TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells.

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Synthesis of galactomannan fragments to help NMR assignment of polysaccharides extracted from lichens.

The synthesis of six model trisaccharides representative of galactomannans produced by lichens was performed through stereoselective glycosylation. These standards include linear and branched galactomannans bearing either galactofuranosyl or galactopyranosyl entities. The complete assignment of 1H and 13C signals for both forms of synthetically reduced oligosaccharides was performed. The resulting NMR data were used to quickly demonstrate the structural characteristics of minor polysaccharides within different extracts of three representative lichens.

Ring-Size Memory of Galactose-Containing MS/MS Fragments: Application to the Detection of Galactofuranose in Oligosaccharides and Their Sequencing.

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.

Fingerprinting of Underivatized Monosaccharide Stereoisomers Using High-Resolution Ion Mobility Spectrometry and Its Implications for Carbohydrate Sequencing.

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 momentum─especially 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.

Gas-Phase Behavior of Galactofuranosides upon Collisional Fragmentation: A Multistage High-Resolution Ion Mobility Study.

Carbohydrates are ubiquitous in nature but are among the least conserved biomolecules in life. These biopolymers pose a particular challenge to analytical chemists because of their high diversity and structural heterogeneity. In addition, they contain many isomerisms that complicate their structural characterization, notably by mass spectrometry. The tautomerism of the constitutive subunits is of particular interest. A given cyclized monosaccharide unit can take two forms: a most common 6-membered ring (pyranose, p) and a more flexible 5-membered ring (furanose, f). The tautomers impact the biological properties of polysaccharides, resulting in interesting properties of the derived oligosaccharides. From an analytical point of view, the impact of tautomerism on the gas-phase behavior of ions has scarcely been described in the literature. In this work, we study the behavior of Galf-containing oligosaccharides, ionized as [M+Li]+ species, under collisional dissociation (CID) conditions using high-resolution and multistage ion mobility (IMS) on a Cyclic IMS platform. In the first part of this work, we studied whether disaccharidic fragments released from Galf-containing (Gal)1(Man)2 trisaccharides (and their Galp counterpart) would match the corresponding disaccharide standards, and─despite the fragments generally being a good match─we showed the possibility of Galf migrations and other unidentified alterations in the IMS profile. Next, we expanded on these unknown features using multistage IMS and molecular dynamics, unveiling the contributions of additional gas-phase conformers in the profile of fragments from a Galf-containing trisaccharide compared with the corresponding disaccharides.

Benefits and Limitations of High-Resolution Cyclic IM-MS for Conformational Characterization of Native Therapeutic Monoclonal Antibodies.

Monoclonal antibodies (mAbs) currently represent the main class of therapeutic proteins. mAbs approved by regulatory agencies are selected from IgG1, IgG2, and IgG4 subclasses, which possess different interchain disulfide connectivities. Ion mobility coupled to native mass spectrometry (IM-MS) has emerged as a valuable approach to tackle the challenging characterization of mAbs' higher order structures. However, due to the limited resolution of first-generation IM-MS instruments, subtle conformational differences on large proteins have long been hard to capture. Recent technological developments have aimed at increasing available IM resolving powers and acquisition mode capabilities, namely, through the release of high-resolution IM-MS (HR-IM-MS) instruments, like cyclic IM-MS (cIM-MS). Here, we outline the advantages and drawbacks of cIM-MS for better conformational characterization of intact mAbs (∼150 kDa) in native conditions compared to first-generation instruments. We first assessed the extent to which multipass cIM-MS experiments could improve the separation of mAbs' conformers. These initial results evidenced some limitations of HR-IM-MS for large native biomolecules which possess rich conformational landscapes that remain challenging to decipher even with higher IM resolving powers. Conversely, for collision-induced unfolding (CIU) approaches, higher resolution proved to be particularly useful (i) to reveal new unfolding states and (ii) to enhance the separation of coexisting activated states, thus allowing one to apprehend gas-phase CIU behaviors of mAbs directly at the intact level. Altogether, this study offers a first panoramic overview of the capabilities of cIM-MS for therapeutic mAbs, paving the way for more widespread HR-IM-MS/CIU characterization of mAb-derived formats.

Specificity of a ß-porphyranase produced by the carrageenophyte red alga Chondrus crispus and implications of this unexpected activity on red algal biology.

The carrageenophyte red alga Chondrus crispus produces three family 16 glycoside hydrolases (CcGH16-1, CcGH16-2, and CcGH16-3). Phylogenetically, the red algal GH16 members are closely related to bacterial GH16 homologs from subfamilies 13 and 14, which have characterized marine bacterial ß-carrageenase and ß-porphyranase activities, respectively, yet the functions of these CcGH16 hydrolases have not been determined. Here, we first confirmed the gene locus of the ccgh16-3 gene in the alga to facilitate further investigation. Next, our biochemical characterization of CcGH16-3 revealed an unexpected ß-porphyranase activity, since porphyran is not a known component of the C. crispus extracellular matrix. Kinetic characterization was undertaken on natural porphyran substrate with an experimentally determined molecular weight. We found CcGH16-3 has a pH optimum between 7.5 and 8.0; however, it exhibits reasonably stable activity over a large pH range (pH 7.0-9.0). CcGH16-3 has a KM of 4.0 ± 0.8 µM, a kcat of 79.9 ± 6.9 s-1, and a kcat/KM of 20.1 ± 1.7 µM-1 s-1. We structurally examined fine enzymatic specificity by performing a subsite dissection. CcGH16-3 has a strict requirement for D-galactose and L-galactose-6-sulfate in its -1 and +1 subsites, respectively, whereas the outer subsites are less restrictive. CcGH16-3 is one of a handful of algal enzymes characterized with a specificity for a polysaccharide unknown to be found in their own extracellular matrix. This ß-porphyranase activity in a carrageenophyte red alga may provide defense against red algal pathogens or provide a competitive advantage in niche colonization.

The Maize Pathogen Ustilago maydis Secretes Glycoside Hydrolases and Carbohydrate Oxidases Directed toward Components of the Fungal Cell Wall.

Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay and pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after reannotation of this data set and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on ß-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called UmGH16_1-A and UmAA3_2-A. We show that UmGH16_1-A displays ß-1,3-glucanase activity, with a preference for ß-1,3-glucans with short ß-1,6 substitutions, and UmAA3_2-A is a dehydrogenase catalyzing the oxidation of ß-1,3- and ß-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model ß-1,3-glucans, we show that the linear oligosaccharide products released by UmGH16_1-A are further oxidized by UmAA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both UmGH16_1-A and UmAA3_2-A are coexpressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role. IMPORTANCE Filamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed toward the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.

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

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

Biorefinery of apple pomace: New insights into xyloglucan building blocks.

Within the apple pomace biorefinery cascade processing framework aiming at adding value to an agroindustrial waste, after pectin recovery, this study focused on hemicellulose. The structure of the major apple hemicellulose, xyloglucan (XyG), was assessed as a prerequisite to potential developments in industrial applications. DMSO-LiCl and 4 M KOH soluble hemicelluloses from pectin-extracted apple pomace were purified by anion exchange chromatography. XyG structure was assessed by coupling xyloglucanase and endo-ß-1,4-glucanase digestions to HPAEC and MALDI-TOF MS analyses. 71.9% of pomaces hemicellulose were recovered with starch. DMSO-LiCl and 4 M KOH soluble XyG exhibited Mw of 19 and 140 kDa, respectively. Besides the XXXG, XLXG, XXLG, XXFG, XLFG and XLLG structures, novel oligosaccharides with degree of polymerization of 6-10 were observed after xyloglucanase digestion. Cellobiose and cellotriose were revealed randomly distributed in XyG backbone and were more present in DMSO-LiCl soluble XyG. Residual pomace remains a potential source of other materials.

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb.

Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis/trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis/trans equilibrium was further tackled by exploiting the IMn slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments.

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Combination of High-Resolution Multistage Ion Mobility and Tandem MS with High Energy of Activation to Resolve the Structure of Complex Chemoenzymatically Synthesized Glycans.

Carbohydrates, in particular microbial glycans, are highly structurally diverse biomolecules, the recognition of which governs numerous biological processes. Of special interest, glycans of known monosaccharide composition feature multiple possible isomers, differentiated by the anomerism and position of their glycosidic linkages. Robust analytical tools able to circumvent this extreme structural complexity are increasing in demand to ensure not only the correct determination of naturally occurring glycans but also to support the rapid development of enzymatic and chemoenzymatic glycan synthesis. In support to the later, we report the use of complementary strategies based on mass spectrometry (MS) to evaluate the ability of 14 engineered mutants of sucrose-utilizing α-transglucosylases to produce type/group-specific Shigella flexneri pentasaccharide bricks from a single lightly protected non-natural tetrasaccharide acceptor substrate. A first analysis of the reaction media by UHPLC coupled to high-accuracy MS led to detect six reaction products of enzymatic glucosylation out of the eight possible ones. A seventh structure was evidenced by an additional step of ion mobility at a resolving power (Rp) of approximately 100. Finally, a Rp of about 250 in ion mobility made it possible to detect the eighth and last of the expected structures. Complementary to these measurements, tandem MS with high activation energy charge transfer dissociation (CTD) allowed us to unambiguously characterize seven regioisomers out of the eight possible products of enzymatic glucosylation. This work illustrates the potential of the recently described powerful IMS and CTD-MS methods for the precise structural characterization of complex glycans.

Ultra-high-performance liquid chromatography charge transfer dissociation mass spectrometry (UHPLC-CTD-MS) as a tool for analyzing the structural heterogeneity in carrageenan oligosaccharides.

Ultra-high-performance liquid chromatography (UHPLC) with charge transfer dissociation mass spectrometry (CTD-MS) is presented for the analysis of a mixture of complex sulfated oligosaccharides. The mixture contained kappa (κ), iota (ι), and lambda (λ) carrageenans that contain anhydro bridges, different degrees of sulfation ranging from one to three per dimer, different positioning of the sulfate groups along the backbone, and varying degrees of polymerization (DP) between 4 and 12. Optimization studies using standard mixtures of carrageenans helped establish the optimal conditions for online UHPLC-CTD-MS/MS analysis. Optimization included (1) UHPLC conditions; (2) ion source conditions, such as the capillary voltage, drying gas and nebulizing gas temperature, and flow rate; and (3) CTD-MS conditions, including data-dependent CTD-MS. The UHPLC-CTD results were contrasted with UHPLC-CID results of the same mixture on the same instrument. Whereas CID tends to produce B/Y and C/Z ions with many neutral losses, CTD produced more abundant A/X ions and less abundant neutral losses, which enabled more confident structural detail. The results demonstrate that He-CTD is compatible with the timescale of UHPLC and provides more structural information about carrageenans compared to state-of-the-art methods like UHPLC-CID analysis.

Systematic comparison of eight methods for preparation of high purity sulfated fucans extracted from the brown alga Pelvetia canaliculata.

Sulfated fucans from brown algae are a heterogeneous group of biologically active molecules. To learn more on their structure and to analyze and exploit their biological activities, there is a growing need to develop reliable and cost effective protocols for their preparation. In the present study, a brown alga Pelvetia canaliculata (Linnaeus) was used as a rich source of sulfated fucans. Sulfated fucan preparation methods included neutral and acidic extractions followed by purification with activated charcoal (AC), polyvinylpolypyrrolidone (PVPP), or cetylpyridinium chloride (CPC). Final products were compared in terms of yield, purity, monosaccharide composition and molecular weight. Acidic extractions provided higher yields compared to neutral ones, whereas the AC purification provided sulfated fucan products with the highest purity. Mass spectrometry analyses were done on oligosaccharides produced by the fucanase MfFcnA from the marine bacterium Mariniflexille fucanivorans. This has provided unique insight into enzyme specificity and the structural characteristics of sulfated fucans.

In-depth structural characterization of oligosaccharides released by GH107 endofucanase MfFcnA reveals enzyme subsite specificity and sulfated fucan substructural features.

The extracellular matrix of brown algae represents an abundant source of fucose-containing sulfated polysaccharides (FCSPs). FCSPs include sulfated fucans, essentially composed of fucose, and highly heterogeneous fucoidans, comprising various monosaccharides. Despite a range of potentially valuable biological activities, the structures of FCSPs are only partially characterized and enzymatic tools leading to their deconstruction are rare. Previously, the enzyme MfFcnA was isolated from the marine bacterium Mariniflexile fucanivorans and biochemically characterized as an endo-α-1 â†’ 4-l-fucanase, the first member of glycoside hydrolase family 107. Here, MfFcnA was used as an enzymatic tool to deconstruct the structure of the sulfated fucans from Pelvetia canaliculata (Fucales brown alga). Oligofucans released by MfFcnA at different time points were characterized using mass spectrometry coupled with liquid chromatography and tandem mass spectrometry through Charge Transfer Dissociation. This approach highlights a large diversity in the structures released. In particular, the analyses show the presence of species with less than three sulfates per two fucose residues. They also reveal species with monosaccharides other than fucose and the occurrence of laterally branched residues. Precisely, the lateral branching is either in the form of a hexose accompanied by a trisulfated fucose nearby, or of a side chain of fucoses with a pentose as the branching point on the polymer. Overall, the results indicate that the structure of sulfated fucans from P. canaliculata is more complex than expected. They also reveal the interesting capacity of MfFcnA to accommodate different substrates, leading to structurally diverse oligofucan products that potentially could be screened for bioactivities.

Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides.

Enzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active [Formula: see text]-transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens.

Molecular Networking of High-Resolution Tandem Ion Mobility Spectra: A Structurally Relevant Way of Organizing Data in Glycomics?

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 that-when it comes to oligosaccharides and their numerous levels of isomerisms-molecular 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.

Charge transfer dissociation of a branched glycan with alkali and alkaline earth metal adducts.

Alkali and alkaline earth metal adducts of a branched glycan, XXXG, were analyzed with helium charge transfer dissociation (He-CTD) and low-energy collision-induced dissociation (LE-CID) to investigate if metalation would impact the type of fragments generated and the structural characterization of the analyte. The studied adducts included 1+ and 2+ precursors involving one or more of the cations: H+ , Na+ , K+ , Ca2+ , and Mg2+ . Regardless of the metal adduct, He-CTD generated abundant and numerous glycosidic and cross-ring cleavages that were structurally informative and able to identify the 1,4-linkage and 1,6-branching patterns. In contrast, the LE-CID spectra mainly contained glycosidic cleavages, consecutive fragments, and numerous neutral losses, which complicated spectral interpretation. LE-CID of [M + K + H]2+ and [M + Na]+ precursors generated a few cross-ring cleavages, but they were not sufficient to identify the 1,4-linkage and 1,6-branching pattern of the XXXG xyloglucan. He-CTD predominantly generated 1+ fragments from 1+ precursors and 2+ product ions from 2+ precursors, although both LE-CID and He-CTD were able to generate 1+ product ions from 2+ adducts of magnesium and calcium. The singly charged fragments derive from the loss of H+ from the metalated product ions and the formation of a protonated complementary product ion; such observations are similar to previous reports for magnesium and calcium salts undergoing electron capture dissociation (ECD) activation. However, during He-CTD, the [M + Mg]2+ precursor generated more singly charged product ions than [M + Ca]2+ , either because Mg has a higher second ionization potential than Ca or because of conformational differences and the locations of the charging adducts during fragmentation. He-CTD of the [M + 2Na]2+ and the [M + 2 K]2+ precursors generated singly charged product ions from the loss of a sodium ion and potassium ion, respectively. In summary, although the metal ions influence the mass and charge state of the observed product ions, the metal ions had a negligible effect on the types of cross-ring cleavages observed.

Oligator: a flexible interface to draw oligosaccharide structures and generate their theoretical tandem mass spectra.

SUMMARY: Oligator is software designed to assist scientists in their exploration of MS/MS experiments, especially for oligosaccharides bearing unreferenced chemical substitutions. Through a graphical interface, users have the total flexibility to build a candidate glycan structure and produce the corresponding theoretical MS/MS spectrum in accordance with the usual ion nomenclature. The structural information is saved using standard notations, in text format, which facilitates the capitalization and exchange of data as well as any other processing of the information. AVAILABILITY AND IMPLEMENTATION: Source code and user manual are freely available at https://github.com/vlollier/oligator. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.