Method for detection and identification of saccharides by mass spectrometry

Abstract

The present disclosure provides a method for the detection and / or identification of reducing monosaccharides or oligosaccharides by mass spectrometry in a sample, wherein the reducing saccharide(s) in the sample is (are) reacted with a ligand of general formula I (I), wherein L is selected from the group comprising, wherein n is from 1 to 3,,,, wherein m is 1 or 2, and, wherein in the general formula I the methoxyphenol group is bound to the carbon atom of the linker L, where R1, R2 and R4 are independently selected from the group comprising hydrogen atom and, wherein R3 is selected from the group comprising hydrogen atom and methyl, to form a conjugate by reaction of the aldehyde group(s) of the reducing monosaccharide(s) and oligosaccharide(s) present in the sample with the NH2 group of the ligand, and the conjugate is subsequently subjected to ionization and mass spectrometry measurement. This method can be used also for detection or identification of microbial polysaccharides by mass spectrometry, wherein prior to the reaction with the ligand, the polysaccharides are cleaved enzymatically or by acid hydrolysis to reducing monosaccharides and / or oligosaccharides.

Description

[0001]Method for detection and identification of saccharides by mass spectrometry Field of Art The present invention relates to a method for detecting and identifying reducing mono- and oligosaccharides by mass spectrometry, and to a method for detecting and identifying bacterial and micromycete polysaccharides by mass spectrometry using the former method. The method of the invention is particularly suitable for detecting cell wall polysaccharides useful for subtyping microbes and identifying microbes from a clinical sample. Background Art Introducing MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight) mass spectrometry (MS) into laboratory diagnostics has significantly changed the taxonomic identification of microbes. This technology has reduced the time required to achieve taxonomic identification of bacteria and micromycetes, as well as rapid identification of microbes from haemocultures and other clinical samples such as urine. Similarly, applications for antibiotic resistance determination have been developed and validated for use in clinical diagnostics. Of these, the determination of beta-lactamase activity by detecting changes in the molecular weight of the indicator, beta-lactam antibiotic, or the detection of polymyxin resistance by lipid A lipopolysaccharide analysis are routinely used. However, in the context of healthcare-associated infections, conventional species identification of microbes, mainly bacteria, does not provide sufficient data for appropriate initial antibiotic therapy, neither epidemiological studies monitoring the spread and identification of sources of infectious agents, nor for vaccine efficacy testing. Although MALDI-TOF mass spectrometry provides efficient and rapid identification to the species level, the use of this method for epidemiological typing directly from the spectra obtained is still difficult, and no reliable method or algorithm has yet been found to allow deeper comparisons of identified species (subtyping). Despite the use of artificial intelligence methods to analyse protein spectra obtained by MALDI-TOF mass spectrometry, the signal lacks information on substances that are difficult to ionise or present in the sample only at very low concentrations. This is because, for the routine use of MALDI-TOF mass spectrometry, the most commonly used whole-cell extract consists mainly of proteins and peptides in the molecular weight range of 2000 to 20000 g / mol. Other methods developed for MALDI-TOF mass spectrometry use cell wall lipid analysis, not only for detection of resistance to polymyxin antibiotics but also for direct identification and typing of some bacteria with lipid-rich cell walls, e.g., Mycobacterium spp. Similarly, periplasmic compartment proteins, e.g., beta-lactamases, can be analysed, which need to be specifically isolated and stabilised with subsequent detection by MALDI-TOF mass spectrometry. The above-mentioned microbial cell wall surface structures (e.g. lipids, proteins) play an essential role in antibiotic resistance, bacterial typing for epidemiological purposes and for the development of new vaccines, as most of them are common targets of the immune response to infection. The most important of these surface structures are lipopolysaccharides (especially in bacteria of the order Enterobacterales, including Escherichia coli, Salmonella spp., Shigella spp., or bacteria of the genus Pseudomonas spp.), polysaccharides (e.g., in Haemophilus influenzae, Neisseria meningitidis, Staphylococcus spp., Streptococcus spp.), or membrane proteins (e.g., in Escherichia coli, Neisseria meningitidis, or Streptococcus pyogenes). Cell wall polysaccharides are important in micromycetes, such as galactomannan in Aspergillus spp. or glucan in yeasts (e.g. Candida spp.). These oligosaccharides and polysaccharides can also be used to detect microbes from a clinical sample. Mass spectrometry analysis of carbohydrates / oligosaccharides / polysaccharides is complicated due to their poor ionization capacity as well as due to their size. In contrast to the analysis of peptides, proteins, and lipids, where a variety of techniques are available, in detecting polysaccharides, these structures need to be specifically cleaved and derivatized. In general, polysaccharides have a vast variation in the composition of individual monosaccharides and a large diversity in binding and branching. Due to their relatively complex structure, there is still no procedure for mass spectrometry analysis that allows specific cleavage of polysaccharides (e.g., universal enzyme, chemical hydrolysis). Also, to achieve the ionization required for MS detection, the cleaved glycan units must be derivatized . This approach can utilize the labeling of the aldehyde group via reductive amination in which the primary amine reacts to form an imine or Schiff base. In the second step, the compound is reduced to a secondary amine. Molecules such as 2-aminobenzamide, 2-aminobenzoic acid, 2-aminopyridine, 2-aminonaphthalene trisulfonic acid, and 1-aminopyrene-3,6,8-trisulfonic acid are widely used in this field (https: / / pubmed.ncbi.nlm.nih.gov / 29553244 / ). Other alternatives for reductive amination are hydrazide reagents such as (carboxymethyl)trimethylammonium chloride hydrazide (Girard's reagent T) (https: / / pubmed.ncbi.nlm.nih.gov / 30598139 / ) or 1-phenyl-3-methyl-5- pyrazolone (https: / / pubmed.ncbi.nlm.nih.gov / 2817366 / ). Extraction of polysaccharides from bacterial cells is usually performed by laboratory-intensive techniques such as hot phenol extraction Similarly, methods have been published that require exposure of a bacterial sample to various enzymes followed by butanol extraction and lyophilization to extract the lipopolysaccharide (https: / / pubmed.ncbi.nlm.nih.gov / 19490391 / ). These methods are time- and laboratory-intensive and not applicable to the diagnostic practice of a routine laboratory. The present invention aims to provide a less laboratory-intensive method for the detection and identification of microbial polysaccharides. This method is also applicable for the direct detection of microbes from a clinical sample, which is extremely important at present. Disclosure of the invention The present invention provides a method for the detection and / or identification of reducing monosaccharides or oligosaccharides by mass spectrometry in a sample, wherein the reducing saccharide(s) in the sample is (are) reacted with a ligand of general formula I (I), wherein L is selected from the group comprising , wherein n is from 1 to 3, wherein in the general formula I the methoxyphenol group is bound to the carbon atom of the linker L, where R1, R2and R4are independently selected from the group comprising hydrogen atom and , wherein R3is selected from the group comprising hydrogen atom and methyl, to form a conjugate by reaction of the aldehyde group(s) of the reducing monosaccharide(s) and oligosaccharide(s) present in the sample with the NH2group of the ligand, and the conjugate is subsequently subjected to ionization and mass spectrometry measurement. The present invention further provides a method for the detection and / or identification of microbial polysaccharides by mass spectrometry, wherein the polysaccharides are cleaved enzymatically or by acid hydrolysis into reducing monosaccharides or oligosaccharides, and a conjugate is prepared by reacting the aldehyde group of the reducing monosaccharides or oligosaccharides and the NH2group of a ligand of general formula I (I), wherein L is selected from the group comprising , wherein n is from 1 to 3, wherein in the general formula I the methoxyphenol group is bound to the carbon atom of the linker L, where R1, R2and R4are independently selected from the group comprising hydrogen atom and wherein R3is selected from the group comprising hydrogen atom and methyl, and the conjugate is subsequently subjected to ionization and mass spectrometry measurement. In one embodiment, L is selected from the group consisting In one embodiment, the ligands of general formula I have the structure of general formula Ia: (Ia), wherein the 2-methoxyphenol-4-yl is bound to the carbon atom of the linker L, wherein the linker L is as defined above. In some embodiments, L in general formula I is a group . In some embodiments, the ligand of general formula Ia has a structure of formula II: In some embodiments, L in the general formula I is a group . In some embodiments, the ligand of general formula Ia has a structure of formula III: In some embodiments, L in the general formula I is a group . In some embodiments, the ligand of general formula Ia has a structure of formula IV: In some embodiments, prior to the cleavage step, the microbial polysaccharides are isolated from the sample comprising microbial cells and / or microbial polysaccharides by extraction. The sample comprising microbial cells may be, for example, a bacterial culture, or a culture of a micromycetes (filamentous fungus or yeast), or a clinical sample collected from a patient. In other embodiments, the microbial polysaccharides may be cleaved directly from the bacterial or micromycete cells or directly in the sample collected from a patient, without any prior isolation step. Extraction of the polysaccharides is preferably performed by cultivating the bacteria in / on a medium designed for bacteria or micromycetes, e.g., selected from Mueller-Hinton agar (MH broth), blood agar, chocolate agar, agar prepared from brain-heart infusion (BHI) medium, and Sabourad agar with dextrose, transferring the bacteria by a bacteriological loop into lysis buffer and resuspending. The lysis buffer comprises a detergent to allow the release of the polysaccharide structures from the cell wall; in a preferred embodiment, the lysis buffer is a sodium acetate buffer containing sodium chloride and a detergent such as lauroyl sarcosinate and / or sodium dodecyl sulfate. The mixture is incubated at 20 to 50 °C, preferably at about 50 °C, preferably with shaking to release the polysaccharide structures effectively. The incubation is typically for 10 to 60 minutes, preferably for about 30 minutes. The mixture is centrifuged, and the cell-free supernatant is used in the next step of cleaving the polysaccharides. The step of cleaving the polysaccharides can preferably be carried out by acid hydrolysis by adding an acid, preferably tartaric acid, hydrochloric acid and / or glyoxalic acid, at a concentration of at least 1M to the supernatant obtained according to the previous polysaccharide isolation procedure and incubating the mixture for at least 20 minutes at a temperature of at least 60 °C, preferably 60 to 100 °C. The mixture is preferably shaken during the incubation to increase the yield of cleavage products. The mixture of cleaved monosaccharides and oligosaccharides is used in further steps of the method. Another possible embodiment is to cleave the polysaccharides enzymatically. Enzymatic cleavage is performed using an enzyme selected from the group alfa-amylase, beta-amylase, lysozyme, and endopeptidase. The enzyme in the buffer is added to the supernatant obtained according to the previous polysaccharide isolation procedure. The buffer is selected so as to be suitable for the activity and pH of the enzyme, for example, the buffer may be selected from HEPES (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid buffer) or MES (2-(N-morpholino)ethanesulfonic acid buffer). Cleavage is carried out at the temperature optimum of the activity of the selected enzyme for at least 20 minutes, preferably for 20 to 60 minutes. The mixture is preferably shaken during the incubation to increase the yield of cleavage products. The mixture of cleaved monosaccharides and oligosaccharides is used in further steps of the method. In a further embodiment, the cleavage of the polysaccharides can be carried out directly from the microbial culture, without the prior step of isolating the polysaccharides, by resuspending the mixture of microbes in a solution of acid, preferably tartaric acid, glyoxalic acid and / or hydrochloric acid, at a concentration of at least 1M and incubating the suspension for at least 10 minutes, preferably at least 30 minutes at a temperature of at least 60 °C, more preferably 60 to 100 °C. After the incubation, centrifugation is performed, and the supernatant containing the cleaved carbohydrates and oligosaccharides is used in further steps of the method. Another embodiment of the step of polysaccharide cleavage is to cleave the polysaccharides using an enzyme selected from the group comprising alfa-amylase, beta-amylase, lysozyme and / or endopeptidase, directly in the microbial culture, without any prior step of isolation of the polysaccharides. Lysozyme or endopeptidase is used when a microbial cell wall component, such as peptidoglycan, is to be detected in the sample by MALDI-TOF MS. The microbial culture is resuspended in a buffer. The buffer is selected so as to be suitable for the activity and pH of the enzyme; for example, HEPES or MES buffers can be used. The enzyme (alfa-amylase, beta-amylase, lysozyme and / or endopeptidase) is then added to the mixture. Cleavage is carried out at the temperature optimum of the activity of the selected enzyme for at least 10 minutes, preferably for 20 to 60 minutes. The mixture is preferably shaken during incubation to increase the yield of cleavage products. The mixture of cleaved monosaccharides and oligosaccharides is used in further steps of the method. Yet another embodiment of the step of the polysaccharide cleavage is the acid cleavage of polysaccharides directly from the sample taken from the patient without any prior step of isolation of the polysaccharides. Such a sample is typically a clinical sample, such as urine, cerebrospinal fluid and / or effluent. An acid, preferably tartaric acid and / or hydrochloric acid at a concentration of at least 1M is added to the sample. The mixture is incubated for at least 10 minutes, preferably for 20 to 60 minutes, at a temperature of at least 60 °C, preferably 60 to 100 °C. If the sample is expected to contain microbes in whole cell form, the sample may be centrifuged and the acid added to the pellet to increase sensitivity. The mixture of cleaved monosaccharides and oligosaccharides is used in further steps of the method. Another embodiment of the polysaccharide cleavage step is the enzymatic cleavage of polysaccharides directly from the sample taken from the patient without any prior step of isolation of the polysaccharides. The enzyme for cleavage is selected from the group of alfa-amylase, beta-amylase, lysozyme, and endopeptidase and added to the sample in a buffer. Lysozyme or endopeptidase is used when a microbial cell wall component, such as peptidoglycan, is to be detected in the sample. The buffer is selected so as to be suitable for the activity and pH of the enzyme, for example, the buffer is selected from HEPES and / or MES buffers. Cleavage of the polysaccharides is carried out at the temperature optimum of the activity of the selected enzyme for at least 10 minutes, preferably for 20 to 60 minutes. The mixture is preferably shaken during incubation to increase the yield of cleavage products. The mixture of cleaved monosaccharides and oligosaccharides is then used in further steps of the method. The ligand binding to the detected carbohydrates and allowing subsequent ionization is prepared by dissolving fuchsin molecule in a suitable solvent such as ethanol in an acidic medium, preferably a medium of hydrochloric acid, tartaric acid, trifluoroacetic acid, formic acid and / or sulfuric acid at a concentration of at least 1 mM, preferably 1 to 10 mM. A ligand ionization agent, preferably vanillin, 3- (4-hydroxyphenyl)acrylaldehyde, and / or 4-hydroxy-3-methoxycinnamic acid aldehyde is added to the mixture at the same molar concentration. The mixture is incubated for 10 to 30 minutes and then evaporated, e.g., by lyophilization, so that the temperature does not exceed 70 °C. The obtained mixture of products is purified, e.g., using a C18 reverse phase carrier, so that a ligand is obtained with only one bound molecule allowing ionization of the ligand, i.e., a vanillin molecule, a 3-(4- hydroxyphenyl)acrylaldehyde molecule, or a 4-hydroxy-3-methoxycinnamic acid aldehyde molecule. In a further embodiment, the ligand binding to the detected carbohydrates and allowing subsequent ionization is prepared using a coupling agent, for example, 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide and / or N,N′-dicyclohexylcarbodiimide, such that, by mixing a molecule of fuchsin dissolved in a solvent such as ethanol or phosphate buffer (pH 6 to 8) with a molar equivalent concentration of vanillic acid, ferulic acid, p-coumaric acid and / or 4-hydroxy-3- methoxycinnamic acid. The mixture is incubated at 4 to 40 °C for at least 20 minutes, preferably for 60 minutes at about 35 °C. The mixture is then evaporated, e.g., by lyophilization, so that the temperature does not exceed 70 °C. The obtained mixture of products is purified, e.g., using a C18 carrier, to obtain a ligand with only one bound molecule allowing ionization of the ligand, i.e., a vanillic acid molecule, a ferulic acid molecule, a p-coumaric acid molecule, or a 4-hydroxy-3-methoxycinnamic acid molecule. The ligand of general formula I is added to the mixture of cleaved monosaccharides and oligosaccharides in a buffer at a pH in the range of 2 to 6, preferably at pH 3. A particularly preferred buffer is acetate buffer with the addition of 25 mM sulfate, wherein the sulfate may be, for example, lithium sulfate, copper sulfate and / or sodium sulfate. The mixture is incubated for at least 10 minutes, preferably for 30 minutes, at a temperature of 20 to 70 °C, more preferably at about 50 °C. At temperatures above 70 °C the ligand of general formula I decomposes. The reducing monosaccharides and oligosaccharides bind their aldehyde group to the primary amine of the general formula I ligand to form a conjugate. An example of a conjugate of a ligand of formula II with a glucose molecule is shown as formula V: Similarly, other monosaccharides and oligosaccharides form conjugates. The ligand of general formula I ionizes very well by itself, thus also providing ionization of the monosaccharides and oligosaccharides bound to it for mass spectrometry. Thus, the ligand itself serves to ionize the conjugate molecule, and there is no need to add any MALDI matrix. The mixture of conjugates, i.e., of monosaccharides and oligosaccharides with the bound ligand, is preferably analyzed by MALDI-TOF mass spectrometry. In a preferred embodiment, for MALDI-TOF mass spectrometry measurements, the sample is purified using a C18 carrier, for example, using ZipTip technology (Merck), by first hydrating the carrier with acetonitrile containing 0.1 vol% formic acid, followed by 0.1 vol% aqueous formic acid solution, then applying the sample to the carrier and washing with 0.1 vol% aqueous formic acid solution. The purified products are eluted with a solution of 80 % vol. acetonitrile with 0,1 % vol. formic acid aqueous solution directly onto the MALDI plate. After drying, measurements are taken. Preferably, it is also possible to analyze the mixture of conjugates, i.e. of monosaccharides and oligosaccharides with bound ligand of general formula I, by electrospray ionization mass spectrometry combined with high pressure liquid chromatography (LC / MS). Microbial identification is preferably performed by comparing the measured mass spectrometry spectra with reference spectra of carbohydrate profiles obtained from known microbes (e.g., bacteria or micromycetes). Known microbes for measuring reference spectra may, for example, be characterized by serotyping or whole genome sequencing. Alternatively, mass spectroscopy spectra of unknown samples may be compared to spectra of polysaccharides of known structures. For the measurement of reference spectra, it is preferable to use samples in which a polysaccharide purification step is performed by extraction prior to the step of cleavage of the polysaccharides. Brief description of drawings Figure 1. Spectrum of unpurified ligand of formula II. Figure 2. Spectrum demonstrating the binding of glucose (a) (m / z 586) and lactose (b) (m / z748) to the ligand of formula II, followed by mass spectrometry measurement. Figure 3: Spectrum of lipopolysaccharide carbohydrates obtained from Escherichia coli strains serotypes O26 (a) and O55 (b) and Salmonella enterica serovar Enteritidis (c). Figure 4: Spectrum obtained from lipid A of Escherichia coli (a) and lipoteichoic acid of Staphylococcus aureus (b). Examples Example 1: Preparation of ligand of formula II Basic fuchsin at a concentration of 20 mM was dissolved in absolute ethanol with hydrochloric acid at a concentration of 0.15 M. Vanillin at a concentration of 20 mM was dissolved in the mixture with shaking. The mixture was incubated for 12 hours at 20 °C. Subsequently, distilled water was added to the mixture to a final mixture concentration of 50 % v / v. The mixture was dried by freeze-drying (lyophilization). The lyophilized mixture was then dissolved in a solution of 20 mM sodium hydroxide containing 20 mM sodium cyanoborohydride. After incubation for 4 hours, the mixture containing basic fuchsin with vanillin bound at one, two or three binding sites containing the primary amine was again dried by lyophilization (MALDI-TOF MS spectrum of the mixture is shown in Figure 1 – the spectrum contains basic fuchsin [m / z 288], basic fuchsin with vanillin bound [m / z 424], basic fuchsin with two vanillin molecules bound [m / z 558] and basic fuchsin with three vanillin molecules bound [m / z 696]). The spectrum demonstrates an unpurified ligand containing bonds at all positions containing the primary amine. The mixture was dissolved in a 5 vol% aqueous solution of acetonitrile containing 0.1 vol% trifluoroacetic acid and purified on a column containing a C18 carrier by a gradient of a 5 - 50 vol% aqueous solution of acetonitrile with 0.1 vol% trifluoroacetic acid. The fractions containing the ligand of formula II (m / z 424) were identified by mass spectrometry and dried by lyophilization. Example 2: Binding of glucose or lactose to the ligand of formula II Glucose or lactose at a concentration of 5 mM dissolved in 25 mM sodium acetate containing 5 mM lithium sulfate at a volume of 25 ^L was mixed with an equivalent amount of 5 mM ligand of Formula II dissolved in 25 mM sodium acetate containing 5 mM lithium sulfate. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. 25 ^L of the mixture was applied to a liquid chromatography tube, and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol. % formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and releasing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile with aqueous 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After the sample was allowed to dry, the measurement was performed. Measurements were performed in both positive and negative linear mode using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. The spectra obtained are shown in Figure 2. Part A of Figure 2 demonstrates the binding of the ligand of formula II with glucose. The molecular weight of the resulting molecule is the simple sum of the molecular weights of the formula II ligand and glucose upon cleavage of the water molecule (m / z 586). Part B demonstrates the binding of the formula II ligand and lactose upon cleavage of the water molecule, with the resulting molecule shown as a signal of m / z 748. Ligands of formula III and IV were tested using the same procedure. In this case, glucose bound to the formula III ligand was detected at m / z 600, and glucose bound to the formula IV ligand was detected at m / z 626. Lactose bound to the formula III ligand was detected at m / z 762, and lactose bound to the formula IV ligand was detected at m / z 788. Example 3: Analysis of known lipopolysaccharide molecules Lipopolysaccharides isolated from Escherichia coli strain - serovar O26 (obtained from Sigma Aldrich - Merck) were diluted to a concentration of 1 mg / mL, 25 ^L of a tartaric acid solution at a concentration of 2 M was added to 25 ^L of the suspension. The mixture was incubated for 30 minutes at 100 °C. After incubation, the mixture was cooled to 50 °C. To the 25 ^L mixture, 50 ^L of a 5 mM solution of the ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate was added. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged, and the supernatant was used for spectrometric measurements.25 ^L of the supernatant was applied to a liquid chromatography tube, and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18 resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and releasing 10 ^L of the sample repeatedly through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile with aqueous 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After the sample was allowed to dry, the measurement was performed. Measurements were performed in both positive and negative linear mode using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. Lipopolysaccharide was also detected in the same way for individual other bacteria, i.e.Escherichia coli serovar O55 and serovar O111, Salmonella enterica serovar Enteritidis and serovar Minnesota, Klebsiella pneumoniae (obtained from Sigma Aldrich - Merck). The spectra obtained are shown in Figure 3. The spectra demonstrate the differences in the different bacterial species based on the different structure of the lipopolysaccharide molecules that were cleaved in the above mentioned manner. In all cases, these are modified carbohydrate residues to which the ligand of formula II has been bound. Example 4: Analysis of a known lipid A lipopolysaccharide molecule Lipid A lipopolysaccharide isolated from a strain of Escherichia coli (obtained from Sigma Aldrich - Merck) was diluted to a concentration of 1 mg / mL, and 25 ^L of tartaric acid solution at a concentration of 2 M was added to 25 ^L of the suspension. After incubation, the mixture was cooled to 50 °C. To 25 ^L of the mixture was added 50 ^L of a 5 mM solution of ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged, and the supernatant was used for spectrometric measurements.25 ^L of the supernatant was applied to a liquid chromatography tube, and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and releasing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile with 1 ^L aqueous formic acid solution on a MALDI plate. After the sample was allowed to dry, the measurement was carried out. Measurements were performed in both positive and negative linear modes using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. The obtained spectra are shown in Figure 4 (a). The specific spectra corresponding to the cleaved lipid A lipopolysaccharide molecule can be seen. When compared with the other spectra, it can be seen that this is a unique profile. Example 5: Analysis of a known lipoteichoic acid molecule isolated from Staphylococcus aureus Lipoteichoic acid isolated from a strain of Staphylococcus aureus (obtained from Sigma Aldrich - Merck) was diluted to a concentration of 1 mg / mL, 25 ^L of tartaric acid solution at a concentration of 2 M was added to 25 ^L of the suspension. The mixture was incubated for 30 minutes at 100 °C. After incubation, the mixture was cooled to 50 °C. To 25 ^L of the mixture was added 50 ^L of a 5 mM solution of ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged, and the supernatant was used for spectrometric measurements.25 ^L of the supernatant was applied to a liquid chromatography tube and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and releasing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile in water with 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After the sample was allowed to dry, the measurement was performed. Measurements were performed in both positive and negative linear modes using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. The obtained spectra are shown in Figure 4 (b). The specific spectra corresponding to the cleaved Staphylococcus aureus lipoteichoic acid molecule can be seen. When compared with the other spectra, it can be seen that this is a unique profile. Example 6: Analysis of bacterial isolates A 1 ^L full bacteriological cell culture of Escherichia coli strain O26 (obtained from the Czech National Type Culture Collection, State Institute of Health), grown for 18 hours at 37 °C on blood agar, was resuspended in 50 ^L tartaric acid solution at a concentration of 2 M. The mixture was incubated for 30 minutes at 100 °C. After incubation, the mixture was cooled to 50 °C and centrifuged. To 25 ^L of the supernatant was added 50 ^L of a 5 mM solution of the ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged, and the supernatant was used for spectrometric measurements. 25 ^L of the supernatant was applied to a liquid chromatography tube and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and releasing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile in water with 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After the sample was allowed to dry, the measurement was performed. Measurements were performed in both positive and negative linear modes using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. Similarly, bacterial cultures of Escherichia coli O55 and Salmonella enterica serovar Enteritidis were analyzed. The spectra obtained were unique and identical to those of the purified lipopolysaccharide of the corresponding bacterial species. The results demonstrate the possibility of differentiation between bacterial taxa. Example 7: Analysis of yeast isolates A 1 ^L full loop of culture of a Candida albicans strain, grown for 24 hours at 37 °C on blood agar, was resuspended in 50 ^L tartaric acid solution at a concentration of 2 M. The mixture was incubated for 30 minutes at 100 °C. After incubation, the mixture was cooled to 50 °C and centrifuged. To 25 ^L of the supernatant, 50 ^L of a 5 mM solution of the ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate was added. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged, and the supernatant was used for spectrometric measurements.25 ^L of the supernatant was applied to a liquid chromatography tube, and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and dispensing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile in water with 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After drying, measurements were taken. Measurements were performed in both positive and negative linear modes using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. Unique spectra were obtained that corresponded to polysaccharides contained in the cell wall of only Candida albicans species. The spectra were compared with other previously measured spectra of bacteria and micromycetes. Consistent with bacteria, no agreement was found for the different taxa. Based on the analysis of the spectra, it can be assumed that the measured signal corresponds to beta- glucan of the cell wall. Example 8: Analysis of filamentous fungi A mycelial culture of Aspergillus fumigatus corresponding to a volume of approximately 1 mm3, grown for 24 hours at 37 °C on blood agar, was resuspended in 50 ^L tartaric acid solution at a concentration of 2 M. The mixture was incubated for 30 minutes at 100 °C. After incubation, the mixture was cooled to 50 °C and centrifuged. To 25 ^L of the supernatant, 50 ^L of a 5 mM solution of the ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate was added. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged, and the supernatant was used for spectrometric measurements. 25 ^L of the supernatant was applied to a liquid chromatography tube, and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and dispensing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile in water with 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After drying, measurements were taken. Measurements were performed in both positive and negative linear modes using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. Unique spectra were obtained that corresponded only to the species Aspergillus fumigatus. The spectra were compared with other previously measured spectra of bacteria and micromycetes. Consistent with bacteria, no agreement was found for the different taxa. Based on the analysis of the spectra, it can be assumed that the measured signal corresponds to cell wall galactomannan. Example 9: Analysis of a urine sample containing Escherichia coli To 25 ^L of a human urine sample containing Escherichia coli strain O111 at 105CFU (colony forming units) / mL was added 25 ^L of a tartaric acid solution at a concentration of 2 M. The mixture was incubated for 30 minutes at 100 °C. After incubation, the mixture was cooled to 50 °C and centrifuged. To the 25 ^L of the supernatant, 50 ^L of a 5 mM solution of the ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate was added. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged and the supernatant was used for spectrometric measurements.25 ^L of the supernatant was applied to a liquid chromatography tube and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and dispensing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile in water with 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After drying of the sample, the measurement was performed. Measurements were performed in both positive and negative linear modes using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. Unique spectra were obtained that matched the spectra of Escherichia coli strain O111 measured from the isolated lipopolysaccharide and spectra obtained from a pure bacterial culture. Example 10: Analysis of a urine sample containing Streptococcus pneumoniae C-polysaccharide To a 25 ^L sample of human urine containing Streptococcus pneumoniae C-polysaccharide at a concentration of 1 mg / mL, a 25 ^L solution of tartaric acid at a concentration of 2 M was added. The mixture was incubated for 30 minutes at 100 °C. After incubation, the mixture was cooled to 50 °C and centrifuged. To the 25 ^L mixture, 50 ^L of a 5 mM solution of the ligand of formula II dissolved in 25 mM sodium acetate with 5 mM lithium sulfate was added. The mixture was heated for 10 min at 50 °C on a shaking thermoblock at 500 rpm. The resulting mixture was centrifuged, and the supernatant was used for spectrometric measurements.25 ^L of the supernatant was applied to a liquid chromatography tube, and the sample was measured using a timsTOF Pro spectrometer (LC / MS). The sample for MALDI-TOF mass spectrometry measurements was purified using a C18 carrier (ZipTip with 0.6 ^L C18resin, Merck) by first hydrating the carrier with acetonitrile (10 ^L) containing 0.1 vol% formic acid by aspirating and dispensing the 10 ^L solution ten times. Subsequently, the carrier was equilibrated by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The sample was applied by pipetting from a tube containing 25 ^L of the sample ten times and dispensing 10 ^L of the sample through the carrier at the tip. The carrier was then washed by aspirating and dispensing 10 ^L of a 0.1 vol% formic acid solution in water ten times. The purified sample was eluted with a solution of 80 vol% acetonitrile in water with 0.1 vol% formic acid in a volume of 1 ^L per MALDI plate. After drying, measurements were taken. Measurements were performed in both positive and negative linear modes using a MALDI-TOF rapiFlex mass spectrometer (Bruker Daltonics) equipped with a UV laser at 355 nm. Parameters were set to measure in the region 200 - 2000 m / z, ion extraction with a pulse of 20 ns, and source voltage of 20 kV. A unique spectrum was obtained that matched the spectrum of Streptococcus pneumoniae C- polysaccharide measured from a bacterial culture of S. pneumoniae.

Claims

CLAIMS 1. A method for the detection and / or identification of reducing monosaccharides or oligosaccharides by mass spectrometry in a sample, wherein the reducing saccharide(s) in the sample is (are) reacted with a ligand of general formula I(I), wherein L is selected from the group comprising, wherein n is from 1 to 3,wherein in the general formula I the methoxyphenol group is bound to the carbon atom of the linker L, where R1, R2and R4are independently selected from the group comprising hydrogen atom and, wherein R3is selected from the group comprising hydrogen atom and methyl, to form a conjugate by reaction of the aldehyde group(s) of the reducing monosaccharide(s) and oligosaccharide(s) present in the sample with the NH2group of the ligand, and the conjugate is subsequently subjected to ionization and mass spectrometry measurement.

2. A method for detecting and / or identifying microbial polysaccharides by mass spectrometry comprising the method according to claim 1, wherein - the polysaccharides are cleaved enzymatically or by acid hydrolysis to reducing monosaccharides and / or oligosaccharides, - then conjugate(s) is / are formed by reacting the aldehyde group of the reducing monosaccharides and / or oligosaccharides with the NH2group of at least one ligand of general formula Iwherein L is selected from the group comprising, wherein n is 1, 2 or 3,a carbon atom of the linker L, wherein R1, R2and R4are independently selected from the group comprising hydrogen atom and, wherein R3is selected from the group consisting of hydrogen atom and methyl, and - the conjugate(s) is / are subsequently subjected to ionization and measured by mass spectrometry.

3. The method according to claim 2, wherein prior to the cleavage step, the microbial polysaccharides are isolated by extraction from the sample comprising microbial cells and / or microbial polysaccharides, preferably by extraction into lysis buffer, and by subsequent centrifugation, wherein the supernatant is used in the further steps.

4. The method according to claim 3, wherein the cleavage of the polysaccharides by acid hydrolysis is carried out by adding an acid to the supernatant, preferably tartaric acid, hydrochloric acid and / or glyoxalic acid, at an acid concentration of at least 1M and incubating the mixture for at least 20 minutes at a temperature of at least 60 °C, preferably 60 to 100 °C, wherein preferably the incubation is carried out under shaking.

5. The method according to claim 3, wherein the enzymatic cleavage of the polysaccharides is carried out by adding an enzyme selected from the group consisting of ^-amylase, ^-amylase, lysozyme, endopeptidase, in a buffer corresponding to the pH optimum of the activity of the enzyme to thesupernatant and by performing the cleavage reaction at the temperature optimum of the activity of the enzyme for at least 20 minutes, preferably for 20 to 60 minutes, wherein preferably the incubation is carried out under shaking.

6. The method according to claim 2, wherein the cleavage of the polysaccharides from microbial culture by acid hydrolysis is carried out by resuspending the microbial culture in a solution of acid, preferably tartaric acid, glyoxalic acid and / or hydrochloric acid, at a concentration of at least 1 M and incubating the mixture for at least 20 minutes at a temperature of at least 60 °C, preferably 60 to 100 °C, wherein preferably the incubation is carried out under shaking.

7. The method according to claim 2, wherein the enzymatic cleavage of the polysaccharides from microbial culture is carried out using an enzyme selected from the group consisting of ^-amylase, ^- amylase, lysozyme, and endopeptidase, wherein the microbial culture is resuspended in a buffer corresponding to the optimum pH of the activity of the enzyme, the enzyme is added, and the cleavage reaction is carried out at the temperature optimum of the activity of the enzyme for at least 20 minutes, preferably for 20 to 60 minutes, wherein preferably the incubation is carried out under shaking.

8. The method according to claim 2, wherein the cleavage of the polysaccharides from a clinical sample by acid hydrolysis is performed by adding an acid, preferably tartaric acid and / or hydrochloric acid, at a concentration of at least 1 M to the clinical sample, and incubating the mixture at a temperature of at least 60 °C, preferably 60 to 100 °C, for at least 20 minutes, wherein preferably the incubation is carried out under shaking.

9. The method according to claim 2, wherein the enzymatic cleavage of the polysaccharides from a clinical sample is performed by adding an enzyme selected from the group consisting of ^-amylase, ^- amylase, lysozyme, and endopeptidase, in a buffer corresponding to the pH optimum of the activity of the enzyme to the clinical sample, and the cleavage is carried out at the temperature optimum of the activity of the enzyme for at least 20 minutes, preferably 20 to 60 minutes, wherein preferably the incubation is carried out under shaking.

10. Method according to any one of claims 1 to 9, wherein the ligand of general formula I is added to the mixture of cleaved monosaccharides and oligosaccharides in a buffer at a pH in the range of 2 to 6, preferably at pH 3, and the mixture is incubated for at least 10 minutes at a temperature of up to 70 °C, preferably 20 to 70 °C.

11. The method according to any one of claims 1 to 10, wherein that the conjugate(s) of monosaccharides and / or oligosaccharides with the ligand of general formula I are subjected to measurement by MALDI- TOF mass spectrometry or by electrospray ionization mass spectrometry combined with high-pressure liquid chromatography.

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