{"paper_id":"2450485c-71c3-41b8-8450-7a2453e8a1dd","body_text":"1 \nMass Spectrometric Profiling of Microbial Polysaccharides Using Laser 1 \nDesorption/Ionization – Time-of-Flight (LDI-TOF) and Liquid 2 \nChromatography/Mass Spectrometry  (LC/MS): A Novel Method for Structural 3 \nFingerprinting and Derivatization 4 \n 5 \nLucia Dadovska1, Veronika Paskova1, Petr Novak2, Jaroslav Hrabak1* 6 \n 7 \n1Department of Microbiology, Biomedical Center, Faculty of Medicine in Pilsen, Charles University, alej Svobody 8 \n76, 323 00 Pilsen, Czech Republic 9 \n1Institute of Microbiology, Czech Academy of Sciences, BIOCEV, Prumyslova 595, 252 50 Vestec, Czech Republic 10 \n 11 \nAUTHOR INFORMATION 12 \nCorresponding Author: Jaroslav Hrabak, Department of Microbiology, Biomedical Center, Faculty of Medicine in 13 \nPilsen, Charles University, alej Svobody 76, 323 00 Pilsen, Czech Republic, E-mail: Jaroslav.Hrabak@lfp.cuni.cz 14 \n 15 \nARTICLE TYPE 16 \nOriginal Article 17 \n 18 \nNumber of Words:  3 726 19 \nNumber of Figures:  7 20 \nNumber of Tables:  1 21 \nNumber of References:  14 22 \n 23 \n 24 \n 25 \n 26 \n 27 \n 28 \n 29 \n 30 \n 31 \n 32 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 2 \nABSTRACT 33 \nOver the last two decades, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-34 \nTOF MS) has been introduced into the routine diagnostic practice of microbiological laboratories for the rapid 35 \ntaxonomic identification of bacteria and yeasts. However, a method that effectively identifies microbes directly 36 \nfrom clinical samples using MALDI-TOF MS has not yet been found.  One of the promising targets is microbial 37 \npolysaccharides, which are abundant structures in bacterial and fungal cells. Their rapid and inexpensive 38 \nanalysis, however, is complicated. This study focused on detecting microbial polysaccharides, such as 39 \nlipopolysaccharides, using MALDI-TOF MS and liquid chromatography-tandem mass spectrometry (LC-MS). We 40 \ndeveloped a method for fingerprinting polysaccharides by acid hydrolysis and enzymatic digestion. The mono- 41 \nand oligosaccharides are then derivatized with a newly developed probe (vanillyl pararosaniline, HD ligand), 42 \nenabling efficient ionization without the use of the MALDI matrix. The esterification of hydroxyl groups by 43 \nformic acid was also optimized for precise analysis of the saccharides. The method was validated using several 44 \nsaccharides as well as Escherichia coli lipopolysaccharides (O26:B6, O55:B5, and O111:B4). Derivatization using 45 \nHD ligand also allows the detection of structures containing amines and phosphate groups in positive ion mode. 46 \nWe also optimize the method using crude bacteria ( Escherichia coli, Salmonella enterica, Shigella dysenteriae, 47 \nShigella boydii , Shigella flexneri, and Legionella pneumophila ). This approach opens the possibility of directly 48 \ndetecting microbial polysaccharides from clinical specimens.  LDI-TOF MS (without a matrix) also allows specific 49 \ndetection of molecules of interest with suppression of the background signal.   50 \n 51 \n 52 \n 53 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 3 \n 54 \nScheme 1. Analysis of polysaccharides by mass spectrometry after digestion and derivatization using a self-55 \nionizable ligand (HD ligand). 56 \n 57 \n 58 \n 59 \n 60 \n 61 \n 62 \n 63 \n 64 \n 65 \n 66 \n 67 \n 68 \n 69 \n 70 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 4 \nINTRODUCTION 71 \nMany innovative applications of matrix-assisted laser desorption/ionization time-of -flight mass spectrometry 72 \n(MALDI-TOF MS) and liquid chromatography coupled with mass spectrometry (LC-MS) have emerged in clinical 73 \ndiagnostics to detect biomarkers and biomolecules, advancing personalized medicine [1, 2]. 74 \nIn diagnostic microbiology, MALDI-TOF MS became a cornerstone in the rapid taxonomic identification of 75 \nbacteria and fungi [2, 3]. Similarly, applications for antibiotic resistance determination have also been 76 \ndeveloped and validated. Among them, beta-lactamase activity determination detecting the molecular mass 77 \nchanges of indicator beta-lactam antibiotics (e.g., carbapenems) has been developed and validated for use in 78 \nroutine laboratory practice [4]. Another method allows the detection of polymyxin resistance using analysis of 79 \nlipid A of lipopolysaccharides and their structural changes (e.g., the addition of phosphoethanolamine or 4-80 \namino-L-arabinose) [5]. 81 \nAs MALDI-TOF MS provides efficient and rapid species identification, there is a key issue whether the method 82 \ncan be used for epidemiological typing directly from spectra obtained directly from a whole-cell lysate. So far, 83 \nno general typing algorithm has been proposed, but only specific peaks representing significant epidemiological 84 \nmarkers have been identified in some species [3]. Artificial intelligence for spectra analysis has recently bee n 85 \ndescribed as a promising tool for predicting antibiotic resistance and epidemiological typing [6].  86 \nDespite the use of artificial intelligence methods for analyzing large-scale data, MALDI-TOF MS should be 87 \nconsidered a biochemical tool allowing precise analysis of molecules based on their molecular weight and 88 \nfragmentation characteristics. Thus, we believe that the scientific community should not resign itself to the 89 \nexact identification of detected molecules/peaks. For such an analysis, it is usually insufficient to analyze crude 90 \nbacterial extract without further processing, i.e., specific extraction and enhancement of MALDI-TOF MS-based 91 \nionization.  92 \nDespite the commonly used taxonomic applications in routine laboratories, methods for identifying microbes 93 \ndirectly from clinical specimens have not yet been successfully developed [3]. Such applications, however, are 94 \nlimited by a poor sensitivity of MALDI-TOF MS and by masking the proteins of interest by other abundant 95 \nmolecules with a high ionization ability. These challenges can be addressed by (a) concentrating microbes or 96 \nmicrobial proteins in the sample and removing host proteins, (b) detecting other molecules (e.g., lipids or 97 \npolysaccharides) that can be explicitly extracted from the sample, and (c) using LC-MS. 98 \nIdentification of microbial polysaccharides represents a challenge in clinical microbiology as those structures can 99 \nbe used for (a) rapid and cheap epidemiological typing (e.g., Salmonella enterica, Shigella spp., Escherichia coli), 100 \n(b) development of polysaccharide vaccines and testing their efficiency, and (c) direct identification of microbes 101 \nfrom clinical specimens. The latter option is challenging as microbial saccharides represent relatively stable 102 \nmolecules that can be directly detected in several clinical specimens (e.g., blood, urine), allowing rapid 103 \nidentification of a causative agent of the infection and thus increasing therapeutic efficiency [7, 8, 9].  104 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 5 \nAs microbial polysaccharides usually contain >10 basic units, their molecular weight is highly above the efficient 105 \nability of MALDI-TOF MS or LC-MS to detect them directly. Therefore, the native molecules must be specifically 106 \ncleaved into smaller units, providing a specific molecule fingerprint. For that purpose, the glycosidic bond is the 107 \ncommon target. Hydrolysis of that bond, however, is challenging due to its stability in some biological 108 \nstructures. Several procedures of polysaccharide fingerprinting, including chemical cleavage by strong acids or 109 \nFenton’s reaction and enzymatic disruption, have been developed and optimized [10, 11, 12].  110 \nEven if monosaccharides or oligosaccharides are available, their ionization using conventional mass 111 \nspectrometry techniques is complex. Compared to peptides and proteins, those molecules are generally more 112 \ncomplicated to ionize and transfer to the vapor phase [12]. Therefore, the typical approach is to derivatize 113 \nmono and oligosaccharides before mass spectrometry. For that purpose, several methods have been proposed, 114 \nincluding the label-assisted laser desorption/ionization approach [13].   115 \nHere, we present a novel method for enzymatic and acidic hydrolysis of bacterial polysaccharides, their 116 \nderivatization, and analysis, where the sample is ionized through the addition of a specific ligand, enabling the 117 \nuse of LDI-TOF (Laser Desorption/Ionization Time-of -Flight) MS and LC-MS (Trapped Ion Mobility 118 \nSpectrometry—TIMS). This approach allows the identification of saccharide fingerprints based on their ion 119 \nmobility and m/z. Additionally, we proposed the esterification of mono- and oligosaccharides by formic acid, 120 \nwhich can aid in their identification.    121 \n 122 \n 123 \nMATERIALS AND METHODS 124 \nReagents  125 \nFor analysis, all chemicals ( D-glucose, D-glucose-1,2-13C2, glucose-6-phosphate, L-glucose, D-fructose, L-rhamnose, 126 \nL-fucose, D-xylose, lactose, maltose, sucrose, raffinose, 5-(hydroxymethyl)furfural), starch, agarose, 127 \nlipopolysaccharides (Escherichia coli O26:B6, E. coli O55:B5, E. coli O111:B4), enzymes (diastase, -amylase, -128 \namylase, lysozyme, chitinase), and other reagents were purchased from Merck (Merck Life Science, Prague, 129 \nCzech Republic). The greater quantity of vanillyl-rosaniline (HD ligand) was synthesized as 2,3-Dichloro- 5,6-130 \ndicyano-1,4-benzoquinone salt by Ratiochem (Brno, Czech Republic).  131 \n 132 \nInstrumentation 133 \nSamples for LC-MS were injected and subsequently desalted online using reverse-phase microtrap column (Neo 134 \nTrap Cartridge 5mm, ThermoFischer Scientific, Prague, Czech Republic) and separated on the reverse-phase 135 \nanalytical column (Bruker TEN, Bruker Daltonics, Bremen, Germany) at 40 °C using Bruker nanoElute 2 HPLC 136 \nsystem at flow rate 0.5 µL/min in water/acetonitrile gradient (mobile phase [A] water with 0.1% formic acid; 137 \nmobile phase [B] acetonitrile with 0.1% formic acid; the gradient started at 10% [B] and reached 50% [B] in 35 138 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 6 \nmin). The eluted analytes were directly analyzed by timsTOF Pro 2 mass spectrometer using a CaptiveSpray for 139 \nionization (Bruker Daltonics, Bremen, Germany). Measurement was performed in positive ion mode over the 140 \nm/z range 100 – 1350 in dia-PASEF® mode (0.75 – 1.60 V.s/cm 2, ramp time 100 ms). Data acquisition and data 141 \nprocessing were performed using DataAnalysis 6.1. 142 \nLDI-TOF MS was performed using a MALDI Biotyper® Sirius mass spectrometer and rapifleX® MALDI-TOF/TOF 143 \nsystem (Bruker Daltonics, Bremen, Germany). The spectra were analyzed using flexAnalysis 4.0 software.  144 \nFor precise identification of molecular mass, the samples were analyzed using a 15T solariX XR FT-ICR mass 145 \nspectrometer (Bruker Daltonics). Mass spectral data were collected in positive broadband mode over the m/z 146 \nrange 150 – 1500, with 1M data points transient and 0.2 s ion accumulation with two averaged scans per 147 \nspectrum. Data acquisition and data processing were performed using ftmsControl 2.1.0 and DataAnalysis 5.0. 148 \n 149 \nSynthesis of Vanillyl-Rosaniline Reagent (HD ligand)  150 \nPararosaniline hydrochloride (basic fuchsin) and vanillin were dissolved in methanol to a 100 mmol/L 151 \nconcentration (both reagents). After dissolving, glacial acetic acid was added to a final concentration of 2.5 152 \nmol/L and mixed well. Methylpyridine borane dissolved in methanol as a reducing agent was added to the 153 \nreaction mixture to a final concentration of 10 mmol/L and incubated at 50 °C with shaking for 2 hrs.  154 \nAfter incubation, the reaction was diluted by deionized water to a final concentration of methanol of 10 % and 155 \ndirectly applied on the NGC Quest Plus System (Bio-Rad, Prague, Czech Republic) equipped with a XSelect® CSH 156 \nC18 OBDTM preparative column (Waters, Gesellschaft m.b.H., Prague, Czech Republic) at flow rate 0.1 mL/min. 157 \nAfter application of the reaction mixture, the column was washed by 50 mL of 5% acetonitrile with 0.1% formic 158 \nacid. Then water/acetonitrile gradient (mobile phase [A] water with 0.1% formic acid; mobile phase [B] 159 \nacetonitrile with 0.1% formic acid; the gradient started at 5% [B] and reached 35% [B] in 60 min) was used for 160 \nsample purification. Two mL fractions were collected and measured using LC-MS after dilution with water 1:10. 161 \nFractions showing purified vanillyl-rosaniline (HD ligand) (Figure 1) were dried by a vacuum concentrator.    162 \n 163 \nSaccharide’s Derivatization and Analysis 164 \nDerivatization was performed in a 1% pyridine buffer, and the pH was adjusted to 4.0 using glacial acetic acid. 165 \nSaccharides were diluted to a concentration of 5 mmol/L with a reaction buffer. Concentrated HD (250 mmol/L) 166 \nsolution diluted in acetonitrile was added to a final 5 mmol/L concentration. The reaction mixture was 167 \nincubated at 50 °C for 20 minutes. 168 \nFor LC-MS, the samples were diluted 1:5 with a pyridine buffer. For LDI-TOF MS, the samples were purified by 169 \nC18-reversed phase silica gel to avoid the “sweet spot” formation on the MALDI target. Purification was 170 \nperformed in the Eppendorf tube. To 5 mg of C18 reverse phase resin, 10 μL of 30% acetonitrile with 0.1% 171 \nformic acid was added. After vortexing, 50 μL of the reaction mixture was added, vortexed for 10 s, and 172 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 7 \ncentrifuged at 14,000 g. The resin was washed with 1 mL of 5% acetonitrile with 0.1% formic acid. The sample 173 \nwas eluted with 25 μL of 50% acetonitrile with 0.1% formic acid. Two μL of the sam ple was directly applied to 174 \nthe MALDI target, allowed to dry, and then measured without adding any matrix. 175 \n 176 \nEnzymatic Digestion of Bacterial Lipopolysaccharides 177 \nTwo milligrams of bacterial lipopolysaccharide were dissolved in 100 μL  of 25 mM EDTA, pH 7.5, with 1 mg/mL 178 \ndiastase (-amylase and -amylase) and 1 mg/mL lysozyme. The reaction was incubated at 50 °C for 30 minutes. 179 \nAfter incubation, the reaction was filtered using an Microcon® - 10 Centrifugal Filters, 10 kDa NMWL (14,000 g , 180 \n30 minutes). To the fraction containing low molecular mass molecules (filtrate collected in the bottom tube), 50 181 \nμL of reaction buffer containing 5 mmol/L HD ligand was added, and saccharides were derivatized at 50°C for 20 182 \nminutes as described above.    183 \n 184 \nAcidic Digestion of Oligo- and Lipopolysaccharides 185 \nSaccharides and lipopolysaccharides were digested by formic acid and its isotopic variant ( 13C). One milligram of 186 \nthe sample was dissolved in 10 μL concentrated formic acid or formic acid -13C and incubated at 98 °C for 10 187 \nminutes in the thermocycler with a heated lid (104 °C). After incubation, 50 μL of 5% pyridine in water was 188 \nadded. The samples were filtered using Microcon® - 10 Centrifugal Filters, 10 kDa NMWL (14,000 g, 30 minutes), 189 \nand the bottom fraction (50 μL) containing digested saccharides was used for derivatization using HD ligand and 190 \nanalysis as described above.      191 \n 192 \nAnalysis of Lipopolysaccharides from Bacterial Cells 193 \nThe bacteria were obtained from the Czech National Collection of Type Cultures (National Institute of Health, 194 \nPrague, Czech Republic): E. coli ATCC25922, E. coli O55:B5 CNCTC5874, E. coli O111:B4 CNCTC5650, Salmonella 195 \nenterica subsp. enterica serovar Enteritidis CNCTC5187, S. enterica  subsp. enterica serovar Montevideo 196 \nCNCTC6279, S. enterica  subsp. arizonae CNCTC6478, Shigella dysenteriae  CNCTC5204, Shigella boydii 197 \nCNCTC6338, Shigella flexneri  serovar 1a I:2,4 CNCTC6370, Shigella flexneri  CNCTC6378 serovar 4a IV:3,4. 198 \nLegionella pneumophila serogroups 1 (sequence type (ST) 1, ST23, ST62), and 10 (ST378) were obtained from 199 \nour collection at the Department of Microbiology, Faculty of Medicine and University Hospital in Pilsen, Czech 200 \nRepublic. All members of the Enterobacterales order were cultivated on Mueller-Hinton agar at 35 °C overnight. 201 \nLegionella pneumophila was cultivated on BCYE agar in an atmosphere with 5% CO2 for 24 hrs. The full loop of 202 \nthe bacteria was resuspended in 1 mL of 96% ethanol, centrifuged, and the pellet was allowed to dry at 98°C for 203 \n5 minutes. Twenty microliters of concentrated formic acid were added to the pellet and incubated at 98°C with 204 \nshaking (1200 rpm) for 15 minutes. After incubation, 100 μL of 5% pyridine was added to adjust the  pH to 3.0. 205 \nThe mixtures were then filtered using an Microcon® - 10 Centrifugal Filters, 10 kDa NMWL (14,000 g, 30 206 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 8 \nminutes), and the bottom filtrate (50 μL) was used for derivatization using HD ligand as described above. For 207 \nmicrobes, further purification of derivatized saccharides is not required. Therefore, one microliter of the 208 \nreaction mixture was directly applied to the MALDI target.      209 \n 210 \n 211 \nRESULTS 212 \nSynthesis of HD Ligand  213 \nInitially, we tested the derivatization of saccharides using rosaniline. Despite the very efficient binding of 214 \naldoses to the rosaniline, the disadvantage of the molecule was the presence of two efficient binding sites (NH 2) 215 \nand the requirement for a matrix for MALDI-TOF MS analysis. Therefore, we had tested several molecules 216 \ncontaining an aldehyde residue to block one of those amines. Using vanillin, we obtained a molecule with an 217 \nexcellent ionization ability that does not require using any matrix in MALDI-TOF MS - an LDI-TOF (laser 218 \ndesorption/ionization time-of -flight) MS approach. It was, however, necessary to enhance the stability of the 219 \nmolecule by reductive amination of Schiff's base (Supplementary Figure 1). Since a conjugated system is 220 \nessential for efficient molecule ionization, we have tested an appropriate reducing agent (e.g., sodium 221 \ncyanoborohydride, borane pyridine complex, 2-methylpyridine borane complex). Finally, we have chosen a 2-222 \nmethylpyridine borane complex as the reaction can proceed in one step. It is also important to note that high 223 \nconcentrations of reducing agents, as recommended for conventional reactions, lead to the formation of leuco 224 \nbase in rosaniline. That molecule has very low ionization ability and requires a classical matrix-based MALDI-TOF 225 \nMS setup. 226 \nThe purity of the HD ligand synthesized in our laboratory was determined using LC-MS, and the structure was 227 \nverified by solariX magnetic resonance mass spectrometry (Bruker Daltonics, Bremen, Germany) 228 \n(Supplementary Figure 2). The quality of commercially synthesized HD ligand available as a 2,3-dichloro- 5,6-229 \ndicyano-1,4-benzoquinone salt (DDQ) was determined by nuclear magnetic resonance (Supplementary Figure 230 \n3). Both variants showed equal results in the following experiments.     231 \n  232 \nDerivatization of Mono- and Disaccharides           233 \nThe reaction was initially optimized using glucose and lactose. The best results were obtained in a low pH (3 - 4) 234 \nand a buffer not containing chlorine ions. Therefore, we selected a pyridine buffer of pH 3.0 for further 235 \nexperiments. Using a MALDI-TOF mass spectrometer, the HD ligand's ionization ability was excellent for 236 \ndetecting relevant signals of the conjugate with saccharides (Figure 1) without using any matrix (LDI-TOF MS).  237 \nIonization was performed in a positive ion mode with the analytes detected as [M+H] + ions. All the saccharides 238 \ntested provide a signal with a mass-to-charge ratio that can be calculated by the following formula (see Table 1): 239 \n 240 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 9 \nm/z = [MHD ligand + MSaccharide - MH2O + H]+, 241 \n 242 \nWhere the M HD ligand is 423.195 (monoisotopic mass), M H2O represents the loss of water (-18.015) during the 243 \nformation of a Schiff base. 244 \nAs demonstrated by glucose-6-phosphate, derivatization with HD ligand enables the detection of molecules with 245 \nstrongly negatively charged groups like phosphates in a positive ion mode as well (Figure 1). 246 \nA purification step using C18 resin can be performed using the resin itself (as described in the methodology) or 247 \nby ZipTips®, which enhances ionization ability in LDI-TOF MS analysis of mono- and oligosaccharides. This step 248 \nremoves unbound sugars that otherwise form a \"sweet spot\" and decreases the method's sensitivity.  249 \nIn LC-MS analysis, derivatized mono-, and di-saccharides have also been detected at the expected mass-to -250 \ncharge ratio (Table 1, Figure 2). The ion mobility of the saccharides allows their further analysis and 251 \nidentification (e.g., discrimination between a disaccharide, lactose, and maltose – see Figure 2). Unfortunately, 252 \nwe could not distinguish between D- and L-glucose by our instrument.   253 \nThe method's sensitivity was tested using diluted glucose and lactose (0.01 mmol/L —100 mmol/L). For both 254 \nmethods (LDI-TOF MS and LC-MS), the sensitivity was determined to be 0.1 mmol/L.  255 \n 256 \nAcidic Digestion and Fischer-Speier Esterification 257 \nUsing acidic digestion of oligosaccharides, we identified peaks that did not correspond with simple derivatized 258 \nmono- and oligosaccharides. Using monosaccharides ( D-glucose, L-fucose, D-xylose), formic acid, and isotopic 259 \nformic acid-13C, the peaks corresponding to esters formed in hydroxyl groups of saccharides can be detected. In 260 \nthis reaction (Fischer-Speier esterification), the peaks are shifted by 28 g/mol, corresponding to formic acid-261 \nderived esters (Figure 3).   262 \nIn glucose, the hydroxyl group at position 6 is almost completely esterified ( m/z 614) with a very low signal of 263 \nderivatized native glucose (m/z 586) (Figure 3). All except one hydroxyl group are also esterified with a different 264 \nratio, showing signals at m/z 642, 670, 698. Similar results were obtained in D-xylose and L-fucose, showing no 265 \nsignal with a non-esterified derivatized saccharide ( m/z 556 and 570). As observed in glucose, variants of all 266 \nesterified hydroxyl groups except one could be detected in LDI-TOF spectra (Figure 3). Based on those 267 \ncharacteristics, we hypothesize that only one hydroxyl group of vicinal diols can be esterified efficiently. The 268 \nesterification mechanism on D-glucose, D-xylose, L-fucose, and lactose was confirmed by precise molecular 269 \nmass determination (<1 ppm) using solariX XR FT-ICR mass spectrometer (Supplementary Figure 4). 270 \nAs hexoses in acidic conditions and high temperatures can be dehydrated to form 5-(hydroxymethyl)furfural, we 271 \nalso focused on identifying this molecule in the reaction. The 5-(hydroxymethyl)furfural can also be recognized 272 \nas a signal at m/z 532 in acidic conditions. In glucose and fructose, the 5-(hydroxymethyl)furfural intermediate 273 \nformed during saccharide dehydration [14] was identified in the spectra at m/z 550 (Figure 4, Figure 5).    274 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 10 \n           275 \nAnalysis of Bacterial Polysaccharides by Acidic Digestion 276 \nInitially, formic acid was tested for non-specific fingerprinting of polysaccharides. As demonstrated in Figure 4 , 277 \nconcentrated hot formic acid (98 °C) can efficiently hydrolyze glycosidic bonds. The same results were obtained 278 \nin raffinose and starch (data not shown). The method also allows the detection of common saccharides of the 279 \nbacterial cell wall, a muramic acid, containing an amine and carboxyl group (m/z 657) (Figure 6).      280 \nBased on those mono- and oligosaccharide results, the method was tested for fingerprinting bacterial 281 \nlipopolysaccharides. Comparing three different bacterial polysaccharides of Escherichia coli, the results 282 \ndemonstrated different patterns. The same results were obtained for Escherichia coli O26:B6, E. coli O55:B5, E. 283 \ncoli O111:B4 purified lipopolysaccharides, and crude bacteria (Figure 7). Similarly, all tested bacteria, including 284 \nSalmonella spp., Shigella spp., and Legionella pneumophila, provided specific patterns showing that the method 285 \ncan be used for bacterial typing.    286 \n 287 \nAnalysis of Polysaccharides by Enzymatic Digestion 288 \nTo analyze polysaccharides and lipopolysaccharides, -amylase and -amylase were tested using starch as a 289 \npositive control. In starch, mono —(m/z 586), di—(m/z 748), and trisaccharide ( m/z 910) can be observed using 290 \nboth methods (LDI-TOF MS and LC-MS). We also detected distinct lipopolysaccharide profiles ( E. coli O26:B6, E. 291 \ncoli O55:B5, E. coli O111:B4) similar to acidic digestion. 292 \n 293 \n 294 \nDISCUSSION         295 \nWe describe here a novel method for derivatizing mono- and oligosaccharides that can be used for the 296 \nidentification and analysis of those molecules, not only restricted to microbial origin. Initially, we focused on 297 \ndeveloping a technique to identify bacterial cell-wall polysaccharides (i.e., lipopolysaccharides). We tested 298 \nseveral derivatizing agents, as saccharides cannot be easily ionized compared to peptides/proteins by MALDI-299 \nTOF mass spectrometry. Inspired by a detection of lactose fermentation using a basic fuchsin in diagnostic 300 \nbacteriology (e.g., Endo agar), we tested that molecule to form a Shiff base between the aldehyde group of 301 \nreducing sugars and the amine of the fuchsin. This complex could be derivatized by MALDI-TOF MS using a 302 \nstandard matrix (e.g., 2,5-dihydroxybenzoic acid). The disadvantage of this process is the presence of two 303 \nefficient amine residues in the molecule, responsible for the polyvalent binding of tested saccharides. However, 304 \nthe ability to modify saccharides with fuchsin and subsequent ionization showed excellent results. Therefore, 305 \nwe decided to focus our research further on modifying this molecule. 306 \nAfter modifying the fuchsin by adding vanillin to one of the amines, a designated HD ligand, allowed the 307 \nionization of the complex with saccharides without using the matrix (LDI-TOF MS). The complex can also be 308 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 11 \nanalyzed using LC-MS with a separation on the C18 reverse phase column. As demonstrated in the results 309 \n(Figure 1), some isomeric saccharides can also be distinguished by their ion mobility using trapped ion mobility 310 \ntechnology. 311 \nThe HD ligand allows the derivatization and analysis of saccharides with different substituents, including 312 \nphosphates, in a positive ion mode. This makes the method universal to detect microbial ol igo- and 313 \npolysaccharides of different origins. However, it is necessary to use optimal reaction conditions for 314 \nderivatization, including a pH between 3 and 5. We also found that a high concentration of chlorine ions inhibits 315 \nthe formation of a Schiff base (data not shown). Among the buffers tested, pyridine in a concentration between 316 \n1 and 10% with a pH adjusted by formic or acetic acid provided the highest binding efficiency.       317 \nAlthough we expected that the fuchsin-based system would also allow the detection of ketones (ketoses), we 318 \ncould not find conditions that would enable efficient, stable binding of the HD ligand. This may be because the 319 \nHD ligand requires specific binding conditions or is unstable upon ionization during mass spectrometry. On the 320 \nother hand, however, ketoses are not common structures in bacterial cells. For analysis, they can be modified to 321 \nfurfurals in acidic conditions that are analyzable by our system as well (see Figure 5).  322 \nWhen different options for the hydrolysis of the glycosidic bond of polysaccharides were tested, we found that 323 \nusing formic acid, spectra containing many ions of different m/z with regular repetitions (+28) were detected. By 324 \ndetailed analysis, including the reaction in 13C formic acid, we identified the signals as Fischer-Speier esters 325 \nformed in the hydroxyl groups of saccharide molecules. This behavior can be further used to identify and 326 \nanalyze saccharides more accurately. For future experiments, the relative intensity of signals representing 327 \nesterified hydroxyl groups can be further verified to determine their position in the molecule. We also tested 328 \nacetic acid for esterification. However, its efficiency was very low compared with formic acid. In glucose , an 329 \nacetic acid-derived ester was formed at position six only (data not shown). Those findings were crucial for 330 \nfurther microbial polysaccharide experiments to understand the reaction. 331 \nAn essential step in the analysis of microbial polysaccharides is to digest the molecule specifically, which usually 332 \npossesses a very high molecular weight. Similarly to derivatization methods, we tested many possibilities, 333 \nincluding specific lipopolysaccharide isolation, e.g., Bligh-Dyer solution and its modifications (data not shown). 334 \nInterestingly, common amylases (  and ) can efficiently digest bacterial polysaccharides to mono-, di-, and 335 \ntrisaccharides. Peptidoglycan-specific enzymes (e.g., lysozyme) can also be used for specific analysis. A similar 336 \napproach can be optimized to analyze other microbial polysaccharides (e.g., galactomannan and glucan in molds 337 \nand yeasts) that are clinically relevant for rapidly diagnosing invasive fungal infection or detecting resistance to 338 \nantifungal drugs.  339 \nFinally, we selected a straightforward method that does not require any specific extraction of cell-wall 340 \npolysaccharides: incubating microbes in concentrated formic acid at 98 °C. This procedure blocks reactive 341 \namines in the crude microbes, and the polysaccharides can be simultaneously digested into mono and 342 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 12 \noligosaccharides in a one-step process without previous specific extraction of the cell walls, which is usually 343 \nlaborious. That approach is a typical example of applying Ockham’s razor and can be easily used in routine 344 \ndiagnostic laboratories.  345 \nIn all applications, however, it is crucial to filter the reaction mixture before the derivatization of saccharides to 346 \nremove undigested polysaccharides (> 3 – 10 kDa). Without this step, the spectra show an insufficient noise-to -347 \nsignal ratio. 348 \n 349 \n 350 \nCONCLUSIONS         351 \nThe method described here can be used to analyze microbial polysaccharides not only for epidemiological 352 \ntyping and polysaccharide vaccine development but also to open the possibility of detecting those structures 353 \ndirectly in clinical specimens. Derivatization of saccharides and related molecules (e.g., furfurals) containing an 354 \naldehyde group using the HD ligand is challenging for further analysis of those important biological structures by 355 \nmass spectrometry (LDI-TOF MS, LC-MS). 356 \n 357 \n 358 \nREFERENCES 359 \n1. Son, A.; Kim, W.; Park, J.; Park, Y.; Lee, W.; Lee, S.; Kim, H. Mass Spectrometry Advancements and 360 \nApplications for Biomarker Discovery, Diagnostic Innovations, and Personalized Medicine. Int. J. Mol. Sci. 361 \n2024, 25, 9880. DOI: 10.3390/ijms25189880 362 \n2. Clark, A.E.; Kaleta E.J.; Arora A.; Wolk D.M. Matrix-assisted laser desorption ionization-time of flight 363 \nmass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin. Microbiol. 364 \nRev. 2013, 26, 547-603. DOI: 10.1128/CMR.00072-12 365 \n3. Kostrzewa, M. Application of the MALDI Biotyper to clinical microbiology: progress and potential. 366 \nExpert. Rev. Proteomics. 2018, 15, 193-202. 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It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 13 \n6. Weis, C.; Cuénod, A.; Rieck, B.; Dubuis, O.; Graf, S.; Lang, C.; Oberle, M.; Brackmann, M.; Søgaard, K.K.; 375 \nOsthoff, M.; Borgwardt, K.; Egli, A. Direct antimicrobial resistance prediction from clinical MALDI-TOF 376 \nmass spectra using machine learning. Nat. Med. 2022, 28, 164-174. DOI: 10.1038/s41591-021-01619-9 377 \n7. Farrington, M.; Rubenstein, D. Antigen detection in pneumococcal pneumonia. J. Infect. 1991, 23, 109-378 \n116. DOI: 10.1016/0163-4453(91)91900-i 379 \n8. Viasus, D.; Gaia, V.; Manzur-Barbur, C.; Carratalà, J. Legionnaires' Disease: Update on Diagnosis and 380 \nTreatment. Infect. Dis. Ther. 2022, 11, 973-986. DOI: 10.1007/s40121-022-00635-7 381 \n9. White, P.L. Progress on nonculture based diagnostic tests for invasive mould infection. Curr. Opin. 382 \nInfect. Dis. 2024, 37, 451-463. DOI: 10.1097/QCO.0000000000001060 383 \n10. Amicucci, M.J.; Nandita, E.; Galermo, A.G.; Castillo, J.J.; Chen, S.; Park, D.; Smilowitz, J.T.; German, J.B.; 384 \nMills, D.A.; Lebrilla, C.B. A nonenzymatic method for cleaving polysaccharides to yield oligosaccharides 385 \nfor structural analysis. Nat. Commun. 2020, 11, 3963. DOI: 10.1038/s41467-020-17778-1 386 \n11. Urakami, S.; Hinou, H. MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria. 387 \nSci Rep. 2024, 14, 12719. DOI: 10.1038/s41598-024-62729-1. 388 \n12. Han, L.; Costello, C.E. Mass spectrometry of glycans. Biochemistry (Mosc).  2013, 78, 710-720. DOI: 389 \n10.1134/S0006297913070031 390 \n13. Lageveen-Kammeijer, G.S.M.; Kuster, B.; Reusch, D.; Wuhrer, M. High sensitivity glycomics in 391 \nbiomedicine. Mass. Spectrom. Rev. 2022, 41, 1014-1039. DOI: 10.1002/mas.21730 392 \n14. Kognou ALM, Shrestha S, Jiang Z, Xu C, Sun F, Qin W. High-fructose corn syrup production and its new 393 \napplications for 5-hydroxymethylfurfural and value-added furan derivatives: promises and challenges. J. 394 \nBioresour. Bioprod.  2022, 7, 148 – 160. DOI: 10.1016/j.jobab.2022.03.004. 395 \n 396 \n 397 \nTABLE 398 \nTable 1. LDI-TOF MS and LC-MS analysis of mono-, disaccharides, and 5-(hydroxymethyl)furfural showing m/z 399 \nand ion mobility of derivatized molecules. 400 \nSaccharide m/z Ion \nMobility \n[V.s/cm2] \nD-glucose 586 1.242 \nL-glucose 586 1.242 \nD-glucose-1,2-13C2 588 1.242 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 14 \nglucose-6-phosphate 666 1.219 \nL-rhamnose 570 1.224 \nL-fucose 570 1.228 \nD-xylose 556 1.210 \nlactose 748 1.503 \nmaltose 748 1.490 \n5-(hydroxymethyl)furfural 532 1.099 \n 401 \n 402 \nFIGURES 403 \nFigure 1 . Vanillyl-rosaniline (HD ligand) mechanism of saccharide’s derivatization, and LDI -TOF MS analysis of 404 \nselected saccharides. Mass spectra of glucose, D-glucose-1,2-13C2, and glucose-6-phosphate derivatized by HD 405 \nligand measured on a rapifleX mass spectrometer in linear positive ion mode. Derivatized glucose is visible at 406 \nm/z 586; D-glucose-1,2-13C is visible as a signal at m/z 588; and glucose-6-phosphate as a signal at m/z 666. 407 \n 408 \nFigure 2 . LC-MS spectra showing differentiation of disaccharide s’ isoforms by ion mobility determination. 409 \nDisaccharides maltose and lactose appear as signals at m/z 612 (HD ligand with the lost vanillin molecule) and 410 \nm/z 748 (intact HD ligand). Maltose complex (m/z 748) shows an ion mobility of 1.490 V.s/cm 2 and lactose 1.503 411 \nV.s/cm2. 412 \n 413 \nFigure 3. Fischer-Speier esterification of glucose using concentrated formic acid at 98 °C and its derivatization by 414 \nHD ligand. Esterification of the hydroxyl group at position 6 (green color) was immediately detected. 415 \nEsterification of other positions was identified in different ratios. LDI-TOF MS spectra of monosaccharides 416 \n(negative control, L-fucose, D-glucose, D-xylose) esterified by concentrated formic acid (+m/z 28). 417 \n 418 \nFigure 4 . LDI-TOF MS spectra of glucose (A), 5-(hydroxymethyl)furfural (B), lactose (C), and sucrose (D) after 419 \ndigestion (lactose, sucrose) and esterification using concentrated formic acid at 98 °C for 10 minutes and 420 \nderivatization using HD ligand. 421 \n 422 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 15 \nFigure 5. LDI-TOF MS spectra show the formation of 5-(hydroxymethyl)furfural intermediate from fructose and 423 \nglucose after heating at 98 °C for 30 minutes ( m/z 550). The molecule was derivatized by HD ligand and 424 \nmeasured using LDI-TOF. 425 \n 426 \nFigure 6. LDI-TOF MS spectra of glucose (A), muramic acid (B), Escherichia coli O26:B6 lipopolysaccharide (C), E. 427 \ncoli O55:B5 lipopolysaccharide (D), and E. coli O111:B4 lipopolysaccharide (E) after digestion and esterification 428 \nusing concentrated formic acid at 98 °C for 10 minutes and derivatization using HD ligand. 429 \n 430 \nFigure 7. LDI-TOF MS spectra of Escherichia coli ATCC25922 (A), E. coli O111:B4 CNCTC5650 (B), E. coli O55:B5 431 \nCNCTC5874 (C), Salmonella enterica  subsp. enterica serovar Enteritidis CNCTC5187 (D), Shigella dysenteriae  432 \nCNCTC5204 (E), and negative control (F) after digestion and esterification using concentrated formic acid at 98 433 \n°C for 10 minutes and derivatization using HD ligand. 434 \n 435 \n 436 \nSUPPLEMENTARY MATERIAL 437 \n Supplementary Figure 1  - Principle of low-scale preparation of Vanillyl-Rosaniline (HD) ligand using 2-438 \nmethylpyridine borane complex as a reduction agent and purification by HPLC system.  439 \n Supplementary Figure 2  - Confirmation of Vanillyl-Rosaniline (HD) ligand structure using 15T solariX XR 440 \nFT-ICR mass spectrometer (Bruker Daltonics).  441 \n Supplementary Figure 3  - Quality control protocol (NMR spectra) of commercially prepared Vanillyl-442 \nRosaniline (HD) ligand.  443 \n Supplementary Figure 4  - Confirmation of esterified D-glucose, D-xylose, L-fucose, and lactose using 15T 444 \nsolariX XR FT-ICR mass spectrometer (Bruker Daltonics). 445 \n 446 \nAUTHOR CONTRIBUTIONS 447 \nL.D. was responsible for laboratory work, interpretation of the results, designing novel derivatization agents, 448 \nand writing the manuscript. V.P. was responsible for laboratory work, the result interpretation, and the 449 \nmanuscript's writing. P.N. was responsible for confirming HD ligand structure and analysis of esterified products. 450 \nJ.H. was responsible for conceptualization, methodology, designing new derivatization agents, data 451 \ninterpretation, and writing the manuscript. 452 \n 453 \nACKNOWLEDGMENT  454 \nWe thank Dana Kralova and Andrea Pospechova for their excellent technical assistance. The study was 455 \nsupported by the Czech Health Research Council grant Nr. NW24-09 -00464, the Charles University Grant Agency 456 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprint (whichthis version posted May 14, 2025. ; https://doi.org/10.1101/2025.05.13.653920doi: bioRxiv preprint \n\n 16 \n(GA UK) project Nr. 550225, and the National Institute of Virology and Bacteriology (Programme EXCELES, ID 457 \nProject No. LX22NPO5103) —funded by the European Union —Next Generation EU. The method has been 458 \npatented, includin g the HD reagent molecule and its variants (Czech National Patent PV 2024-48, 459 \nPCT/CZ2025/050014). 460 \n.CC-BY 4.0 International licenseavailable under a \nwas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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