyclic traveling-wave IM separator integrated in a quadrupole/time-of-flight mass spectrometer. This system allows for multipass ion separations and for pre-, intra-, and post-IM fragmentation. In the present study, we utilize this system to explore a complex mixture of oligoporphyrans derived from the enzymatic digestion of the cell wall of the red alga P. umbilicalis. We are able to deduce their complete structure using IM arrival times and the m/z of specific fragments. This approach was successfully applied for sequencing of oligoporphyrans of up to 1500 Da and included the positioning of the methyl ether and sulfate groups. The structures defined in this study by IM-MS/MS agree with those found in the past but use much more time-consuming analytical approaches. This study also revealed some so far undescribed structures, present at very low abundance. In addition, the results made it possible to compare the abundance of the different isomers released by the enzyme and to draw further conclusions on the specificity of β-porphyranase and more particularly on its accommodation tolerance of anhydro-bridges in subsites. Finally, a separation of two isomers with very similar mobility was obtained after 58 passes around the cIM, with an estimated resolving power of 920 for these triply charged species, confirming the structures attributed to these two isomers.
Although carbohydrates are the most abundant biopolymers on Earth, there is currently no streamlined method to elucidate their complete sequence. Mass spectrometry (MS) alone is blind to many cases ...of isomerism and thus gives incomplete information for carbohydrates. Notably, the coexistence of numerous stereoisomeric monosaccharide subunits is of special concern. Over the last 10 years, the coupling of ion mobility spectrometry (IMS) with MS has kept gaining momentumespecially with the advent of high-resolution (HR) IMS devices such as cyclic IMS (cIMS). In fact, IMS is sensitive to the gas-phase conformations of molecules and, thus, to stereoisomerisms. In this article, we present innovative ion mobility methods on a cIMS instrument that allowed us to build a database of HR-IMS fingerprints for various underivatized monosaccharide stereoisomers. The conditions were fully compatible with MS/MS fragmentation approaches. We further verify that these fingerprints afford the identification of monosaccharidic fragments released upon collisional fragmentation of oligosaccharides. Overall, these results pave the way toward direct sequencing of carbohydrates at the monosaccharide level using HR-IMS.
Data organization through molecular networks has been used in metabolomics over the past years as a way to efficiently mine the massive amount of structural information produced by tandem mass ...spectrometry (MS). However, glycomics lags a step behind: carbohydrate structures involve numerous levels of isomerism, making MS and tandem MS blind to many key structural features of glycans. This roadblock can in part be alleviated with gas-phase ion mobility spectrometry (IMS), a method highly sensitive to isomerism. In this work, we propose a novel strategy for structural glycomics: molecular networking of high-resolution IMS/IMS spectra. We combine the cutting-edge strategies of tandem IMS and molecular networking of spectral data. We demonstrate thatwhen it comes to oligosaccharides and their numerous levels of isomerismsmolecular networks based on IMS/IMS spectra are widely superior to MS/MS-based networks to sort and organize molecules with a high degree of structural relevance.
Carbohydrates are complex structures that still challenge analysts today because of their different levels of isomerism, notably the anomerism of the glycosidic bond. It has been shown recently that ...anomerism is preserved upon gas-phase fragmentation and that high-resolution ion mobility (IMS) can distinguish anomers. However, these concepts have yet to be applied to complex biological products. We have used high-resolution IMS on a cyclic device to characterize the reaction products of Uhgb_MS, a novel mannoside synthase of the GH130 family. We designed a so-called IMS n sequence consisting of (i) separating and isolating specific IMS peaks, (ii) ejecting ions to a pre-array store cell depending on their arrival time, (iii) inducing collisional activation upon reinjection, and (iv) performing multistage IMS analysis of the fragments. First, we applied IMS2 sequences to purely linked α1,2- and β1,2-mannooligosaccharides, which provided us with reference drift times for fragments of known conformation. Then, we performed IMS n analyses of enzymatically produced mannosides and, by comparison with the references, we succeeded in determining the intrachain anomerism of a α1,2-mannotriose and a mix-linked β/α1,2-mannotetraosea first for a crude biological medium. Our results show that the anomerism of glycosides is maintained through multiple stages of collisional fragmentation, and that standalone high-resolution IMS and IMS n can be used to characterize the intrachain anomerism in tri- and tetrasaccharides in a biological medium. This is also the first evidence that a single carbohydrate-active enzyme can synthesize both α- and β-glycosidic linkages.
The analysis of discovery proteomics experiments relies on algorithms that identify peptides from their tandem mass spectra. The almost exhaustive interpretation of these spectra remains an ...unresolved issue. At present, an important number of missing interpretations is probably due to peptides displaying post-translational modifications and variants that yield spectra that are particularly difficult to interpret. However, the emergence of a new generation of mass spectrometers that provide high fragment ion accuracy has paved the way for more efficient algorithms. We present a new software, SpecOMS, that can handle the computational complexity of pairwise comparisons of spectra in the context of large volumes. SpecOMS can compare a whole set of experimental spectra generated by a discovery proteomics experiment to a whole set of theoretical spectra deduced from a protein database in a few minutes on a standard workstation. SpecOMS can ingeniously exploit those capabilities to improve the peptide identification process, allowing strong competition between all possible peptides for spectrum interpretation. Remarkably, this software resolves the drawbacks (i.e., efficiency problems and decreased sensitivity) that usually accompany open modification searches. We highlight this promising approach using results obtained from the analysis of a public human data set downloaded from the PRIDE (PRoteomics IDEntification) database.
Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective 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 oxidative 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.
Analysis of glycans remains a difficult task due to their isomeric complexity. Despite recent progress, determining monosaccharide ring size, a type of isomerism, is still challenging due to the high ...flexibility of the five-membered ring (also called furanose). Galactose is a monosaccharide that can be naturally found in furanose configuration in plant and bacterial polysaccharides. In this study, we used the coupling of tandem mass spectrometry and infrared ion spectroscopy (MS/MS-IR) to investigate compounds containing galactofuranose and galactopyranose. We report the IR fingerprints of monosaccharide fragments and demonstrate for the first time galactose ring-size memory upon collision-induced dissociation (CID) conditions. The linkage of the galactose unit is further obtained by analyzing disaccharide fragments. These findings enable two possible applications. First, labeled oligosaccharide patterns can be analyzed by MS/MS-IR, yielding full sequence information, including the ring size of the galactose unit; second, MS/MS-IR can be readily applied to unlabeled oligosaccharides to rapidly identify the presence of a galactofuranose unit, as a standalone analysis or prior to further sequencing.
The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These ...very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.
This work provides the first use of helium charge transfer dissociation (He-CTD) tandem mass spectrometry (MS/MS) in negative polarity mode. Three sulfated oligosaccharides of natural origin were ...chosen as representative structures that are difficult to solve by conventional MS/MS approaches. Negative polarity He-CTD provided a full set of structurally informative fragments, which permitted the unambiguous determination of the complete structures of these molecules, including the characterization of labile sulfated functional groups. Despite close structural features, the three molecules led to distinct fragmentation patterns depending on the position of the sulfate group in the heterocycle. The observed fragments showed a consistent radical-initiated mechanism of dissociation, which shares similarities with fragment types produced in electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), or extreme UV photodissociation (XUV-PD). Short times of data collection and the fact that the technique can be affordably implementable in any standard laboratory and with a classical ion trap mass spectrometer were other remarkable characteristics of negative polarity He-CTD.
Pectins are natural polysaccharides made from galacturonic acid residues, and they are widely used as an excipient in food and pharmaceutical industries. The degree of methyl-esterification, the ...monomeric composition, and the linkage pattern are all important factors that influence the physical and chemical properties of pectins, such as the solubility. This work focuses on the successful online coupling of charge transfer dissociation–mass spectrometry (CTD-MS) with ultrahigh-performance liquid chromatography (UHPLC) to differentiate isomers of oligogalacturonans derived from citrus pectins. This work employed CTD fragmentation of the pectin mixtures in data-dependent acquisition mode. Compared to the UHPLC with collision-induced dissociation mass spectrometry (UHPLC-CID-MS), UHPLC-CTD-MS yielded fewer ambiguous ions and more structurally informative results. The developed UHPLC-CTD-MS method resulted in abundant cross-ring cleavagesand especially 1,4X n , 1,5X n , and 2,4X n ionswhich helped to identify most of the isomers. The Gal A isomers differed only in the methyl group position along the galacturonic acid backbone. The combination of CTD in real time with UHPLC provides a new tool for the structural characterization of complex mixtures of oligogalacturonans and potentially other classes of oligosaccharides.
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