Method for analyzing free sugar chain in blood sample

Abstract

This method for analyzing a free sugar chain in a blood sample comprises: a preparation step for preparing a blood sample; a sample preparation step for purifying the free sugar chain from the blood sample to obtain a sample for analysis; and an analysis step for analyzing the sample for analysis through use of a mass spectrometer, a liquid chromatograph, or an electrophoresis apparatus. Before the sample preparation step, a pretreatment step for pre-treating the prepared blood sample may be included, and the pretreatment step does not include a protein precipitation treatment.

Description

Method for analyzing free glycans in blood samples

[0001] The present invention relates to a method for analyzing free sugar chains in a blood sample.

[0002] In recent years, it has been reported that free oligosaccharides (fOS) with structures similar to N-linked glycans bound to proteins are present in serum (Non-Patent Documents 1 to 4). Non-Patent Document 1 detects 12 types of fOS (mainly complex fOS) from serum samples treated with ultrafiltration and solid-phase extraction, and discloses that biantennary glycans containing two sialic acids are most abundant. On the other hand, Non-Patent Document 2 detects 13 types of fOS from the supernatant of ethanol precipitation, and discloses that they are mainly high-mannose types.

[0003] K. Iwatsuka et al. , J. Chromatogr. B, 2013, 928, 16-21. Y. Yoshida et al. , Proteomics, 2016, 16, 2747-2758. J. Seino et al. , Glycobiology, 2016, 26, 1072-1085. C. Huang et al. , Glycobiology, 2022, 32, 314-332.

[0004] Considering that glycans are potential biomarkers for various diseases, it is expected that free glycans in the blood may also be useful biomarkers. However, there are many unknowns, such as the types of free glycans present in the blood, the mechanism of their production, and the effect of disease on the structure of free glycans. For example, it is still unknown whether GN0-type free glycans exist in the blood.

[0005] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for analyzing free sugar chains in a blood sample with high sensitivity.

[0006] As a result of intensive research, the inventors discovered that free glycans in blood samples can be detected and analyzed with high sensitivity by eliminating the protein precipitation treatment, which has been considered an almost essential pretreatment in the analysis of glycans in blood samples, and thus completed the present invention.

[0007] A first aspect of the present invention relates to a method for analyzing free glycans in a blood sample, comprising: a preparation step of preparing a blood sample; a sample preparation step of purifying the free glycans from the blood sample to obtain an analytical sample; and an analysis step of analyzing the analytical sample using a mass spectrometer, a liquid chromatograph, or an electrophoresis device, wherein the method may further comprise a pretreatment step of pretreating the prepared blood sample prior to the sample preparation step, and wherein the pretreatment step does not include a protein precipitation treatment.

[0008] According to the present invention, it is possible to provide a method for analyzing free sugar chains in a blood sample with high sensitivity.

[0009] Figure 1 is a flow diagram showing the pretreatment and sample preparation steps in Experiment 1. Figure 2 is a portion of the mass spectrum obtained by MALDI-TOF MS measurement in Experiment 1. The horizontal axis indicates the m / z value, and the vertical axis indicates the detection intensity (left: relative signal intensity, right: absolute signal intensity). Figure 3 is an extracted ion chromatogram of a GN2-type glycan, a GN1-type glycan, and a GN0-type glycan having two sialic acids. The horizontal axis indicates the retention time (minutes), and the vertical axis indicates the relative amount. Figure 4 is a mass spectrum corresponding to Peaks 1 to 9 in Figure 3. The horizontal axis indicates the m / z value, and the vertical axis indicates the detection intensity (relative signal intensity). Figure 5 is an MS / MS spectrum of a GN2-type glycan having two sialic acids (a), a GN1-type glycan (b), and a GN0-type glycan (c) in Experiment 2. The horizontal axis represents the m / z value, and the vertical axis represents the detection intensity (relative signal intensity). FIG. 6 is a flow diagram showing the pretreatment and sample preparation steps in Experiment 3. FIG. 7 is an extracted ion chromatogram (left) and mass spectrum (right) of a GN0-type glycan having two sialic acids in Experiment 3. In the extracted ion chromatogram, the horizontal axis represents the retention time (minutes), and the vertical axis represents the relative amount. In the mass spectrum, the horizontal axis represents the m / z value, and the vertical axis represents the detection intensity. FIG. 8 is a flow diagram showing the pretreatment, sample preparation, and analysis steps in Experiment 4. FIG. 9 is an extracted ion chromatogram (left) and mass spectrum (right) of a GN0-type glycan having two sialic acids in Experiment 4. In the extracted ion chromatogram, the horizontal axis represents the retention time (minutes), and the vertical axis represents the relative amount. In the mass spectrum, the horizontal axis represents the m / z value, and the vertical axis represents the detection intensity. 10 shows a portion of the mass spectrum obtained by MALDI-TOF MS measurement in Experiment 5. The horizontal axis indicates the m / z value, and the vertical axis indicates the detected intensity.

[0010] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0011] <<Method for Analyzing Free Glycans in a Blood Sample>> A first aspect of the present invention is a method for analyzing free glycans in a blood sample, comprising: a preparation step of preparing a blood sample; a sample preparation step of purifying the free glycans from the blood sample to obtain an analytical sample; and an analysis step of analyzing the analytical sample using a mass spectrometer, liquid chromatograph, or electrophoresis device, and may include a pretreatment step of pretreating the prepared blood sample before the sample preparation step, and the pretreatment step does not include protein precipitation treatment.

[0012] <Preparation Step> In this step, a blood sample is prepared. In this embodiment, "blood sample" refers to plasma or serum. Plasma refers to a liquid component obtained by removing blood cell components from blood. Serum refers to a liquid component obtained by removing blood clots from blood. In one aspect of this embodiment, the blood sample is a sample derived from blood, and can also be understood as a sample that is transparent to the naked eye.

[0013] Examples of animals from which blood samples are derived include humans, mice, rats, guinea pigs, hamsters, rabbits, dogs, birds, and fish. The blood sample can be collected by any known method without any particular limitations. In one aspect of this embodiment, the blood sample may be a blood sample that has been collected in advance.

[0014] <Pretreatment Step> In this step, the prepared blood sample is pretreated. In this embodiment, the pretreatment step does not include a protein precipitation treatment. In one aspect of this embodiment, the analysis method can also be understood as not including a protein precipitation treatment in the blood sample.

[0015] The analysis method according to this embodiment may or may not include a pretreatment step. In one aspect of this embodiment, the analysis method preferably does not include the pretreatment step.

[0016] The blood sample may be pretreated by any method without particular limitation, including, for example, removal of low molecular weight compounds using an ultrafiltration filter (e.g., Ultrafiltration device Amicon Ultra 0.5 ml cut-off 3000, manufactured by Merck Millipore) and removal of proteins using a C18 desalting column (e.g., MonoSpin C18 column).

[0017] Examples of protein precipitation treatments not included in the pretreatment step include protein precipitation treatment using an organic solvent, protein precipitation by heating, protein precipitation by salting out, etc. Precipitants used in such precipitation treatments include, for example, salts such as ammonium sulfate, organic solvents such as acetone, acetonitrile, and chloroform, alcohols such as methanol, propanol, and ethanol, acids such as trichloroacetic acid (TCA), hydrochloric acid, and metaphosphoric acid, water-soluble polymers such as polyethylene glycol and dextran, and combinations thereof.

[0018] <Sample Preparation Step> In this step, the free glycan is purified from the blood sample to obtain an analytical sample. In this embodiment, "glycan" refers to a polymer compound in which various sugars are polyglycosylated by glycosidic bonds. "Free glycan" refers to a glycan that is not bound to a protein or lipid in vivo. Examples of the free glycan include a GN2 type glycan, a GN1 type glycan, and a GN0 type glycan. In one aspect of this embodiment, the free glycan preferably contains at least one sialic acid.

[0019] In one aspect of this embodiment, it is preferable that the sample preparation step is carried out immediately after the preparation step.

[0020] In this embodiment, "purifying free glycans" means selectively recovering free glycans using beads or the like that specifically capture glycans. Examples of solid phase supports (beads, columns, etc.) that specifically capture glycans include solid phase supports having hydrazide groups or aminooxy groups. Examples of solid phase supports having hydrazide groups include BlotGlyco (manufactured by Sumitomo Bakelite Co., Ltd.). Free glycans may also be purified by adsorbing them to a support for hydrophilic interaction chromatography (hereinafter also referred to as HILIC).

[0021] In one aspect of this embodiment, when the free glycan contains at least one sialic acid, the sample preparation step preferably includes neutralizing the free glycan. "Neutralizing the free glycan" means generating a new compound derived from the free glycan by esterification, amidation, or both of the sialic acid that constitutes the free glycan. The neutralization of the free glycan may be performed by linkage-specific modification of sialic acid (sometimes referred to as "SALSA"). Here, "linkage-specific modification of sialic acid" and "linkage-specific modification of sialic acid" refer to a modification reaction that acts on sialic acid, in which the chemical structure generated by the modification reaction differs when the sialic acid is α2,3-sialic acid (or α2,8-sialic acid or α2,9-sialic acid) and when the sialic acid is α2,6-sialic acid. Specifically, the linkage-specific modification of sialic acid preferably comprises a first reaction for lactonizing sialic acid and a second reaction for amidating the lactone structure produced by the first reaction. The first and second reactions are described below.

[0022] (First Reaction) In the first reaction, when sialic acid is bound to the above-mentioned free sugar chain, the sialic acid is selectively lactonized depending on the binding mode. In the first reaction, preferably, in addition to α2,3-sialic acid in the free sugar chain, α2,8-sialic acid and α2,9-sialic acid are lactonized.

[0023] The first reaction can be carried out by contacting a sample containing the free glycans (e.g., a solid-phase carrier on which the free glycans have been captured) with a solution for linkage-specific lactonization of sialic acid (hereinafter also referred to as a "lactonization reaction solution"). One example of a contacting method is to add the lactonization solution to a sample containing the free glycans. The lactonization reaction solution preferably contains a dehydrating condensing agent. Simultaneously with the first reaction, α2,6-sialic acid is preferably subjected to a modification other than lactonization, preferably amidation or esterification. In this case, the lactonization reaction solution preferably contains, in addition to the dehydrating condensing agent, a nucleophile containing at least one selected from the group consisting of alcohols, amines, and salts thereof.

[0024] The types and concentrations of the dehydration condensation agent and nucleophile may be adjusted to selectively induce dehydration or nucleophilic reactions based on the bond mode of sialic acid. The lactone produced by intramolecular dehydration of the carboxy group of α2,3-sialic acid is a six-membered ring, while the lactone produced by intramolecular dehydration of the carboxy group of α2,6-sialic acid is a seven-membered ring. α2,3-sialic acid, which produces a six-membered ring that is more stable than a seven-membered ring, is more easily lactonized than α2,6-sialic acid. Furthermore, since the carboxy group of α2,3-sialic acid is located in a position that is relatively more sterically hindered than the carboxy group of α2,6-sialic acid, large molecules are less likely to react with α2,3-sialic acid than with α2,6-sialic acid. Based on the differences in molecular structure due to the bond mode of sialic acid, the types and concentrations of the dehydration condensation agent and nucleophile are adjusted to achieve different modifications depending on the bond mode of sialic acid.

[0025] (Second Reaction) The second reaction may be carried out by contacting the product (intermediate) from the first reaction with a solution for amidation (hereinafter also referred to as the "amidation reaction solution"). The first reaction can differentiate between α2,3- and α2,6-linkages of sialic acid, but the lactone structure generated from α2,3-sialic acid may be unstable and tends to revert to the original carboxylic acid structure upon hydrolysis. The second reaction specifically amidates the lactone structure, stabilizing the lactone structure. At the same time, the second reaction results in linkage-specific modification of sialic acid with different masses, making it possible to differentiate between α2,3-sialic acid and α2,6-sialic acid. By carrying out the second reaction, sialic acid can be more quickly and specifically modified in a linkage-specific manner. An example of the second reaction may be aminolysis. Aminolysis is a reaction based on the interaction between an amino group and a lactone structure. The aminolysis is different from hydrolysis because it is favorable even under anhydrous conditions. In this specification, the ring-opening and amidation of a lactone structure with ammonia, an amine, or a salt thereof, which is possible even under anhydrous conditions, is referred to as "aminolysis." In one aspect of this embodiment, "stabilization of a lactone structure" and "stabilizing a lactone structure" can also be understood as replacing an unstable lactone structure with another stable structure (such as a methylamide group) by performing aminolysis.

[0026] The above-mentioned bond-type-specific modification of sialic acid may be carried out using, for example, SialoCapper-iD Kit (product name, manufactured by Shimadzu Corporation).

[0027] In one aspect of this embodiment, the sample preparation step preferably includes labeling the released glycans. Here, "labeling the released glycans" refers to modifying the reducing ends of the released glycans with a labeling compound to enable fluorescent or UV detection. Examples of the labeling compound include 2-aminobenzoic acid (2-AA), 2-aminopyridine (PA), 8-aminopyrene-1,3,6-trisulfonic acid (APTS), and 2-aminobenzamide (2-AB), which are used in the Examples. Labeling with 2-AA is sometimes referred to as "AA labeling," and labeling with 2-aminopyridine is sometimes referred to as "PA labeling."

[0028] In addition to the above-mentioned operations, the sample preparation step may also include purification using an amide tip or a carbon tip. An example of an amide tip is GL-Tip Amide (product name) manufactured by GL Sciences. An example of a carbon tip is GL-Tip GC (product name) manufactured by GL Sciences.

[0029] <Analysis Step> In this step, the analysis sample is analyzed using a mass spectrometer, a liquid chromatograph, or an electrophoresis device. In the analysis step, the analysis sample is preferably analyzed using a high-performance liquid chromatograph, a liquid chromatograph-mass spectrometer, or a matrix-assisted laser desorption / ionization mass spectrometer.

[0030] In this step, the method of ionization using a mass spectrometer is not particularly limited, and matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), nano-electrospray ionization (nano-ESI), etc. can be used. In this embodiment, the ionization method is preferably nano-ESI. Ionization in mass spectrometry may be performed in either positive ion mode or negative ion mode. Mass spectrometry may be performed in multiple stages.

[0031] Examples of the mass spectrometer include a time-of-flight (TOF) mass spectrometer, a Fourier transform mass spectrometer, a quadrupole-time-of-flight (Q-TOF) mass spectrometer, a triple quadrupole mass spectrometer, an ion trap mass spectrometer, a matrix-assisted laser desorption / ionization mass spectrometer (MALDI mass spectrometer), etc. In this embodiment, from the viewpoint of analyzing known components accurately in a short time, the mass spectrometer is preferably a Q-TOF mass spectrometer.

[0032] In this step, the liquid chromatograph (LC) utilizes the difference in affinity of each component in the blood sample for the mobile phase and the stationary phase to separate the components and elute them at different retention times. Examples of the liquid chromatograph that can be used include nano LC, micro LC, high performance liquid chromatograph (HPLC), and ultra high performance liquid chromatograph (UHPLC).

[0033] In this embodiment, the mobile phase is not particularly limited, but for example, when a reverse-phase column is used as the stationary phase, examples of the mobile phase A include a 0.1% formic acid aqueous solution and a 0.1% formic acid acetonitrile solution as the mobile phase B. When a HILIC-based amide column is used as the stationary phase, examples of the mobile phase A include an ammonium formate aqueous solution (pH 4.5) and the mobile phase B includes acetonitrile.

[0034] In this embodiment, the stationary phase is not particularly limited, but examples thereof include silanes supported on a carrier such as silica gel to which a straight-chain hydrocarbon such as C8 or C18 is bonded, and the stationary phases described in the Examples below. In one aspect of this embodiment, a HILIC column (e.g., an amide column, a ZIC-HILIC column, a diol column, etc.) may be used as the stationary phase.

[0035] In one aspect of this embodiment, the liquid chromatograph and the mass spectrometer may be used in succession for analysis. For example, a liquid chromatograph-tandem mass spectrometer may be used. The liquid chromatograph-tandem mass spectrometer is typically a liquid chromatograph-triple quadrupole mass spectrometer or a liquid chromatograph-quadrupole / time-of-flight (Q-TOF) mass spectrometer. In such a liquid chromatograph-tandem mass spectrometer, analysis conditions are typically set to maximize detection sensitivity, i.e., to achieve the best possible conditions, for each component in the eluate separated by the liquid chromatograph.

[0036] The electrophoresis method used in this step is not particularly limited as long as it is a known method, and any method can be used. In one aspect of this embodiment, the electrophoresis method is preferably at least one selected from the group consisting of capillary electrophoresis, microchip electrophoresis, two-dimensional electrophoresis, and isoelectric focusing, and more preferably at least one selected from the group consisting of capillary electrophoresis and microchip electrophoresis. The analytical equipment and analytical conditions used for the electrophoresis method are not particularly limited, and known analytical equipment and analytical conditions can be used.

[0037] The method for analyzing free glycans according to this embodiment has been described above. Conventionally, protein precipitation has been considered an almost essential pretreatment in the analysis of glycans in blood samples. The present inventors, unconstrained by this common technical knowledge, discovered that free glycans in blood samples can be detected and analyzed with high sensitivity by preparing an analytical sample from a blood sample without protein precipitation, and thus completed the present invention. In particular, the present inventors are the first to successfully detect GN0-type free glycans. The present inventors believe that, in the past, protein precipitation treatments resulted in many free glycans precipitating along with protein aggregates, leaving almost no residue in the supernatant, making GN0-type free glycans, which are particularly low in abundance, below the detection limit and therefore undetectable.

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0039] [Experiment 1] Investigation of pretreatment steps Human plasma was prepared as a blood sample (preparation step), and the following three types of pretreatment were investigated (Figure 1). In Method 1 (Example 1), human plasma (20 μL) was directly subjected to glycan capture beads (product name: BlotGlyco, manufactured by Sumitomo Bakelite Co., Ltd.) (the sample preparation step was proceeded to without performing the pretreatment step).

[0040] In Method 2 (Example 2), human plasma (40 μL) was first filtered using an ultrafiltration filter (product name: Ultrafiltration device Amicon Ultra 0.5 ml cut-off 3000, manufactured by Merck Millipore) to remove low molecular weight compounds. The concentrated fraction (20 μL) was then subjected to BlotGlyco (Method 2a), or the concentrated fraction (20 μL) was subjected to a C18 desalting column (product name: MonoSpin C18 column, manufactured by GL Sciences), and the flow-through fraction was collected and subjected to BlotGlyco (Method 2b). Both Methods 2a and 2b involved a pretreatment step before proceeding to the sample preparation step.

[0041] In Method 3 (prior art, comparative example), human plasma (20 μL) was first diluted 2-fold with water to 40 μL, to which ethanol (160 μL) was added and stirred. The stirred sample was then incubated at −25°C for 60 minutes to precipitate proteins (protein precipitation treatment). The sample was centrifuged at 17,000 g for 10 minutes, and the supernatant was collected, concentrated to approximately 20 μL, and subjected to BlotGlyco (a pretreatment step including protein precipitation was performed before proceeding to the sample preparation step).

[0042] In all of Methods 1 to 3, the sample preparation process was performed as follows (Figure 1). Pretreated human plasma was subjected to BlotGlyco, and free glycans were captured on beads. Next, the sialic acids of the glycans on BlotGlyco were derivatized (linkage-specific amidation) using the SialoCapper-iD Kit (product name, manufactured by Shimadzu Corporation) to neutralize the sialic acids. Subsequently, the glycans were re-released from BlotGlyco according to the manual provided with BlotGlyco, and the reducing ends were labeled (2-aminobenzoic oxidation, AA). The labeled free glycans were purified sequentially using an amide tip (product name: GL-Tip Amide, manufactured by GL Sciences) and a carbon tip (product name: GL-Tip GC, manufactured by GL Sciences). The purified free glycans were then measured using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF MS) under the following conditions:

[0043] (MALDI-TOF MS Measurement Conditions) The measurement apparatus used was an AB SCIEX TOF / TOF 5800 (AB SCIEX). 2,5-dihydroxybenzoic acid and methylenediphosphonic acid were dissolved in 50% aqueous acetonitrile to final concentrations of 5 mg / mL and 1 mg / mL, respectively, to prepare a matrix solution. Equal amounts of the sample solution and the matrix solution were mixed to obtain a mixed solution. 2 μL of the obtained mixed solution was sampled and applied to a μFocusplate, 700 μm (Hudson Surface Technology), allowed to dry naturally, and then subjected to mass spectrometry. Measurements were performed in the negative ion mode of a reflectron, with a measurement range of m / z 1000 to 5000.

[0044] Figure 2 shows a portion of the mass spectrum obtained by MALDI-TOF MS measurement. In Figure 2, diamonds represent sialic acid (here, N-acetylneuraminic acid), white circles represent galactose, white squares represent N-acetylglucosamine, and gray circles represent mannose. In Method 1 (Example 1), in which the sample preparation step was performed without a pretreatment step, these two types of GN0-type free glycans (m / z 1686.7 and 2018.8) were clearly detected (Figure 2(a)). Furthermore, in Method 2 (Example 2), in which the pretreatment step was performed using ultrafiltration (Amicon Ultra) and a C18 desalting column, GN0-type glycans were also detected (Figure 2(b) and (c)). On the other hand, in Method 3 (Comparative Example), in which protein precipitation with ethanol was performed, GN0-type glycans were not detected (Figure 2(d)). These results demonstrate that free glycans in blood samples can be detected and analyzed with high sensitivity by preparing analytical samples without protein precipitation. Furthermore, they demonstrate that free glycans in blood samples can be detected and analyzed with even higher sensitivity by preparing analytical samples directly from blood samples without pretreatment.

[0045] Figure 3 shows extracted ion chromatograms for the m / z values ​​(theoretical values) of doubly protonated molecules corresponding to GN2-type glycans, GN1-type glycans, and GN0-type glycans, which distinguish between α2,3-linked sialic acid and α2,6-linked sialic acid when similarly treated samples were measured by LC / MS / MS. Figure 4 shows the mass spectra for the retention times of Peaks 1 to 9 shown in Figure 3. As a result, all measurements were consistent with the theoretical values ​​(mass accuracy within 1 ppm), revealing that GN2-type free glycans, GN1-type free glycans, and GN0-type free glycans consist of isomers with only α2,3-linkages, only α2,6-linkages, and a mixture of α2,3-linkages and α2,6-linkages. Thus, by using the sialic acid linkage specific modification (SALSA) method as a modification method for sialic acids, it is possible to distinguish between α2,3-linkages and α2,6-linkages, thereby enabling analysis with increased information content.

[0046] (LC / MS / MS Measurement Conditions) Analysis was performed using a nanoLC-ESI-MS / MS system combining an UltiMate 3000RSLCnano and a Q Exactive (Thermo Fisher Scientific). A nano HPLC capillary column (75 μm capillary inner diameter, 3 μm packing, 15 cm length, manufactured by Nikkyo Technos) was used as the analytical column. Glycan separation was performed using 0.1% formic acid aqueous solution (mobile phase A) and 0.1% formic acid acetonitrile (mobile phase B) at a flow rate of 300 nL / min, with the concentration of mobile phase B increasing from 2% to 40% over 30 min. The injection volume was equivalent to 2 μL of plasma. Analysis was performed in positive ion mode. High-energy collisional dissociation (HCD) was used for MS / MS analysis, with ions fragmented at a normalized collision energy (NCE) of 15.

[0047] [Experiment 2] Structural analysis of GN0-type glycans Figure 5 shows the MS / MS spectra of the GN2-type glycan (Figure 5(a)), GN1-type glycan (Figure 5(b)), and GN0-type glycan (Figure 5(c)), which are biantennary glycans having two α2,6-linked sialic acids, obtained by LC / MS / MS measurement of an analytical sample obtained by treating human plasma in the same manner as in Method 1 (Example 1) above. The LC / MS / MS measurement conditions were the same as above. In the MS / MS of the GN0-type glycan, peaks derived from fragment ions at m / z 302, 464, and 626, corresponding to Hex1 to Hex3 at the reducing end derivatized with AA, were detected (Figure 5(c)). On the other hand, fragment ion peaks derived from the non-reducing end were detected at m / z 315 (dehydrated α2,6-linked sialic acid), 333 (α2,6-linked sialic acid), 366, and 698. These four fragment ion peaks were commonly detected in GN2, GN1, and GN0 glycans (Figure 5 (a), (b), and (c)). These results indicated that the GN0 glycans contained in plasma are free glycans with a novel structure in which two GlcNAc residues on the reducing end side are missing, resulting in Man as the reducing end.

[0048] [Experiment 3] Investigation of sialic acid modification (neutralization) and glycan reducing end modification (labeling) Next, we investigated the cases where sialic acid modification was not performed in the sample preparation process, and where different glycan modifications were performed at the glycan reducing end. Figure 6 shows a flow diagram of the cases where sialic acid modification was performed using the SialoCapper-ID Kit, and where AA or PA was performed as labeling of the glycan reducing end. Each procedure in this flow diagram was performed in accordance with Method 1 in Experiment 1.

[0049] Figure 7 shows the extracted ion chromatograms and respective mass spectra for the m / z values ​​(theoretical values) of doubly protonated molecules corresponding to GN0-type glycans with two sialic acids. The measurement conditions were the same as those for the experiments corresponding to Figures 3 and 4. As a result, measurements were obtained that agreed with the theoretical values ​​(mass accuracy within 1 ppm) in all cases, demonstrating that GN0-type free glycans can be detected without sialic acid modification or any other modification at the reducing end, as long as protein precipitation with ethanol is not performed before glycan purification.

[0050] [Experiment 4] Investigation of Glycan Purification Using BlotGlyco To investigate whether treatment with BlotGlyco is essential, an experiment was conducted without using BlotGlyco (Figure 8). Figure 8 shows a flow diagram for investigating the effects of ultrafiltration, C18 desalting column treatment, BlotGlyco treatment, and sialic acid modification using the SialoCapper-ID Kit. In Method 11 in Figure 8, low molecular weight compounds were removed by ultrafiltration, and the pass-through fraction from the C18 desalting column was directly subjected to AA conversion and analyzed by LC-MS / MS. In Method 12, glycans were purified with BlotGlyco after ultrafiltration and C18 desalting column treatment. In Method 13, the procedure of Method 12 was followed by sialic acid modification using the SialoCapper-ID Kit. Method 13 has the same flow as Method 2 above. In Method 14, the plasma was directly subjected to BlotGlyco without ultrafiltration or C18 desalting column treatment. In Method 15, the plasma was further modified with sialic acid using the SialoCapper-ID Kit in addition to the procedure of Method 14. Method 15 has the same flow as Method 1 above. The LC-MS / MS measurement conditions were the same as above.

[0051] Figure 9 shows the extracted ion chromatograms and their respective mass spectra for the m / z values ​​(theoretical values) of doubly protonated molecules corresponding to GN0-type glycans having two sialic acids. The measurement conditions were the same as those for the experiments corresponding to Figures 3 and 4. GN0-type glycans were also detected using Method 11, which does not use BlotGlyco, demonstrating that glycan purification using BlotGlyco is not essential. Furthermore, after AA conversion in Method 11, purification was performed using a HILIC-type column to remove reagents. However, this process can be considered as a purification process in which glycans are actively adsorbed to a carrier. Therefore, it was considered important not to perform protein precipitation before purification by actively adsorbing or binding glycans using BlotGlyco or HILIC.

[0052] [Experiment 5] Investigation of blood samples (human plasma, human serum, rat plasma, mouse plasma) Method 1 (Example 1) of Experiment 1 was performed on four types of blood samples: human plasma, human serum, rat plasma, and mouse plasma. Figure 10 shows a portion of the mass spectrum obtained by MALDI-TOF MS measurement. The measurement conditions were the same as those of Experiment 1. GN0-type glycans (m / z 2019, 1687) were detected in both human plasma and human serum. In addition, human plasma and human plasma from different origins such as gender and race were also tested, and GN0-type glycans were similarly detected. The same GN0-type glycans were also detected in rat plasma. In the case of mouse plasma, the type of sialic acid is Neu5Gc (N-glycolylneuraminic acid) rather than Neu5Ac (N-acetylneuraminic acid), so there is a shift of 16 Da to the higher m / z side per sialic acid, but GN0-type glycans (m / z 2051, 1703, etc.) could be detected without any problems. From the above, it was found that the analytical method according to the present invention is capable of analyzing free glycans not only in human serum and plasma, but also in serum and plasma of other organisms.

[0053] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments and examples described above are examples of the following aspects.

[0054] (Item 1) A method for analyzing free glycans according to one embodiment is a method for analyzing free glycans in a blood sample, comprising: a preparation step of preparing a blood sample; a sample preparation step of purifying the free glycans from the blood sample to obtain an analytical sample; and an analysis step of analyzing the analytical sample using a mass spectrometer, liquid chromatograph, or electrophoresis device, and may comprise a pretreatment step of pretreating the prepared blood sample prior to the sample preparation step, where the pretreatment step does not include a protein precipitation treatment. The method according to item 1 makes it possible to provide a method for analyzing free glycans in a blood sample with high sensitivity.

[0055] (Item 2) In the method according to item 1, the free glycans include GN0-type free glycans. According to the method according to item 2, GN0-type free glycans in a blood sample can be analyzed with high sensitivity.

[0056] (Item 3) The method according to item 1 or 2, which does not include the pretreatment step. According to the method according to item 3, free sugar chains in a blood sample can be analyzed with even higher sensitivity and in a simpler manner.

[0057] (Item 4) In the method according to any one of Items 1 to 3, the sample preparation step is carried out immediately after the preparation step. According to the method according to Item 4, free sugar chains in a blood sample can be analyzed with even higher sensitivity and in a simpler manner.

[0058] (Item 5) In the method according to any one of items 1 to 4, the blood sample is serum or plasma. According to the method according to item 5, free sugar chains in serum and plasma can be analyzed with high sensitivity.

[0059] (Item 6) In the method according to any one of Items 1 to 5, the free glycan contains at least one sialic acid, and the sample preparation step further comprises neutralizing the purified free glycan. According to the method according to Item 6, structural information of the free glycan can be analyzed in detail.

[0060] (Item 7) In the method according to any one of Items 1 to 6, the analysis step involves analyzing the analysis sample using a high-performance liquid chromatograph, a liquid chromatograph-mass spectrometer, or a matrix-assisted laser desorption / ionization mass spectrometer. According to the method according to Item 7, a large number of free glycans in a blood sample can be simultaneously detected and analyzed.

[0061] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0062] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

Claims

1. A method for analyzing free glycans in a blood sample, Preparation steps for preparing blood samples, A sample preparation step of purifying the free glycans from the blood sample to obtain a sample for analysis, The analytical step includes analyzing the aforementioned analytical sample using a mass spectrometer, liquid chromatograph, or electrophoresis apparatus. The sample preparation step may include a pretreatment step in which the prepared blood sample is pretreated, The aforementioned pretreatment step does not include protein precipitation. The free glycans include GN0 type free glycans. Analysis method.

2. The analytical method according to claim 1, which does not include the aforementioned pretreatment step.

3. The analytical method according to claim 1 or claim 2, wherein the sample preparation step is performed immediately after the preparation step.

4. The analytical method according to claim 1 or claim 2, wherein the blood sample is serum or plasma.

5. The free sugar chain comprises at least one sialic acid, The analytical method according to claim 1 or claim 2, further comprising neutralizing the purified free sugar chain in the sample preparation step.

6. The analytical method according to claim 1 or claim 2, wherein in the analytical step, the analytical sample is analyzed using a high-performance liquid chromatograph, a liquid chromatograph-mass spectrometer, or a matrix-assisted laser desorption / ionization mass spectrometer.