Deciphering branched galactomannan structures via multistage ion mobility MS and MSn fragmentation (Multistage IMSn)

Deciphering branched galactomannan structures via multistage ion mobility MS and MSn fragmentation (Multistage IMSn) | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Rapid Communications in Mass Spectrometry This is a preprint and has not been peer reviewed. Data may be preliminary. 22 April 2025 V1 Latest version Share on Deciphering branched galactomannan structures via multistage ion mobility MS and MSn fragmentation (Multistage IMSn) Authors : Rania BENAZZA 0000-0002-9616-1225 , Mathieu FANUEL , Hélène ROGNIAUX , and David ROPARTZ [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174535564.44097969/v1 Published Rapid Communications in Mass Spectrometry Version of record Peer review timeline 335 views 222 downloads Contents Abstract Introduction Material and methods Results and Discussion Conclusion Acknowledgments Bibliography Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Rationale Hemicelluloses are abundant carbohydrate components in plant cell walls. They play a crucial structural role in binding cellulose microfibrils, in addition to other biological functions. Their high structural variability directly influences their biological properties, making it important to establish a structure–function relationship. In the case of galactomannans, this complexity relies in branching, large size, in addition to isomerism, which makes their characterization challenging. In this context, we have demonstrated that cyclic ion mobility spectrometry (IMS), combined with porous graphitic carbon (PGC) chromatography, mass spectrometry (MS) and multistage MS/MS fragmentation (IMS n ), is a powerful tool for the detailed elucidation of galactomannan structures. Methods In this study, we show that our multistage IMS n sequencing approach, previously validated for homo-linear oligosaccharides, can be successfully applied to hetero-branched hemicelluloses with careful adjustments. Our approach consists of building a database library of high resolution IMS profiles of di- and tri-saccharidic fragments. Then, the sequence of the oligosaccharide of interest is retrieved by comparing the HR-IMS profile of its di- and tri-saccharide fragments with the profiles from the DB library. Results In fact, our IMS n experiments on galactomannan reveal ATD profiles matching with known reference structures, confirming the co-existence of multiple isomers. In addition, we proved that this approach could be improved by incorporating trisaccharidic fragments to our DB library, serving in the characterization of higher DP structures (DP4 in this case). Conclusions Overall, this work paves the way for the characterization of even more complex oligosaccharides, which can be potentially used for bio-based material conception. Deciphering branched galactomannan structures via multistage ion mobility MS and MS n fragmentation (Multistage IMS n ) Rania BENAZZA 1,2 , Mathieu FANUEL 1,2 , Hélène ROGNIAUX 1,2 , David ROPARTZ 1,2 1 INRAE, UR BIA, F-44316 Nantes, France 2 INRAE, PROBE Research Infrastructure, BIBS Facility, F-44316, Nantes, France Corresponding author : David ROPARTZ Email : [email protected] Address : INRAE. UR BIA. La Géraudière, F-44316 NANTES. Telephone : +33 (0)2 40 67 50 34 Graphical abstract: Abstract Rationale Hemicelluloses are abundant carbohydrate components in plant cell walls. They play a crucial structural role in binding cellulose microfibrils, in addition to other biological functions. Their high structural variability directly influences their biological properties, making it important to establish a structure–function relationship. In the case of galactomannans, this complexity relies in branching, large size, in addition to isomerism, which makes their characterization challenging. In this context, we have demonstrated that cyclic ion mobility spectrometry (IMS), combined with porous graphitic carbon (PGC) chromatography, mass spectrometry (MS) and multistage MS/MS fragmentation (IMS n ), is a powerful tool for the detailed elucidation of galactomannan structures. Methods In this study, we show that our multistage IMS n sequencing approach, previously validated for homo-linear oligosaccharides, can be successfully applied to hetero-branched hemicelluloses with careful adjustments. Our approach consists of building a database library of high resolution IMS profiles of di- and tri-saccharidic fragments. Then, the sequence of the oligosaccharide of interest is retrieved by comparing the HR-IMS profile of its di- and tri-saccharide fragments with the profiles from the DB library. Results In fact, our IMS n experiments on galactomannan reveal ATD profiles matching with known reference structures, confirming the co-existence of multiple isomers. In addition, we proved that this approach could be improved by incorporating trisaccharidic fragments to our DB library, serving in the characterization of higher DP structures (DP4 in this case). Conclusions Overall, this work paves the way for the characterization of even more complex oligosaccharides, which can be potentially used for bio-based material conception. Keywords : Hemicellulose, oligosaccharides, IMS, multistage IMS n , galactomannan Introduction Cell wall polysaccharides are classically divided into cellulose, hemicellulose and pectin, which represent the majority of the plant cell wall, along with proteins and, in the case of secondary cell walls, of phenolic compounds 1 . Hemicelluloses are β(1-4)-linked sugars in equatorial configuration, forming structurally distinct classes such as xyloglucans, xylans, mannans, glucomannans, and mixed β(1-4), (1-3)-glucans. Although, they are abundant in many plant species, they show very distinct structural details in different species and different plant cell types 1 . The major role of hemicelluloses is to strengthen the cell wall by tethering cellulose microfibrils 2 , but they are also responsible of – or involved in – many other biological functions in primary and secondary cell walls, like cell expansion 2,3 , seed storage and molecules signalling 1 . Beyond their structural and biological roles in plant cell wall, plant-based polysaccharides are increasingly recognized for their potential as promising eco-friendly and sustainable bio-materials. Due to their abundance, biocompatibility and biodegradability, they are replacing synthetic materials in biomedical engineering, food packaging and agricultural or industrial purpose for a greener chemistry 4 . The functions and properties of polysaccharides for the aforementioned industrial applications are highly related to their structures, although these structures vary considerably at different levels. The variability can occur in the number of constituent subunits, the nature of these subunits, and in the nature of their linkage. In addition to their isomeric heterogeneity, the presence of chemical modifications and branched chains on polysaccharides adds a supplementary level of complexity to their structure. It is therefore necessary to finely characterize these polysaccharides, in order to better understand the impact of all these structural details on their function, so as to be able to control them closely and adjust their properties to targeted uses. Common analytical tools used currently for the characterization of oligosaccharides lack speed and sensitivity. Nuclear magnetic resonance spectroscopy (NMR), although highly informative, requires a large amount of material, in addition to extensive analysis time 5 . Mass spectrometry (MS)-based approaches provide a valuable information on the direct measurement of oligosaccharides, however cannot fully resolve their structures due to the isomers’ inherent complexity 6 . MS can be coupled to liquid chromatography, such as hydrophilic interaction LC (HILIC-MS) 7–9 and porous graphitic carbon (PGC-MS) 10–12 for neutral oligosaccharides, to separate isomers prior to their MS analysis., However, due to the limitation of classical MSMS approach (Collision induced dissociation, CID) to produce informative fragments, the method often fail to enable the structural characterization of the separated isomers. On the other hand, ion mobility (IM) is a robust an reproducible method that has the ability to tackle the isomerism challenges, if the modifications impact the conformation of the ion in gas phase. IM is based on measuring the mobility of ions in a buffer gas and under the effect of an electric field, and thus depends on the ions’ conformation under these conditions 13 . Current state-of-the art IM instruments, achieve high resolutions, making it possible to decipher the challenges encountered in separating isomeric molecules. SLIM-based TWIMS developed by the group of R. D. Smith 14 can theoretically reach a resolving power (Rp) of 600 when travelling the 13m drift tube 100 times. Giles et al 15 also presented Cyclic IMS as the highest resolution commercially available IM device, using the TWIMS method as well. It has been proved that a Rp up to 900 for oligosaccharides characterization can be reached upon increasing the number of passes in the 1m cyclic racetrack 16 . In addition to access to very high-resolution ion mobility, the clear advantage of this instrument is that it can combine ion mobility and MSMS on several stages, making the cyclic IMS a powerful tool in glycosciences. Notably, our group used cyclic IMS to sequence linear homo-oligosaccharides using a multistage IMS n approach. The first step is to build up a database of HR-IMS profiles of mono- and di-saccharides. Then, the sequence of the oligosaccharide of interest is retrieved by comparing the HR-IMS profile of its monosaccharide fragments (to define the sequence in terms of building blocks) or disaccharide fragments (to define the sequence in terms of branching pattern, including anomerism and positional isomerism) with the profiles stored in the databank. The benefits of this approach allowed mainly to tackle the isomerism challenges within carbohydrates, which helped understanding the gas phase behaviour of galactofuranosides in a study by Ollivier et al. 17 and was expanded to the structural elucidation of enzymatically produced mannosides 18 . Additionally, Van de Put et al. used a similar strategy, based on cyclic IMS separation and pre-store CID fragmentation, to determine the monosaccharide composition and the anomeric configuration of disaccharide isomers within prebiotic galactooligosaccharides 19 . There studies highlight the many advantages of cyclic IMS in overcoming the difficulties of the challenging characterization of oligosaccharides. However, hemicelluloses in nature are much more heterogeneous than synthetic oligosaccharides. For instance, xyloglucans, which represent the majority of the hemicelluloses of the plants primary cell walls, have a high structural complexity due to their branched carbohydrate chains. They are in fact composed of a glucosyl backbone with xylosyl side chains, that can be substituted by a galactose that can potentially be acetylated and/or substituted by a fucose. β(1-4)-linked mannans are also challenging as they can be found as a linear backbone consisting of mannose units, called mannans. They can be substituted by galactose, referred to as galactomannans. They can consist as well of nonrepeating mannose and glucose units, known as glucomannans. They can also be acetylated as in the case of mannans and glucomannans 1 . Each of these structural variations, in addition to isomerism, can influence the physical and biological properties of oligosaccharides, and push us in advancing analytical tools to characterize these complex structures and better answer these questions. In this study, we focus on galactomannan structures, which are quite complex due to the presence of galactose ramifications. Accordingly, the aim of this study is to i) characterise the ramified structures and demonstrate the applicability of our multistage IMS n approach, and ii) to assess its ability to handle more complex and larger oligosaccharides structures. Material and methods Samples Branched galactomannan oligosaccharides including (Galα1-6)-Manβ1-4Man (code MAN213, hereafter referred to as GM) and the mixture of β(1-4) linked D-mannopyranosyl units substituted by an α(1-6) D-galactopyranosyl (code MAN215) were purchased from Elicityl (Crolles, France). Branched Manβ1-4(Galα1-6)-Man (code GMM3, hereafter referred to as MG) as well as linear mannotriose (Manβ(1-4)Manβ(1-4)Man, code O-MTR, hereafter referred to as M3) were obtained from Megazyme (Wicklow, Ireland). The structure of the samples following Haworth projection is represented in Figure S1 . Chemicals HPLC grade methanol (MeOH), acetonitrile (ACN) and water (H 2 O) were purchased from Carlo-Erba (Val de Reuil, France). Lithium chloride (LiCl) was obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). IMS-MS experiments IMS-MS analyses were performed on a SELECT SERIES Cyclic IMS instrument (Waters, Wilmslow, UK). Oligosaccharides were infused at a concentration of 0.5-25 µg/mL in 50:50 H 2 O/MeOH depending on the purity of the sample, through a syringe pump set at a flowrate of 5 µL/min. Lithium adducts were generated upon samples doping with 0.1 mM LiCl. The Z-spray ESI source parameters were set as following: the capillary voltage at 2.8 kV, the cone voltage at 80V, the source temperature at 100°C, the desolvation temperature at 300°C and the nebulizer pressure at 6 bar. Of note, the capillary voltage and cone voltage were both increased to 3 kV and 160V, respectively, to boost higher DP structures ionization. IM separations were performed in static mode with a cyclic and array traveling wave (TW) velocity both at 350 m/s and a TW static height of 16V. The IMS-MS parameters are detailed in the SI ( Text S1 ). Top & Tail (TT) experiments were carried out in the IMS to clean the samples and obtain distinct IM profile for each structure ( Figure S2 ). Oligosaccharides sequencing was performed as well in the pre-store where an optimized collisional energy (CID) for each sample was applied on the slices of interest, to release the fragments of interest ( Table SI ). LC-IMS-MS experiments. Acquisitions were performed with the same IMS and MS parameters than for classical IMS-MS experiments previously detailed. Here, the mass spectrometer was coupled with an Acquity H-Class UHPLC system (Waters, Manchester, UK) equipped with a Hypercarb Porous Carbon Graphite (PGC) column (100 × 1 mm, particle size 3 µm from Thermo-Fischer Scientific, Courtaboeuf, France) heated at 80°C. Elution was performed at a flowrate of 0.165 mL/min. The gradient was optimized in order to separate the different conformers of DP3 and DP4 species ( Figure S3 ) and was as follows: 0−33 min at 0−20 % B; 33−34 min at 20−80 % B; 34−38 min at 80 % B; 38−45 min, 80-0 % B. In LC-IMS-MS/MS experiments, the energy in the trap cell was optimized depending on the nature of the fragments of interest ( Table S2 ). Data analysis. All obtained spectra were recorded using the Quartz software on the cIMS instrument (Waters Embedded Analyser release 9, Waters, Wilmslow, UK). Data processing was performed on Masslynx v4.2 and Driftscope v2.9 (both from Waters, Wilmslow, UK). Arrival times (t A ) were corrected to give drift times (t D ) to compare the effective separation time of the fragments with the reference standards. This was done by simply subtracting the duration of all events prior to the separation time. ATDs smoothing was performed using the Stravizky-Golay algorithm (window size = 1, number of smooths = 2). Gaussian fitting of the extracted ATDs was done on CIUSuite2 software with the following parameters: minimum peak amplitude = 0.01, maximum components = 6, expected peak width = 2 and peak width tolerance = 1). Results and Discussion Enrichment of the data base library: addition of new fingerprints In order to produce an efficient IMS database library for fragments, we chose to dope our samples with LiCl and we focused on [M+Li] + ions in all experiments, because it is reported that Li adducts yield higher intensity fragments upon CID experiments of oligosaccharides, compared to Na adducts for instance 17 . The three trisaccharides representative of the different branching patterns found in galactomannans are commercially available (DP3 (β1,4)Man-Man (linear backbone, M3), a DP2 (β1,4)Man-Man with (α1,6)Gal branched on the reducing end (MG) and a DP2 (β1,4)Man-Man with (α1,6)Gal branched on the non-reducing end (GM) ( Figure S1 ). We used these trisaccharides i) to register disaccharides’ and monosaccharides’ fragments IMS fingerprints to complete our database, and ii) to validate the fact that the fingerprints measured are independent from the lateral branching of the galactose in (α1,6) in the different position. In a first step, each trisaccharide was analysed independently. We selected the cIMS parameters ( Text S1) that were able to differentiate between the trisaccharidic standards (3 isomers) where the number of separations was set to eight passes ( Figure S4 A ). The three standards (M3, MG and GM) exhibited each a distinct distribution after eight passes, upon selection of a mass corresponding to DP3 species ([M+Li] + , m/z 511.2). However, we show that each species exhibits multiple IMS peaks. Considering that, each species can have several conformations, we cannot conclude whether the conformations come from a single species or whether there is a contamination by the presence of several isomers (as exemplified for M3 that presents two IMS peaks at 124.20 ms and 126.44 ms, and for GM that presents a peak shoulder before the major IMS peak at 132.48 ms). To validate the specific fingerprints, we therefore carried out PGC-IM-MS analysis on the DP3 species ( Figure S5 ). The aim of this experiments is i) to determine the number of real isomers with the chromatographic scale and ii) to obtain the structural information through IM/MS. Indeed, when using PGC-IM-MS ( Figure 1 ), the M3 presents a bimodal distribution with two specific features at 124.11 ms and 126.35 ms (corrected t D ), thus two conformations instead of only one that was observed in IMS-MS experiments ( Figure S4 ). GM presents also a bimodal distribution with baseline resolved peaks at 127.14 ms and 132.42 ms. Here, we notice the disappearing of the peak shoulder observed in direct infusion preceding the major peak, with the appearance of the supplementary conformation observed when using PGC, at 127.14 ms. This conformation has a t D close to that from M3 (~126.44 ms in M3, Figure S4 ), and thus was lost at the pre-purification step via top and tail experiments. For MG, a single peak at 130.44 ms was observed ( Figure 1 A ), in-line with the results from IMS-MS. Overall, the observed features for M3 and MG using PGC-IMS-MS were comparable with those observed through direct infusion. However, in the case of GM, when using PGC-IM-MS, we noticed the presence of a distinct feature at 127.14 ms that was not observed in direct infusion. This feature could have been lost during slicing experiments ( Figure S2 ), where the sample containing GM isomer was present in the mixture with other structures that overlapped with its second feature. This step, highlighted the complementarity of PGC to ion mobility when it comes to building a database library of IM profiles. Thus, we chose to keep the ATD profiles obtained through PGC-IM-MS for the DP3 species. Each standard was then fragmented and the signature of the product ions ( i.e. Y and B product ions) was registered. A CID energy of 100-120 eV was applied on the three species in the pre-store of the IMS cell, followed by the IM measurement of the resulting fragments, following a strategy previously described by our group 17 . In theory, the fragmentation of oligosaccharides ions under CID condition, leads to the production of fragments which contain the reducing ( Y- and Z- type ions) and the non-reducing ( B- and C- type ions) ends 20 . This can be a problem because these fragments can be isobaric due to the symmetry of the molecule ( m/z of fragment Y = m/z of fragment C , and m/z of fragment Z = m/z of fragment B ). However, in the cyclic IMS, previous studies 17 showed that, when working with nitrogen as a collision gas in the transfer, trap or pre-store cell, the C and Z fragment ions are minor (100-fold lower (if any) than Y- and B- type ions), which leads us to focus only on the latter fragments to determine the IM profiles ( Figure S6 ). Specific sequence of separation was applied for each fragment. In a first step, we attempted to produce B3 fragments by applying a pre-store energy on DP3 species. However, since B3 fragments are a dehydrated form of intact Y3 ions, they appear with very low intensity which makes their detection difficult after ion mobility separation (data not shown). To address this limitation, B3 fragments were obtained via fragmentation of pure DP3 structures in the trap cell where the energy was finely tuned with increasing slightly the cone voltage in order to induce pseudo- B 3 formation ( Figure 1 B ). The three species exhibited each distinct B3 fragment ([M+Li] + at m/z 493.2) profiles after four passes in the cyclic IMS. M3 presents a multimodal distribution with peaks at 57.71 ms, 60.75 ms, 62.60 ms and 65.90. GM presents, as well, a multimodal distribution with features at 57.71 ms, 63.13 ms and 67.09 ms. For MG, the profile presents a bimodal distribution with peaks at 63.79 ms and 69.07 ms with minor contribution of other conformations, which allows to distinguish this fragment from the prior characterized ones. In general, the pseudo-fragmentation of DP3 species produced distinct profiles enabling the differentiation between the three isomers. Next, the product ions which correspond to the disaccharides are analysed ( i.e specificity of the branching pattern in our sequencing strategy) and are their signatures are presented in Figure 1C and D . Y 2 fragment at m/z 349.1 and B 2 fragment at m/z 331.1 were respectively submitted to 8 and 10 passes in the cyclic cell. The Y 2 fragment at ([M+Li] + at m/z 349.1) produced from the fragmentation of the M3 showed a bimodal distribution (peaks at t D = 80.71 ms and 84.14 ms). The B 2 fragment was also detected at ([M+Li + ] at m/z 331.1), and showed a multimodal distribution with peaks at 95.95 ms, 97.93 ms and 101.23 ms (corrected t D ), corresponding to the mannobiose structure at the non-reducing end. The observed matches of the Y 2 and B 2 fragments with the IM profiles of the mannobiose structures stored in the database confirm i) the structure of the starting M3, and ii) the validity of the sequencing approach. For GM, the same IMS signature was observed for the Y 2 fragment at ([M+Li] + at m/z 349.1), confirming its mannobiose unit at the reducing end. However, a new B 2 fragment signature was detected with peaks at 96.45 ms, 98.43 ms and 101.86 ms (corrected t D ), which gives a new specific signature of the (α1,6)Gal-Man unit at the non-reducing end that allows to add a new fingerprint to our DB library. From the analysis of the MG, no B 2 ions signal was obtained. However, a complex Y 2 fragments profile that correspond to the measurement of the [M+Li] + at m/z 349.1 was observed. To clarify this point, slicing experiments on the different Y 2 IMS peaks were performed. This revealed two distinct signatures; the first corresponding to the fingerprint of the mannobiose with peaks at 80.81 ms and 84.14 ms (corrected t D ), the second is a new signature with a single IMS peak at 86.12 ms. From the structure of the MG, the fragment that corresponds to the mannobiose is a Yα resulting from the loss of the reducing end galactose. From that, the new signature obtained is the real fingerprint of the (α1,6)Gal-Man. Remarkably, this sample exemplifies the challenges that can be encountered upon the fragmentation of ramified oligosaccharides. Whereas multiple Y n fragments were generated and confirmed the ramified structure, the lack of B 2 fragments that should reinforce our interpretation, shows that branching information could be lost due to the consecutive fragmentation pathways. Overall, the features observed at both trisaccharide extremities, show distinct fingerprints that allow to confirm their structure and can serve as new reference fingerprints. Even if signatures seem to be complex (as shown for the B 2 fragments of the M3 and GM), their deconvolution upon gaussian fitting of their t D profiles ( Figure S7 ) exhibit distinct multimodal distributions that allow their differentiation with sufficient clarity. In a final step, the product ions which correspond to the monosaccharides are analysed, as shown in Figure S8 . Y 1 fragment at m/z 187.1 and B 2 fragment at m/z 169.1 were respectively submitted to 6 passes (including 4 static and 2 ramped waves) and 2 passes in the cyclic cell. M3 fragmentation produced mannose Y 1 ([M+Li] + , m/z 187.1) and B 1 ([M+Li] + , m/z 169.1) fragments, as expected. The Y 1 fragment exhibited a single peak at t D = 39.44 ms, while B 1 showed a bimodal distribution with a major peak at 14.25 ms and a minor one at 15.70 ms (corrected t D ). As expected, the IMS profiles correlated perfectly with mannose units at both extremities as stored in the library ( Figure S8 ). Surprisingly, For GM and MG, the same fragmentation patterns were observed as in the case of M3, which would indicate the presence of mannose units at both-extremities. From these results, the branched galactose units are mainly not observed upon CID fragmentation. Our hypothesis is that the lack of information on the galactose branching is due to the lithium adduct being preferentially coordinated on the mannose main chain. The fragmentation would then mainly produce a “neutral loss” of galactose and thus would prevent the complete assignment of monosaccharidic fragments. However, it is worth noting that the B 1 fragment stemming from the dissociation of each reference, presents a slightly different profile. This difference is reflected in the relative intensity of the second feature measured at 15.70 ms for the M3, 15.93 ms for the GM and 15.82 for the MG. Indeed, this feature is signature of the mannose B 1 fragment, which can be different according to the position of the double-bond formed based on the particular nature of the glycosidic-bond cleaved 21 . To conclude in this part, these results led to a modification in our strategy because of the lack of information provided by monosaccharides for hetero-branched OS. However, the use of disaccharides fragments does not appear to be sufficient due to the possibility of a neutral loss of the laterally branched galactose (as exemplified for the MG structure). We showed that the trisaccharide standards exhibit unique IMS fingerprints, allowing a confident structural discrimination, thus we decided to complete the database signature with the profiles of Y 3 (the profiles of the trisaccharides) and B 3 for the study of larger species. Insights from multistage IMSexperiments To validate our approach, glycan structures that consist of enzymatic digestion of a galactomannan oligosaccharide were studied by IMS n experiments. MAN215 (from Elicityl) contains multiple DP species (from DP2 to DP9) with potential isomers for each DP depending of the branching of galactopyranosyl units ( Figure S1 ). The DP4 of the MAN215 were studied by IMS-MS in direct infusion. We selected in the quadrupole a mass corresponding to DP4 species ([M+Li] + , m/z 673.2), after injecting the sample in similar MS conditions than the previously analysed DP3 species. Figure S9A shows the mobilogram obtained after 6 passes in the cyclic IMS. In a first place, we performed slicing experiments to separate the different conformers, which were subjected to 6 passes in the mobility cell. This resulted in four distinct IMS peaks depicted in Figure S9B-E with C1, C2, C3 and C4 at 132.02 ms, 133.77 ms, 136.64 ms and 140.87 ms, respectively. As demonstrated for the M3 ( Figure 1 and Figure S5 ), a single structure can adopt different conformations so providing different peak in IMS. To correlate the number of conformations detected and the real number of structures in the sample, the PGC-IM-MS approach was used for DP4 species analysis ( Figure 2 ). Indeed, at the chromatographic scale, the separation reveals three baseline resolved peaks, hence three different isomers. The first isomer is observed at 14.6 min and presented one conformation measured at 133.08 ms (P1) by IMS. The second isomer eluting at 15.4 min presented two different conformations with IMS peaks at 131.10 ms and 133.08 ms (P2). Finally, a last isomer eluted at 20.6 min and presented a bimodal IMS peak distribution at 136.38 ms and 140.01 ms (P3) ( Figure 2A ). These observations highlight the complementarity of PGC and IMS to address the problem of the structural study of complex mixture of oligosaccharides. Indeed, the conformations C1 and C2 observed in IMS-MS experiments ( Figure S9B-C ) correspond to the P1 isomer observed in PGC, confirming the presence of a first distinct DP4 structure. However, the C2 feature observed at 133.77 ms in IMS-MS ( Figure S9C ) could also match with the one corresponding to the P2 isomer in PGC that elutes at 15.4 min. This poses a real challenge, since both isomers (P1 and P2 in Figure 2 ) share a common feature at 133.08 min. In contrast, the conformations C3 and C4 obtained through direct infusion ( Figure S9D-E ) correspond clearly to the isomer eluting at 20.6 min (P3), highlighting the complementarity of both approaches. From these experiments we obtain the specific IM signatures of each DP4 isomers with the contribution of all conformations, allowing the production of comprehensive product ion profiles. Can we go further and sequence higher DP species? We then applied our sequencing strategy on each DP4 efficiently separated by PGC. Figure 3 shows the mobilograms obtained from the sequencing of MAN215 DP4 species ([M+Li] + , m/z 673.2) compared to reference fragments. At the reducing extremity, there are mainly peaks corresponding to mannobiose observed for P1, P2, and for P3. P1 exhibits an additional peak corresponding to the galactosyl-mannose unit. On the non-reducing end, B 2 fragments matched mainly with mannobiose disaccharide for all isomers, i.e., P1, P2 and P3. In these experiments, disaccharidic fragments showed very similar features, with slight differences regarding the Y 2 fragment for the first conformer (P1) at 85.70 ms. Therefore, the high similarity in the profiles of the B 2 and Y 2 fragments do not allow a definitive assignment of the tetrasaccharidic structures of P1, P2 and P3. Once again, as discussed for the trisaccharides sequencing, the disaccharide product ions are not sufficient. We considered trisaccharidic fragment profiles previously registered for the characterization of DP4 structures. Alignments are shown in Figure 4 . The IMS profile obtained for the Y 3 of P1 revealed the contribution of two species. First, a doublet at 124.5 ms and 127.01 ms that can be attributed to the Y 3 of M3, and a single peak at 131.10 ms that is attributed to the Y 3 of MG. From that, P1 was identified as a linear M3 backbone with a galactose branched at the reducing extremity. The absence of the B 3 product ion support this attribution, as explained for the results presented Figure 1 (absence of B 2 on the MG standard that has the galactose branched on the reducing end mannose). For P2, the IMS profile obtained for the Y 3 corresponds to the signature of M3 (doublet at 124.64 ms and 126.88 ms). Of note, a minor peak detected at 131.10 ms was unexpectedly observed at the reducing end. This signal may originate from the preceding P1 structure, as slight carryover given the heterogeneity of the sample. The IMS profile of the B 3 matches with the pseudo B 3 GM standard distribution (57.71 ms, 63.13 ms and 66.82 ms). All these results show that P2 is a linear M3 backbone with a galactose branched at the non-reducing extremity. Finally, the Y 3 signature of the P3 exhibited two co-existent conformations at the reducing extremity corresponding to M3 (124.37 ms and 126.88 ms) and GM (126.88 ms and 132.69 ms), with a B 3 fragment signature that corresponds to the one from MG (63.52 ms and 69.20 ms). All these measurements show that P3 is a linear M3 backbone with galactose branching at the central mannose. To conclude, our sequencing strategy enabled the characterization of branched DP4 structures. The optimized LC gradient, combined with high resolution IM measurements, provided clear information on the number of isomers present in a complex biological sample, with distinct IMS profiles. While disaccharidic fragments offered limited structural differentiation for DP4 species, the trisaccharidic fragments provided highly informative insights, revealing the identity of each isomer. In summary, we could prove via our sequencing strategy the variability in DP4 structures in the MAN215 sample, which correspond here to linear mannobiose bearing galactose branching either at the reducing extremity (P1), non-reducing extremity (P2) or at the central position (P3). Conclusion Throughout this study, we expanded the applications of cyclic IMS to the determination of HR-IMS profiles of oligosaccharides derived from branched polysaccharides. This work has highlighted several limitations that make, in our hands, the characterization of ramified oligosaccharides rather tricky through these IMS n strategies. One of the main challenges is the potential loss of lateral chain. We approached this difficulty by focusing instead on di- and tri-saccharidic fragments, that provide valuable information (contrary to the monosaccharide signatures). The reference fingerprints were then compared with the unknown structures to define their identity. We note that, our study is the first, to our best knowledge, to focus on branched oligosaccharides characterization, completing our previous ground-breaking advances on linear oligosaccharides sequencing using multistage IMS n . Our aim is to extend our refined approach, to the characterization of other structurally complex hemicelluloses, such as xyloglucans and glucomannans, which can also occur in acetylated forms 1 .To strengthen our characterization capability and fulfil this ambition, we could consider, in addition to IMS, higher-energy fragmentation data. Indeed, methods such as electron-based dissociation ( ExD 22 or He-CTD 23,24 ) and photon-based dissociation ( XUVPD 25 or VUV 26 ) can pave the way to better understanding oligosaccharides structures and enhance the ability to characterize complex structures. Acknowledgments This work is powered by the AMETHYST project of the PEPR DIADEM, with the financial support from France 2030 program (project number ANR-22-PEXD-0015). 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Keywords galactomannan hemicellulose ims multistage imsn oligosaccharides Authors Affiliations Rania BENAZZA 0000-0002-9616-1225 UR BIA View all articles by this author Mathieu FANUEL UR BIA View all articles by this author Hélène ROGNIAUX UR BIA View all articles by this author David ROPARTZ [email protected] UR BIA View all articles by this author Metrics & Citations Metrics Article Usage 335 views 222 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Rania BENAZZA, Mathieu FANUEL, Hélène ROGNIAUX, et al. Deciphering branched galactomannan structures via multistage ion mobility MS and MSn fragmentation (Multistage IMSn). Authorea . 22 April 2025. DOI: https://doi.org/10.22541/au.174535564.44097969/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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