Method for producing glycopeptide samples

Abstract

Provided is a more efficient method for producing a glycopeptide sample having a high ratio of glycopeptides to non-glycopeptides. The method for producing a glycopeptide sample includes: (3) a step of contacting a particle that retains a protein on a surface with a protease; (4) a step of adding an organic solvent after the step (3); and (5) a step of washing the particle obtained in the step (4), the particle being a cellulose-coated particle, and / or an ion pair reagent is added together with the organic solvent in the step (4).

Description

Technical Field

[0001] This invention relates to methods for manufacturing glycopeptide samples, etc. Background Technology

[0002] In the field of bottom-up proteomics research, various pretreatment methods have been studied to improve data quality (protein identification count, yield stability, etc.), increase efficiency, and automate the process. One example is the SP3 (single-pot, solid-phase-enhanced sample preparation) method, which uses magnetic particles with diameters of tens of nanometers to several micrometers to prepare proteomics samples in a single tube (Non-Patent Literature 1). Proteins in the sample solution are held and held by agglutination on the magnetic particles using organic solvents (ethanol, acetonitrile, etc.). After washing, protein digestion is performed using digestive enzymes. The purification efficiency using magnetic particles is high, as strong denaturing agents can be used and subsequently removed, improving digestion efficiency. Peptides, the digestion product, can also be held and purified on the magnetic particles using acetonitrile.

[0003] On the other hand, in glycoproteomics, a branch of proteomics that focuses on glycan modifications of proteins, it is difficult to obtain comprehensive data when analyzing total peptides prepared from biological samples using conventional proteomics methods. This is because the detection sensitivity of mass spectrometry analysis of peptides containing glycan modifications (glycopeptides) is generally lower compared to non-glycan addition peptides.

[0004] To improve the data quality of glycopeptide analysis, it is necessary to remove non-glycan addition peptides from the total peptide mixture produced by protein digestion and separate the glycopeptides. In the selective purification of glycopeptides, the most commonly used purification method is hydrophilic interaction liquid chromatography (HILIC), which utilizes the principle of separating the glycan addition moieties on the glycopeptides. The glycopeptides are held and washed on a hydrophilic stationary phase under conditions of high organic solvents (mainly acetonitrile), and then eluted with a solvent with a high water ratio. However, protein pretreatment and specific purification of glycopeptides are performed independently, which is not suitable for efficient processing, especially for automated schemes used in large-scale analysis.

[0005] Existing technical documents Non-patent literature Non-patent literature 1: Mol Syst Biol. 2014 Oct 30;10(10):757. doi: 10.15252 / msb.20145625. PMID: 25358341; PMCID: PMC4299378. Summary of the Invention

[0006] The technical problem that the invention aims to solve The technical problem of this invention is to provide a more efficient method for manufacturing glycopeptide samples with a high ratio of glycopeptides to non-glycopeptides.

[0007] Technical solutions for solving the problem The inventors of this invention conducted in-depth research in view of the above-mentioned technical problems and found that the above-mentioned technical problems can be solved by a method for manufacturing glycopeptide samples as follows: (3) a step of contacting particles with proteins on their surface with a protease, (4) a step of adding an organic solvent after step (3), and (5) a step of washing the particles obtained in step (4), wherein the particles are cellulose-coated particles, and / or an ion-pairing reagent is added together with the organic solvent in step (4). Based on this discovery, the inventors of this invention conducted further research and completed this invention. That is, this invention includes the following methods.

[0008] Item 1. A method for manufacturing a glycopeptide sample, comprising: (3) The process of bringing the protein particles on the surface into contact with the protease. (4) The process of adding organic solvents after process (3), and (5) The process of cleaning the particles obtained in step (4), The aforementioned particles are hydrophilic polymer-coated particles, and In the above step (4), an ion-pairing reagent is added together with the above organic solvent.

[0009] Item 2. The manufacturing method as described in Item 1, wherein the hydrophilic polymer-coated particles are polysaccharide-coated particles.

[0010] Item 3. The manufacturing method as described in Item 2, wherein the polysaccharide-coated particles are cellulose-coated particles.

[0011] Item 4. The manufacturing method as described in Item 1, wherein it includes: (2) a step prior to step (3) of contacting the protein with the particles in a solution containing an organic solvent to retain the protein on the surface of the particles.

[0012] Item 5. The manufacturing method as described in Item 4, wherein step (2) includes a step of dispensing a pre-prepared particle suspension containing the above-mentioned particles and a thickener.

[0013] Item 6. The manufacturing method as described in Item 5, wherein the thickener is glycerin.

[0014] Item 7. The manufacturing method as described in Item 1, wherein the hydrophilic polymer-coated particles are carboxylic acid polymer-coated particles.

[0015] Item 8. The manufacturing method as described in Item 1, wherein the hydrophilic polymer-coated particles are magnetic particles.

[0016] Item 9. The manufacturing method as described in Item 1, wherein the ion-pairing reagent is an organic acid.

[0017] Item 10. The manufacturing method as described in Item 9, wherein the ion-pairing reagent comprises a halogenated short-chain fatty acid.

[0018] Item 11. The manufacturing method as described in Item 10, wherein the ion-pairing reagent is a mixture comprising halogenated short-chain fatty acids and non-halogenated short-chain fatty acids.

[0019] Item 12. The manufacturing method as described in Item 1, wherein the organic solvent is selected from at least one of alcohols, acetonitrile, and acetone.

[0020] Item 13. The manufacturing method as described in any one of items 1 to 12, wherein all processes are carried out in the same container.

[0021] Item 14. The manufacturing method as described in any one of Items 1 to 12, wherein the glycopeptide sample is a sample for mass spectrometry analysis.

[0022] Item 15. A glycopeptide sample obtained by any one of items 1 to 12.

[0023] Item 16. An analytical method for glycan modification of a protein, comprising: a step of performing mass spectrometry analysis on the glycopeptide sample described in Item 15.

[0024] Invention Effects According to the present invention, a more efficient method for manufacturing a glycopeptide sample with a high ratio of glycopeptides to non-glycopeptides can be provided. Furthermore, an analytical method for glycan modification of proteins using the glycopeptide sample can also be provided. Attached Figure Description

[0025] Figure 1 This section outlines the experimental procedures for Example 1.

[0026] Figure 2 This represents the comparison results of the number of peptides identified by Byonic search in Experiment Example 1.

[0027] Figure 3 This represents the total amount of glycopeptides containing N-binding glycan modifications detected in Experiment Example 1.

[0028] Figure 4 This represents the total amount of non-glycopeptides (excluding N-binding glycan modifications) detected in Experiment Example 1.

[0029] Figure 5 This is a representative example showing the quantitative values ​​of peptides derived from major blood glycoproteins in Experiment 1. The amino acid sequences in the figure correspond to sequence numbers 1–6 in the sequence listing.

[0030] Figure 6 This section outlines the experimental procedures for Example 2.

[0031] Figure 7 This represents the total amount of non-glycopeptides (excluding N-binding glycan modifications) detected in Experiment Example 2.

[0032] Figure 8 This is a photograph showing what the glycopeptide in Experiment Example 2 looks like during HILIC purification.

[0033] Figure 9 Microscopic photographs of the carboxyl-modified polymer magnetic particle suspension and the cellulose magnetic particle suspension in Experimental Example 3, as well as photographs of the appearance of the containers 1 minute, 6 minutes, and 10 minutes after stirring.

[0034] Figure 10 This indicates the composition of the HILIC washing solution in Example 4 and the number of N-bound glycopeptides identified from untreated serum.

[0035] Figure 11 This figure shows the comparison results of the recovery of N-bound glycopeptides prepared from serum (untreated, HAS and Ig removed) in Experiment Example 4. The amino acid sequences in the figure correspond to sequence numbers 7-8 in the sequence listing. Detailed Implementation

[0036] In this specification, expressions for “containing” and “including” include the concepts of “containing”, “including”, “substantially constituted by” and “consistent only by”.

[0037] In one aspect, the present invention relates to a method for manufacturing a glycopeptide sample, comprising: (3) a step of contacting particles with proteins on their surface with a protease; (4) a step of adding an organic solvent after step (3); and (5) a step of washing the particles obtained in step (4), wherein the particles are cellulose-coated particles, and / or in step (4), an ion-pairing reagent is added together with the organic solvent (sometimes referred to in this specification as "the manufacturing method of the present invention").

[0038] The protein can be any protein for which fragmented peptides need to be analyzed; there are no particular limitations. In a preferred embodiment, the protein is a protein derived from a biological sample of the test subject. Examples of biological species that can be used as test subjects and protein sources include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, with humans being a preferred example. As for the biological sample, any biological sample containing protein is acceptable; there are no particular limitations, and examples include bodily fluids, skin, mucous membranes, and internal tissues. From the viewpoint of ease of collection and low invasiveness, bodily fluids, skin, and mucous membranes are preferred, with bodily fluids being more preferred. Examples of bodily fluids include blood, follicular fluid, menstrual blood, saliva, cerebrospinal fluid, synovial fluid, urine, tissue fluid, sweat, and tears. Examples of mucous membranes include oral mucosa and nasal mucosa. Biological samples are typically subjected to appropriate pretreatment, preferably treatment to remove certain components, protein concentration, and / or purification. In one aspect of the invention, from the viewpoint of effectively processing multiple samples, biological samples are preferably biological samples that have not undergone treatment to remove abundance proteins (e.g., albumins (especially serum albumin) and / or immunoglobulins (especially immunoglobulin-G)). Examples of such removal treatment include processes using chromatography, centrifugation, etc., based on differences in affinity, adsorption, solubility, etc., depending on the type of abundance protein. This removal treatment can remove, for example, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the abundance protein.

[0039] In one aspect of the present invention, the protein can be a protein contained in a blood sample or a sample obtained by removing some components contained in the blood (such as albumin, IgG, antibodies, etc.).

[0040] From the viewpoint of utilizing the hydrolysis efficiency of proteases and the suitability for mass spectrometry analysis, proteins are preferably treated with denaturation, reduction, alkylation, etc. From this viewpoint, the manufacturing method of the present invention preferably includes: (1) a step of performing at least one treatment selected from denaturation, reduction and alkylation on the protein before step (3) (in a preferred embodiment, before step (2)).

[0041] Denaturation is a process that opens the folded structure of a protein. There are no particular limitations on this process, but it is preferable to use a protein denaturing agent such as a surfactant. Examples of surfactants used for denaturation include anionic surfactants such as dodecyl sulfate, deoxycholate, and lauroyl sarcosinate. In the manufacturing method of the present invention, since the protein is treated while remaining on the particle surface as in step (3), solution displacement can be performed. Therefore, it is possible to use components with strong denaturing effects that have a strong influence on protease activity. Therefore, alkyl sulfates such as dodecyl sulfate (e.g., sodium salt) are also preferred among the above-mentioned components.

[0042] Reduction treatment is the process of breaking disulfide bonds within and / or between proteins. There are no particular limitations on this type of treatment, but it is preferable to use a reducing agent. Examples of reducing agents used in reduction treatment include tris(2-carboxyethyl)phosphonic acid hydrochloride, dithiothreitol, 2-mercaptoethanol, and tributylphosphonic acid. Among these, tris(2-carboxyethyl)phosphonic acid hydrochloride is preferred considering the intensity of the denaturing effect.

[0043] Alkylation is the irreversible alkylation of cysteine ​​residues in a protein. There are no particular limitations on the type of alkylation, but an alkylating agent is preferred. Examples of alkylating agents used in alkylation include halogenated acetamides such as chloroacetamide and iodoacetamide. Chloroacetamide is a preferred example among these.

[0044] More specifically, step (1) can be carried out, for example, by reacting the protein in a solution containing at least one selected from surfactants, reducing agents, and alkylating agents. In one manner (particularly when a phosphine-based reducing agent is used as the reducing agent), a solution containing both a reducing agent and an alkylating agent (and, if necessary, a surfactant) can be used. In another manner (particularly when a reducing agent based on a thiol group (dithiothreitol, 2-mercaptoethanol, etc.) is used), an alkylating agent can be added after a solution containing a reducing agent (and, if necessary, a surfactant).

[0045] When the solution in step (1) contains a surfactant, its concentration is, for example, 0.1–10 w / v%, preferably 0.2–5 w / v%, more preferably 0.5–2 w / v%. When the solution in step (1) contains a reducing agent, its concentration is, for example, 1–100 mM, preferably 2–50 mM, more preferably 5–20 mM. When the solution in step (1) contains an alkylating agent, its concentration is, for example, 5–200 mM, preferably 10–100 mM, more preferably 20–60 mM. Water is typically used as the solvent.

[0046] The processing temperature of step (1) is, for example, 15-100°C, preferably 40-100°C, more preferably 60-100°C, and even more preferably 80-98°C. The processing time can be set appropriately according to the processing temperature, for example, 2 minutes to 3 hours. If the processing is carried out at high temperature (e.g., above 90°C), it is preferably 2 minutes to 1 hour, more preferably 2-30 minutes, and preferably 5-15 minutes.

[0047] There are no particular limitations on the particles, as long as they can retain proteins and peptides on their surface in an organic solvent. The material of the particles is not particularly limited, and examples include particles made of metals such as gold, silver, copper, iron, aluminum, nickel, manganese, titanium, and their oxides; particles made of resins such as polystyrene and latex; and silica particles. The shape of the particles is not particularly limited, and examples include spheres, cuboids, cubes, triangular pyramids, or shapes similar to these. The particles can be coated with other molecules or modified with functional groups (e.g., epoxy, amino, carboxyl, amide, etc.). The average particle size is not particularly limited, for example, from 100 nm to 500 μm. In one embodiment, the average particle size is, for example, from 100 nm to 10 μm, more preferably from 300 nm to 5 μm, and even more preferably from 500 nm to 3 μm. In another embodiment, the average particle size is, for example, from 500 nm to 300 μm, preferably from 1 μm to 200 μm, and more preferably from 10 μm to 100 μm.

[0048] In the manufacturing method of the present invention, the particles are preferably coated with a hydrophilic polymer, more preferably with polysaccharides and / or carboxylic acid polymers. As the hydrophilic polymer, there are no particular limitations as long as it has hydrophilic functional groups such as carboxyl or hydroxyl groups. As the polysaccharide, there are no particular limitations; examples include cellulose, dextran, and agarose. As the carboxylic acid polymer, examples include styrene-acrylic acid copolymers and glycidyl methacrylate polymers.

[0049] In the glycopeptide purification process of step (4), since the hydroxyl and / or carboxyl groups constituting the polysaccharide molecule can be utilized as HILIC hydrophilic groups on the hydrophilic stationary phase, the glycopeptide sample can be obtained more effectively depending on the presence or absence of sugar chains (difference in hydrophilic interactions). From the viewpoint of obtaining glycopeptide samples with a higher ratio of glycopeptides to non-glycopeptides more effectively, and from the viewpoint that such an effect can be obtained without using highly corrosive reagents (such as TFA) as ion-pairing reagents, the particles are preferably polysaccharide-coated particles, and cellulose-coated particles are particularly preferred.

[0050] In the manufacturing method of the present invention, the particles are preferably magnetic particles. Magnetic particles can be electromagnet particles. This allows for simple and rapid replacement of the solution surrounding the particles, and further makes the manufacturing method of the present invention more suitable for automated solutions used in large-scale analysis.

[0051] The granules can be used directly from commercially available products, or they can be manufactured according to or based on known methods.

[0052] The particles that retain protein on the surface can be obtained according to or based on known methods, such as by protein aggregation caused by the addition of an organic solvent, which can retain the protein on the particle surface. From this point of view, the manufacturing method of the present invention preferably includes: (2) a step prior to step (3) of contacting the protein with the particles in a solution containing an organic solvent to retain the protein on the particle surface.

[0053] In step (2), by dispensing the protein and particles into the same container, they can come into contact within the container. During the research, attention was paid to the problem that the particles precipitate in the container before dispensing, making the dispensing ineffective. This problem is particularly pronounced when processing multiple samples (e.g., 10 or more, 20 or more, 50 or more, 70 or more, 90 or more) in an automated dispensing machine. Therefore, in order to suppress particle precipitation before dispensing, it is preferable to include a step in step (2) to dispense a pre-prepared particle suspension containing the aforementioned particles and a thickener. This suppresses particle precipitation in the container before dispensing, avoids / suppresses the operation of stirring the particles before dispensing to ensure uniform particle distribution, and thus enables effective dispensing.

[0054] There are no particular limitations on the thickener, but from the viewpoint of suppressing the influence on glycopeptide preparation, it is preferable to avoid using polymer-based thickeners (synthetic polymers, thickening polysaccharides, etc.). From the viewpoint of suppressing particle settling velocity and ease of removal, the thickener is preferably a polyol with a relatively low molecular weight, more preferably a polyol with a molecular weight of 50 to 300 (further preferably 50 to 200, 60 to 150, 70 to 120, 70 to 100). The polyol is, for example, a diol or triol. Glycerol and propylene glycol are preferred examples of such polyols, with glycerol being particularly preferred. The concentration (volume %) of the thickener in the pre-prepared particle suspension is not particularly limited as long as it is viscous enough to suppress particle settling velocity and make the particle suspension viscous enough for dispensing operations. For example, in the case of glycerol, it is preferably 5 to 90%, more preferably 10 to 90%, further preferably 20 to 85%, even more preferably 30 to 85%, particularly preferably 40 to 80%, and especially preferably 50 to 80%.

[0055] The organic solvent used in step (2) can be any solvent that can retain the protein on the particle surface through protein aggregation, and there are no particular limitations. Examples include alcohols with 1 to 4 carbon atoms such as ethanol (preferably ethanol and 2-propanol), acetonitrile, acetone, etc., with alcohols with 1 to 4 carbon atoms and acetonitrile being preferred. The concentration of the organic solvent in the solution of step (2) is, for example, 40 to 90 v / v%, preferably 70 to 90 v / v% when it is ethanol, more preferably 75 to 85 v / v%, and preferably 40 to 60% when it is acetonitrile, more preferably 45 to 55 v / v%.

[0056] The processing temperature of step (2) is, for example, 10 to 40°C, preferably 15 to 30°C, and the processing time is, for example, 2 to 30 minutes, preferably 5 to 15 minutes. During the processing, it is preferred to stir the solution, and more preferably to oscillate (e.g., 500 to 2000 rpm).

[0057] The particles that retain protein on the surface are preferably cleaned after step (2) and before step (3). The solution used in step (2) can be used as the cleaning solution. The cleaning is preferably performed multiple times (e.g., 2 to 5 times, preferably 2 to 3 times).

[0058] After step (2) or cleaning, it is preferable to remove the solvent by drying or the like.

[0059] The protease used in step (3) can be any protease that can fragment the protein, and there are no special restrictions. Examples include endopeptidases such as trypsin, Asp-N, Lys-C, and Glu-C, and exopeptidases such as aminopeptidases and carboxypeptidases.

[0060] Process (3) is usually carried out in solution.

[0061] The concentration of protease in the solution of step (3) is, for example, 0.01 to 0.5 μg / μL, preferably 0.002 to 0.1 μg / μL, and more preferably 0.01 to 0.1 μg / μL. In the case of trypsin, the ratio of protease to protein is preferably, for example, 1:1000 to 1:10 (v / v), preferably 1:200 to 1:50. To promote digestion, and to facilitate redissolution of the sample during drying in step (2) or after washing, the solution preferably contains a surfactant, for example, 2 to 30 mM, preferably 5 to 20 mM. As a surfactant, anionic surfactants with carboxyl groups that have relatively weak protein denaturing effects (e.g., deoxycholate, lauroyl sarcosinate, etc.) are preferred. The solution may also contain buffers, metal salts necessary for protease activity, etc.

[0062] The processing temperature of step (3) is, for example, 20-40°C, preferably 30-40°C, more preferably 35-39°C, particularly preferably 36-38°C, and the processing time is, for example, 30 minutes to 48 hours, preferably 8-24 hours. During the processing, it is preferred to stir the solution, and more preferably to oscillate (e.g., 500-2000 rpm).

[0063] Through process (3), the particulate protein is fragmented to generate peptides (glycopeptides and non-glycopeptides).

[0064] In step (4), an organic solvent is added after step (3).

[0065] As the organic solvent used in step (4), acetonitrile is mainly used in the solvent shown in step (2).

[0066] The amount and concentration of the organic solvent added in step (4) are not particularly limited as long as the concentration of the organic solvent in the container is such that the peptide remains at the particle level (e.g., 70-100 v / v%, preferably 75-90 v / v%).

[0067] In step (4), from the viewpoint of more effectively obtaining glycopeptide samples with a higher ratio of glycopeptides to non-glycopeptides, it is preferable to add an ion-pairing reagent together with an organic solvent. As an ion-pairing reagent, any reagent that can be used in peptide analysis is acceptable, without particular limitations. Organic acids, for example, can be widely used as ion-pairing reagents. Fatty acids are preferred as organic acids, with short-chain fatty acids (fatty acids with 5 or fewer carbon atoms) being particularly preferred. Examples of short-chain fatty acids include formic acid, acetic acid, propionic acid, isobutyric acid, butyric acid, etc., with formic acid being particularly preferred. From the above viewpoint and the viewpoint of ease of removal, halogenated short-chain fatty acids are preferred as ion-pairing reagents. Halogenated short-chain fatty acids are particularly preferred, with fluoroacetic acid (especially trifluoroacetic acid) being particularly preferred. Alternatively, conventional (non-halogenated) short-chain fatty acids (formic acid, acetic acid, etc.) can also be used. By using both halogenated and non-halogenated short-chain fatty acids, especially when using biological samples that have not undergone abundance protein removal treatment, the recovery rate of hydrophobic glycopeptides can be improved. The ratio of non-halogenated short-chain fatty acids to halogenated short-chain fatty acids (mass of non-halogenated short-chain fatty acids / mass of halogenated short-chain fatty acids) at this time is, for example, 1 to 500, preferably 2 to 300, more preferably 5 to 200, further preferably 10 to 100, more preferably 20 to 80, further preferably 30 to 70, and even more preferably 40 to 60. When adding an ion-pairing reagent, the concentration of the ion-pairing reagent in the container can be added, for example, in a manner of 0.05 to 10 v / v, preferably 0.1 to 5 v / v. More preferably, in the case of non-halogenated short-chain fatty acids such as formic acid, it is added in a manner of 1-5% v / v, and in the case of halogenated short-chain fatty acids such as trifluoroacetic acid, it is added in a manner of 0.1-1% v / v.

[0068] The processing temperature of step (4) is, for example, 10 to 40°C, preferably 15 to 30°C, and the processing time is, for example, 5 minutes to 2 hours, preferably 15 to 45 minutes. During the processing, it is preferred to stir the solution, and more preferably to oscillate (e.g., 500 to 2000 rpm).

[0069] In step (5), the particles obtained in step (4) are cleaned. The solution used in step (2) can be used as the cleaning solution. Cleaning is preferably performed multiple times (e.g., 2 to 5 times, preferably 2 to 3 times). In the final stage of cleaning, it is preferable to clean with acetonitrile at a high concentration (preferably 90 v / v% or more, particularly preferably 95 v / v% or more).

[0070] After step (5), it is preferable to elute the peptide from the particles. As the eluent, a solution with a relatively low concentration of the aforementioned organic solvent (e.g., 50 v / v% or less, preferably 40 v / v% or less) can be used. For elution, compared to carrying out under acidic conditions, carrying out under neutral conditions can suppress the hydrolysis of sialic acid. The eluent preferably contains, for example, 3 to 30 mM, preferably 5 to 15 mM, ammonium formate. The elution treatment temperature is, for example, 10 to 40°C, preferably 15 to 30°C, and the treatment time is, for example, 2 minutes to 1 hour, preferably 5 to 30 minutes. During treatment, it is preferable to stir the solution, and preferably to shake (e.g., 500 to 2000 rpm).

[0071] After step (5) or elution, the solvent is preferably removed by drying or the like.

[0072] All steps of the manufacturing method of the present invention can be performed within the same container. Therefore, it is particularly suitable for automation solutions used in large-scale analysis.

[0073] The glycopeptide sample obtained by the manufacturing method of the present invention has a higher proportion of glycopeptides relative to non-glycopeptides. Therefore, the glycopeptide sample obtained by the manufacturing method of the present invention is suitable as a sample for analytical methods, particularly mass spectrometry analysis, where the detection sensitivity of glycopeptides is generally low compared to non-glycopeptides. Thus, in one aspect of the present invention, mass spectrometry analysis can be performed on the glycopeptide sample obtained by the manufacturing method of the present invention.

[0074] Mass spectrometry analysis can be performed according to or based on known methods. Liquid chromatography-mass spectrometry is preferred because it is suitable for the analysis of glycopeptides.

[0075] There are no particular limitations on ionization (ionization) methods in mass spectrometry. Examples of peptide ionization methods include laser desorption / ionization (LDI) and electrospray ionization (ESI).

[0076] There are no particular restrictions on the methods for determining the mass-to-charge ratio in mass spectrometry analysis. For example, time-of-flight type, magnetic field deflection type, quadrupole type, ion trap type, Fourier transform ion cyclotron resonance type, tandem type, etc. can be used.

[0077] The technology of this invention can be used to explore glycopeptide biomarkers in biological samples (e.g., plasma, urine, cerebrospinal fluid, saliva, autopsy tissue, etc.). Furthermore, glycoproteomics is an essential technology in the quality management of biopharmaceuticals such as antibody drugs. The technology of this invention can also enable high-throughput evaluation of antibody drugs.

[0078] Example The present invention will now be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0079] Experimental Example 1. Comparison of protein pretreatment and glycopeptide purification conditions using magnetic particles <1-1. Methods> The types of digestion, HILIC purification, and magnetic particles were compared. As the starting sample, 10 μL of plasma that had undergone albumin / IgG removal and centrifugation was used. Triple samples were used in parallel for each condition. A summary of each condition is shown in Table 1, and a summary of the experimental procedures is shown in Table 2. Figure 1 .

[0080] [Table 1] <1-1-1. Protein denaturation, reduction, and alkylation> As the starting sample, 10 μL of plasma, after albumin / IgG removal and centrifugation drying, was used. The procedure was performed in 1.5 mL polypropylene tubes (ProteinLobind, Eppendorf) of the protein-low adsorption type. A solution for protein denaturation, disulfide bond reduction, and alkylation of the sample was added (1% (w / v) sodium dodecyl sulfate (SDS), 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), 40 mM chloroacetamide (CAA)). The sample was redissolved by shaking at 1000 rpm at 25 °C for 1 min using a temperature-controlled stirrer, followed by denaturation, reduction, and alkylation reactions via heat treatment (95 °C, 10 min).

[0081] <1-1-2. Cleaning of proteins using magnetic particles> After cooling the sample solution to room temperature, add 30 μL of a 50 μg / μL magnetic particle mixture. The magnetic particles vary depending on the conditions. Under conditions A and C, Sera-Mag Speedbead, Carboxylate-modified, Hydrohilic [E7] / Hydrophobic [E3] = 1:1 (25 μg / μL each, equivalent to 1.5 mg of magnetic particles; manufactured by Cytiva) is used. Under conditions E and G, ReliaPrep Resin (equivalent to 1.5 mg of magnetic particles; manufactured by Promega) is used. The mixture is shaken at 1000 rpm at 25°C for 1 min using a thermostatic stirrer. Then, to induce protein aggregation on the beads, add 520 μL of ethanol (final concentration 80% v / v) and shake at 1000 rpm at 25°C for 10 min using a thermostatic stirrer. The test tube containing the sample solution was placed on a magnetic support and allowed to stand for 2 minutes. The magnetic particles were then collected on the test tube wall using the magnet, and the supernatant was removed using a pipette. For further cleaning, 800 μL of 80% (v / v) ethanol was added, allowed to stand for 2 minutes, and then the supernatant was removed. This cleaning process was repeated twice. For the next cleaning step, 800 μL of acetonitrile was added, allowed to stand for 2 minutes, and then the supernatant was removed. With the test tube cap open, the tube was placed on an aluminum dry block heater at 45°C for 15 minutes to allow the acetonitrile to evaporate and the beads to dry.

[0082] <1-1-3. Digestion of Proteins on Beads> Next, 50 μL of a buffer solution containing surfactants to promote sample redissolution and digestion was added at a 2-fold concentration (4.8 mM SDC, 4.8 mM SLS, 200 mM Tris-HCl, pH 8.5). The sample was resuspended by shaking at 25°C and 2000 rpm for 5 min using a temperature-controlled stirrer (in many cases, the protein-bead clumps are not completely dispersed at this point). 50 μL of an enzyme solution for protein digestion (0.02 μg / μL trypsin / Lys-C mix, 4 mM CaCl2, 2 mM acetic acid (acetic acid derived from the enzyme redissolution solution)) was added, and the digestion reaction was carried out at 37°C and 1000 rpm for 16 h using a temperature-controlled stirrer (the protein-bead clumps were substantially completely dispersed by stirring after the addition of the digestive enzyme).

[0083] <1-1-4. Purification of Glycopeptides> ※ The magnetic particles added in the previous stage can also be used directly for glycopeptide purification.

[0084] First, the droplets are sank to the bottom by momentary centrifugation (using a small benchtop centrifuge).

[0085] First, add 200 μL of glycopeptide loading solution to hold the glycopeptides on the beads to stop the digestion reaction. As the glycopeptide loading solution (depending on conditions), acetonitrile (ACN) containing 1% (v / v) trifluoroacetic acid (TFA) is used under conditions A and E, while acetonitrile containing 3% (v / v) formic acid (FA) is used under conditions C and G.

[0086] Add 10 μL of a standard glycopeptide solution (1 μM sialic acid glycopeptide, 1 μM 2-peptide; manufactured by Tokyo Chemical Industry Co., Ltd.) and stir at 1000 rpm at 25°C for 30 seconds in a thermostatic mixer.

[0087] Next, glycopeptide loading solutions corresponding to each condition were added. The additional volumes were set as follows: Condition A: 900 μL, Condition C: 550 μL, Condition E: 400 μL, and Condition G: 240 μL. Glycopeptides were captured by shaking at 1500 rpm, 25°C, and 30 min in a constant-temperature stirrer. The droplets were then settled to the bottom by brief centrifugation. The test tube containing the sample solution was placed on a magnetic support, allowed to stand for 2 min, and then the supernatant was removed.

[0088] Next, a wash solution was added as a cleaning treatment. The solution was shaken at 1500 rpm at 25°C for 30 min. The droplets were then settled to the bottom by momentary centrifugation. The test tube containing the sample solution was placed on a magnetic support and allowed to stand for 2 min. The supernatant was then removed. This operation was repeated twice. The wash solution was 800 μL, and its composition varied depending on the conditions: Condition A: 90% acetonitrile containing 1% TFA; Condition C: 85% acetonitrile containing 3% formic acid; Condition E: 82.5% acetonitrile containing 1% TFA; Condition G: 77.5% acetonitrile containing 3% formic acid (concentration is volume percentage (v / v)).

[0089] Next, with the sample-bead test tube placed on the magnet holder, 800 μL of TFA Rinse Sol (98% (v / v) ACN) was added to remove TFA around the beads. After standing for 2 min, the supernatant was removed. Then, to elute the glycopeptides from the beads, 300 μL of neutral elution solution (30% (v / v) acetonitrile containing 20 mM ammonium formate) was added, and the mixture was shaken at 1000 rpm and 25°C for 10 min in a constant-temperature stirrer. After standing on the magnet holder for 5 min, the supernatant separated from the beads was collected, transferred to a new low-adsorption 1.5-mL polypropylene test tube, and dried by centrifugation.

[0090] <1-1-5. Redissolution of Glycopeptides and LC / MS Determination (Common Conditions)> The dried glycopeptide sample was redissolved in 60 μL of 0.5% (v / v) acetic acid aqueous solution and transferred to the inside of a filter (Millex LH 45 μm, 4 mm, Merck Millipore) installed on a low-adsorption 0.5-mL PP test tube. The sample was centrifuged at 2000×g, 25℃, for 2 min, and then filtered. 40 μL of 0.1% (v / v) acetic acid aqueous solution was then introduced into the filter for rinsing and sample dilution. The sample solution was then mixed using a vortex mixer to prepare a 1 / 10 diluted plasma glycopeptide solution (0.1 μL plasma / μL).

[0091] During the assay, a 1 / 10-dilute plasma glycopeptide solution was transferred into a 0.3-mL Proteosave-coated (AMR) HPLC sample vial made of low-adsorption TPX resin, and then diluted with 4 times the volume of 0.1% (v / v) formic acid aqueous solution to prepare a 1 / 50-dilute plasma glycopeptide solution, which was then placed in the automated sampler of the high-performance liquid chromatography (HPLC) system.

[0092] Analysis of the glycopeptide samples was performed using a liquid chromatography-mass spectrometry (LC-MS) method. The apparatus used was a nano-flow high-performance liquid chromatography (HPLC) system (UltiMate 3000 RSLCnano HPLC system, Thermo Fisher Scientific) and a quadrupole-Orbitrap mass spectrometer (Orbitrap Exploris 240, Thermo Fisher Scientific).

[0093] In HPLC, the mobile phases used were: A: ultrapure water containing 0.1% (v / v) formic acid; and B: acetonitrile containing 0.1% (v / v) formic acid. An integrated emitter-type nano-HPLC capillary column (C18, 0.075 × 150 mm, 3 μm; manufactured by Nikkyo Technos) was used. The flow rate for sample separation was set at 0.30 μL / min, and the column temperature was set at 40 °C. The proportion of mobile phase B was fixed at 0% for elution time 0–2 min, linearly increased to 1% for 2–2.5 min, linearly increased to 40% for 2.5–35 min, and linearly increased to 95% for 35–40 min.

[0094] In the mass spectrometer, the ion source settings are set as follows: Spray Voltage: 1800V, Ion Transfer Tube Temperature: 275℃.

[0095] Full MS scan settings: Orbitrap resolution: 120000, scan range: 500-1800 m / z, RF-Lens: 70%, normalized AGC target value: 300%, maximum injection time: 50 ms, number of microscans: 1, polarity: positive ion. Data dependent on MS. 2 The filter settings were set to MIPS: peptide, intensity threshold: 1.0e4, charge state: 2-8, dynamic exclusion duration: 20s, dynamic exclusion quality tolerance: high-10, low-10 ppm. Data-dependent MS2 (ddMS2) acquisition settings were set to data-related mode: cycle time (1.5s), isolation window: 1.6 m / z, collision energy type: normalized, HCD collision energy: 20, 30, 40 (%), Orbitrap resolution: 15000, scan range: 150-2000 m / z, normalized AGC target value: 200%, maximum injection time: 50ms.

[0096] Measurements were performed using Full MS and data-dependent MS, respectively. 2 For both ddMS2 and Full MS only, the sample injection volume was set at 5 μL (equivalent to 0.1 μL of plasma) for the former (data-dependent analysis: Full MS and ddMS2) and 2.5 μL (equivalent to 0.05 μL of plasma) for the latter (Full MS only).

[0097] The raw data files of the obtained LC / MS data were retrieved using Byonic (Protein Metrics), a peptide analysis software corresponding to glycopeptides, for glycopeptide identification. The data were then analyzed in conjunction with the data from Progenesis QI for Proteomics (Waters) for glycopeptide identification and quantitative comparison.

[0098] <1-2. Results> <1-2-1. Comparison of the number of peptides identified by Byonic search> Figure 2 This represents the result of a comparison of the number of peptides identified using Byonic search. Figure 2In the figure, the number of peptides identified based on data-dependent MS2 assays under each condition is represented by glycopeptide (a peptide with N-binding glycan modification; blue bar) and non-glycopeptide (a peptide without N-binding glycan modification; red bar).

[0099] Overall, the number of glycopeptides detected using cellulose magnetic particles (E, G) showed slightly higher values ​​compared to the number of glycopeptides detected using carboxyl-modified magnetic particles (A, C).

[0100] In addition, in glycopeptide detection, the number of non-glycopeptides (peptides without added N-binding glycans) that act as interfering substances showed a lower value when using cellulose magnetic particles (E, G), indicating that higher glycopeptide purification was achieved.

[0101] In HILIC purification using carboxyl-modified magnetic particles, the addition of TFA (A) showed a higher number of glycopeptide identifications and a lower number of non-glycopeptide identifications compared to the addition of formic acid (C), indicating that higher glycopeptide purification was achieved.

[0102] In HILIC purification using cellulose magnetic particles, the addition of TFA (E) showed a slightly higher number of glycopeptide identifications compared to the addition of formic acid (G), but no significant difference was observed.

[0103] Furthermore, the concentration of acetonitrile (ACN) used varies depending on the magnetic particles (carboxyl-modified polymer magnetic particles or cellulose magnetic particles) and the added acid (1% TFA or 3% FA). The acetonitrile concentration is set to an optimal (presumably close to) concentration to achieve higher glycopeptide recovery and higher non-glycopeptide removal rates.

[0104] Washing with a lower concentration of acetonitrile improves the removal efficiency of non-glycopeptides and enhances the purification of glycopeptides.

[0105] On the other hand, if the acetonitrile concentration decreases, the retention of glycopeptides is also more likely to weaken.

[0106] Compared to carboxyl-modified magnetic particles, cellulose magnetic particles may exhibit a greater difference in acetonitrile concentration between non-glycopeptides and glycopeptides, potentially leading to improved glycopeptide purification.

[0107] <1-2-2. Comparison of Peptide Detection Amounts> Data from data-dependent analyses and Full MS-only assays under various conditions were compared using Progenesis QI for proteomics. A Byonic search was used to retrieve peptides containing N-binding glycan modifications, enabling peptide identification and quantification of Full MS-only assays. Based on these quantifications, quantitative comparisons were performed between the different conditions.

[0108] Figure 3 This represents the total amount of glycopeptides detected, including those modified with N-binding glycans.

[0109] No significant difference was observed in the total values ​​of the glycopeptide detection signals.

[0110] Overall, the detection values ​​were slightly higher when using cellulose magnetic particles (E, G) compared to when using carboxyl-modified magnetic particles (A, C), indicating that higher glycopeptide recovery was achieved.

[0111] Figure 4 This represents the total amount of non-glycopeptides detected without N-binding glycan modifications. In glycopeptide detection, this is the total detection signal of non-glycopeptides that act as interfering substances.

[0112] Significant differences were observed based on the conditions.

[0113] Overall, the detection values ​​were lower when using cellulose magnetic particles (E, G) compared to when using carboxyl-modified magnetic particles (A, C), indicating that higher glycopeptide purification was achieved (by being of higher purification, it is believed to lead to an increase in the number of glycopeptide identifications in data-dependent MS2 assays).

[0114] This indicates that, under TFA-HILIC purification conditions following on-bead digestion, cellulose magnetic particles (E) achieve a higher purification level compared to carboxyl-modified magnetic particles (A).

[0115] When using cellulose magnetic particles, the detection levels of non-glycopeptides are often lower. Specifically, the levels are slightly lower under TFA-HILIC purification conditions (E) following bead digestion compared to HILIC conditions (G) using FA (formic acid).

[0116] Figure 5 A representative example of the quantitative value of peptides derived from major blood glycoproteins.

[0117] like Figure 5As shown, under conditions A and E, respectively, bead digestion and TFA-based HILIC purification using carboxyl-modified magnetic particles and cellulose magnetic particles resulted in no significant difference in the overall glycopeptide yield. Even when individual glycopeptides from major glycoproteins in the blood were observed, the differences were small (ac). On the other hand, under conditions A and C with carboxyl-modified magnetic particles, the yield was low, while under conditions E and G with cellulose magnetic particles, higher levels of glycopeptides were observed (df). Furthermore, the non-glycopeptide fraction from the glycoprotein (fibrinogen alpha chain) was abundant under conditions A and C with carboxyl-modified magnetic particles, but less abundant under conditions E and G with cellulose magnetic particles, particularly under condition E with TFA, where the detection amount was very low (g).

[0118] Furthermore, in the case of carboxyl-modified magnetic particles, higher acetonitrile concentrations are required for the retention of (glyco)peptide molecules. On the other hand, high acetonitrile concentrations have been reported to readily cause glycan aggregation, sometimes resulting in their loss during HILIC purification. This is particularly true for glycopeptides with large glycan chains and peptides with a high number of hydrophilic residues. Therefore, it is possible that these peptides may not be retained on the magnetic particles and may instead form fine aggregates that are removed during washing. The solubility of peptides is TFA > formic acid, so in the case of formic acid, more hydrophilic glycans may be lost (d: Oligomannose, f: Triantennary). Moreover, cellulose magnetic particles are operated at lower acetonitrile concentrations, and therefore such aggregation is considered less likely to occur.

[0119] Experimental Example 2. Comparison of cellulose digestion by magnetic particles, beads, and solution. <2-1. Methods> The digestion methods were compared. Three samples were used in parallel for each condition. A summary of each condition is shown in Table 2, and a summary of the experimental procedures is shown in [Table 3]. Figure 6 .

[0120] [Table 2] The bead-on protein digestion and glycopeptide purification under condition E were performed in the same manner as in Example 1. The re-dissolution of the glycopeptides and LC / MS determination were also performed in the same manner as in Example 1.

[0121] Protein digestion and glycopeptide purification in solution under condition F were performed as follows.

[0122] <2-1-1. Protein denaturation, reduction, and alkylation> As the starting sample, 10 μL of plasma, after albumin / IgG removal and centrifugation drying, was used. The procedure was performed in 1.5-mL polypropylene tubes (Protein Lobind, Eppendorf) of the low-absorption protein type. 50 μL of a surfactant-containing buffer was added to promote sample redissolution and digestion via denaturation, in a 2-fold concentrated state (4.8 mM sodium deoxycholate (SDC), 4.8 mM sodium lauroyl sarcosinate (SLS), 200 mM Tris-HCl, pH 8.5). 10 μL of 100 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl) for disulfide bond reduction and 10 μL of 400 mM chloroacetamide (CAA) for alkylation were added, along with 28 μL of ultrapure water for dilution. After redissolution, the sample was subjected to denaturation, reduction, and alkylation reactions by heat treatment (95 °C, 10 min) at 1000 rpm at 25 °C for 1 min.

[0123] <2-1-2. Digestion of proteins in solution> After cooling the sample solution to room temperature, add 10 μL of 20 mM CaCl2 for enzyme stabilization and 2 μL of enzyme solution (0.5 μg / μL trypsin / Lys-C mix, 50 mM acetic acid). Shake at 1000 rpm at 25°C for 1 min in a constant temperature stirrer. After stirring the sample, allow it to stand at 37°C for 16 h for enzyme digestion.

[0124] <2-1-3. HILIC purification of glycopeptides> First, the droplets were briefly centrifuged (using a benchtop centrifuge) to allow them to settle to the bottom. Then, 20 μL of a 50 μg / μL magnetic particle mixture was added. The magnetic particles used were ReliaPrep Resin (equivalent to 1 mg of magnetic particles; manufactured by Promega).

[0125] First, 200 μL of glycopeptide loading solution (acetonitrile (ACN) containing 1% (v / v) trifluoroacetic acid (TFA)) was added to hold the glycopeptides on the beads, stopping the digestion reaction. Then, 10 μL of a standard glycopeptide solution (1 μM sialic acid glycopeptide, 1 μM G2-glycopeptide; manufactured by Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was stirred at 1000 rpm at 25°C for 30 seconds in a temperature-controlled stirrer.

[0126] Next, 510 μL of glycopeptide loading solution was added. The glycopeptides were captured by shaking at 1500 rpm, 25°C, and 30 min in a constant-temperature stirrer. The droplets were then settled to the bottom by momentary centrifugation. The test tube containing the sample solution was placed on a magnetic support and allowed to stand for 2 min before the supernatant was removed.

[0127] Then, as a cleaning treatment, 800 μL of washing solution (82.5% (v / v) acetonitrile containing 1% TFA) was added, and the mixture was shaken at 1500 rpm at 25°C for 30 min. After a brief centrifugation, the droplets were allowed to settle to the bottom. The test tube containing the sample solution was placed on a magnet holder and allowed to stand for 2 min. The supernatant was then removed, and this operation was repeated twice.

[0128] Next, with the sample-bead test tube placed on the magnet holder, 800 μL of TFA Rinse Sol (98% (v / v) ACN) was added to remove TFA around the beads. After standing for 2 min, the supernatant was removed. Then, to elute the glycopeptides from the beads, 300 μL of neutral elution solution (30% (v / v) acetonitrile containing 20 mM ammonium formate) was added, and the mixture was shaken at 1000 rpm at 25°C for 10 min in a constant-temperature stirrer. After standing on the magnet holder for 5 min, the supernatant separated from the beads was collected and transferred to a new low-adsorption 1.5-mL polypropylene test tube, and the solution was dried by centrifugation.

[0129] <2-2. Results> Figure 7 This represents the total amount of non-glycopeptides detected without N-binding glycan modifications.

[0130] In the detection of glycopeptides, among the total detection signals of non-glycopeptides that act as interfering substances, the value of digestion on beads (condition E) is lower than that of digestion in solution (F).

[0131] Figure 8 This photo shows what the glycopeptide looks like during HILIC purification.

[0132] Protein digestion was performed in deep-well plates (Protein LoBind Plates, Eppendorf). The digestion reaction was stopped by adding 200 μL of a glycopeptide loading solution (1% (v / v) trifluoroacetic acid (TFA) in acetonitrile (ACN)). Then, 10 μL of a standard glycopeptide solution was added, followed by a final addition of 310 μL of the glycopeptide loading solution. The mixture was then agitated at 1500 rpm in a temperature-controlled stirrer. No overflow occurred in the deep wells despite the agitation at 1500 rpm.

[0133] Test Example 3. Particle Sedimentation Test Suspensions of carboxyl-modified polymer magnetic particles and cellulose magnetic particles were added dropwise to plastic petri dishes and observed using an inverted microscope. The magnified microscopic image is shown below. Figure 9Compared to carboxyl-modified polymer magnetic particles with a particle size of about 1 μm, cellulose magnetic particles form larger particle blocks.

[0134] A container containing a suspension of cellulose magnetic particles (Table 1 of Experimental Example 1) was placed on a magnet holder. The supernatant (water, the original preservation solution, containing <0.1% sodium azide) was separated and removed. After removal from the magnet holder, glycerol aqueous solutions of various concentrations were added and stirred. Photographs of the container's appearance after stirring for 1 minute, 6 minutes, and 10 minutes are shown in the table. Figure 9 The addition of glycerol inhibits particulate precipitation. Furthermore, the higher the glycerol concentration, the greater the effect of inhibiting particulate precipitation.

[0135] Experimental Example 4. Study on the combined effect of ion-pair reagents As the starting sample, 2.5 μL of untreated serum (commercially available, purchased from Cosmo Bio) and a sample treated with serum albumin and immunoglobulin removal resin (High-Select HSA / Immunoglobulin Depletion Resin; purchased from Thermo Fisher) (HSA & Ig-dep serum, equivalent to 10 μL) were used.

[0136] [Protein denaturation, reduction, and alkylation] The samples were centrifuged and dried before preparation using 1.5-mL polypropylene tubes (Protein Lobind, Eppendorf) of low protein adsorption type. A solution for protein denaturation, disulfide bond reduction, and alkylation was added (1% (w / v) sodium dodecyl sulfate (SDS), 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP-HCl), 40 mM chloroacetamide (CAA)). The samples were reconstituted by shaking at 1000 rpm at 25°C for 1 min in a thermostatic stirrer, followed by denaturation, reduction, and alkylation reactions via heat treatment (95°C, 10 min).

[0137] [Protein Cleaning via Magnetic Particles] After cooling the sample solution to room temperature, add 50 μg / μL of cellulose magnetic particle suspension (Relia Prep Resin; Promega). The amount added is approximately 10 times the weight of the sample protein, with 40 μL (equivalent to 2 mg) for untreated serum and 30 μL (equivalent to 1.5 mg) for HSA & Ig-dep serum. Mix the solution by shaking at 1000 rpm at 25°C for 1 min in a thermostatic stirrer. Next, to induce protein aggregation on the beads, add ethanol (final concentration 80% v / v; 560 μL for untreated serum and 520 μL for HSA & Ig-dep serum), and shake at 1000 rpm at 25°C for 10 min in a thermostatic stirrer. After placing the test tube containing the sample solution on a magnetic support and allowing it to stand for 2 min, remove the supernatant using a pipette while the magnetic beads have aggregated on the test tube wall. To perform the cleaning, 800 μL of 80% (v / v) ethanol was added, and the mixture was allowed to stand for 2 minutes before removing the supernatant. This cleaning procedure was repeated 3 times.

[0138] [Digestion of proteins on magnetic particles] Next, 50 μL of a surfactant-containing buffer solution, used to promote sample redissolution and digestion, was added at a 2-fold concentration (4.8 mM SDC, 4.8 mM SLS, 200 mM Tris-HCl, pH 8.5). The sample was resuspended by shaking at 2000 rpm at 25°C for 5 min in a thermostatic stirrer. Then, 50 μL of an enzyme solution for protein digestion (0.02 μg / μL trypsin / Lys-C mix, 4 mM CaCl2, 2 mM acetic acid (acetic acid derived from the enzyme redissolution solution)) was added, and the digestion reaction was carried out at 37°C for 16 h in a thermostatic stirrer at 1000 rpm.

[0139] [Purification of Glycopeptides] The composition of the glycopeptide loading solution used to hold the glycopeptides on the beads and the washing solution used in the washing process varies depending on the ion-pairing reagents added therein.

[0140] The HILIC purification solutions used to compare the ion pairs of untreated serums with added washing solution reagents are shown in Table 3.

[0141] (1) Add 1% TFA.

[0142] (2) Add 5% formic acid.

[0143] (3) Add 5% formic acid and 0.1% TFA (for glycopeptide loading solution, only formic acid is added).

[0144] [Table 3] The HILIC purification solutions used to compare untreated serum and HSA&Ig-removed serum with added ion-pairing reagents are shown in Table 4.

[0145] (1) Add 1% TFA.

[0146] (2) Add 5% formic acid and 0.1% TFA.

[0147] [Table 4] First, the droplets are sank to the bottom by momentary centrifugation (using a small benchtop centrifuge).

[0148] First, add 100 μL of glycopeptide loading solution corresponding to the various conditions used to hold the glycopeptides on the beads to stop the digestion reaction. Then add 10 μL of standard glycopeptide (sialic acid glycopeptide; Tokyo Chemical Industries) solution (untreated serum: 1 μM, HSA & Ig-dep serum: 2 μM) and stir at 1000 rpm at 25°C for 30 seconds in a temperature-controlled stirrer.

[0149] Then, additional glycopeptide loading solutions corresponding to each condition were added. The total amount added is shown in Tables 3 and 4. Glycopeptides were captured by shaking at 1500 rpm, 25°C, and 30 min in a constant-temperature stirrer. The droplets were settled to the bottom by momentary centrifugation, and the test tube containing the sample solution was placed on a magnetic support. After standing for 2 min, the supernatant was removed.

[0150] Then, as a cleaning treatment, a cleaning solution was added, and the mixture was shaken at 1500 rpm, 25°C, and 30 min. The droplets were then settled to the bottom by instantaneous centrifugation. The test tube containing the sample solution was placed on a magnet support, and after standing for 2 min, the supernatant was removed. This operation was repeated twice.

[0151] Next, with the sample-bead test tube placed on the magnet holder, 800 μL of TFA Rinse Sol (98% (v / v) CAN) was added to remove TFA around the beads. After standing for 2 min, the supernatant was removed. Then, to elute the glycopeptides from the beads, 300 μL of neutral eluent (30% (v / v) acetonitrile containing 20 mM ammonium formate) was added, and the mixture was shaken at 1000 rpm, 25°C, for 10 min in a constant-temperature stirrer. After standing on the magnet holder for 5 min, the supernatant separated from the beads was collected and transferred to a new low-adsorption 1.5-mL polypropylene test tube, and the solution was dried by centrifugation.

[0152] [Redissolution of glycopeptides, LC / MS determination (common under all conditions)] The dried glycopeptide sample was redissolved in 50 μL of a 1% (v / v) acetic acid and 2% (v / v) acetonitrile aqueous solution. The solution was then transferred to the filter (Millex LH 45 μm, 4 mm, Merck Millipore) mounted on a low-adsorption 0.5-mL PP tube and centrifuged at 2000×g, 25℃, for 2 min to separate the solution and filter. For assaying, the glycopeptide solution was transferred to a 96-well plate (Sarstedt) for low-adsorption protein PCR and diluted with 0.1% (v / v) formic acid aqueous solution to prepare a 1 / 50 diluted plasma glycopeptide solution, which was then loaded into an automated sampler for high-performance liquid chromatography (HPLC).

[0153] The analysis of glycopeptide samples was performed using a liquid chromatography-mass spectrometry (LC-MS) based method. The apparatus used was a Vanquish-Neo UHPLC system (Thermo Fisher Scientific) and an Orbitrap quadrupole-Orbitrap mass spectrometer (Orbitrap Exploris 240, Thermo Fisher Scientific).

[0154] In HPLC, the mobile phases used were: A: ultrapure water containing 0.1% (v / v) formic acid; and B: acetonitrile containing 0.1% (v / v) formic acid. An integrated emitter-type nano-HPLC capillary column (C18, 0.075 × 150 mm, 3 μm; manufactured by Nikkyo Technos) was used. The flow rate for sample separation was set at 0.30 μL / min, and the column temperature was set at 40 °C. The proportion of mobile phase B was fixed at 0% for elution time 0–2 min, linearly increased to 1% for 2–2.5 min, linearly increased to 44.5% for 2.5–60.5 min, and linearly increased to 95% for 60.5–65.5 min.

[0155] In the mass spectrometer, the settings for the ion source are: spray voltage: 1800V, ion transfer tube temperature: 275℃.

[0156] Full MS scan settings: Orbitrap resolution: 120000, scan range: 500-1800 m / z, RF-Lens: 70%, normalized AGC target value: 300%, maximum injection time: 50 ms, microscans: 1, polarity: positive ion. Data-dependent MS2 filter settings: MIPS: peptide, intensity threshold: 1.0e4, charge state: 2-8, dynamic exclusion duration: 20 s, dynamic exclusion mass tolerance: high -10, low -10 ppm. Data-dependent MS2 (ddMS2) acquisition settings: Data-related mode: cycle time (1.5 s), isolation window: 1.6 m / z, collision energy type: normalized, HCD collision energy: 20, 30, 40 (%), Orbitrap resolution: 30000, scan range: 150-2000 m / z, normalized AGC target value: 200%, maximum injection time: 75 ms. The raw LC / MS data files were used for glycopeptide identification using peptide analysis software corresponding to the glycopeptides and byonic (Protein Metrics) search. Furthermore, for glycopeptide identification and quantitative comparison, data comparison using Proteome Discoverer (Thermo Fisher) combined with Byonic search was performed.

[0157] [result] • As for the identification number of glycopeptides, the washing solution with only TFA (1%) as the ion-pairing reagent had the highest number, while the washing solution with only formic acid (5%) had the lowest number. The washing solution with a mixture of formic acid and TFA (5% and 0.1%) had a higher identification number than the washing solution with only formic acid. Figure 10 ).

[0158] Regarding the recovery rate of glycopeptides, firstly, in the samples where abundance proteins (serum albumin and immunoglobulins) were removed (HSA & Ig-dep), the type of ion-pairing reagent had little effect on the HILIC cleaning solvent. Figure 11 (a) and (b), portions of HSA & Ig-dep.

[0159] • In untreated serum, for glycopeptides with highly hydrophilic peptide chains, the ion pair reagent has little effect on the washing solvent (e.g., complement C3 [74-94] Hex2HexNAc6; Figure 11 (a)).

[0160] On the other hand, it was found that glycopeptides containing many hydrophobic residues were poorly recovered in washing solvents with only TFA added, and the recovery rate was significantly improved by using a mixed washing solvent of formic acid and TFA (e.g., α1-antitrypsin [94-125] Hex5HexNAc4NeuAc2; Figure 11 (b)).

[0161] The results are summarized below.

[0162] (1) By using an acetonitrile-based HILIC cleaning solvent in which formic acid and TFA are mixed, the recovery rate of glycopeptides prepared from untreated serum / plasma was improved compared with that of a cleaning solvent in which only TFA is added as an ion-pairing reagent.

[0163] (2) The synergistic effect of combining TFA and formic acid was observed for the first time in the analysis of non-depleted serum.

[0164] (3) Although serum and plasma after abundance protein removal treatment have the advantage of easily improving the depth of analysis, they also have the problems of large amount of operation required for preparation and high cost.

[0165] (4) The analysis of untreated serum and plasma can be directly carried out from the sample to the glycopeptide preparation process, with high throughput and low cost, making it suitable for large-scale analysis.

[0166] (5) Through the study of ion-pairing reagents, the reliability of sample preparation for analysis of untreated serum and plasma has been further improved.

Claims

1. A method for manufacturing a glycopeptide sample, characterized in that, include: (3) The process of bringing the protein particles on the surface into contact with the protease. (4) The process of adding organic solvents after process (3), and (5) The process of cleaning the particles obtained in step (4), The particles are hydrophilic polymer-coated particles, and In step (4), an ion-pairing reagent is added together with the organic solvent.

2. The manufacturing method as described in claim 1, characterized in that: The hydrophilic polymer-coated particles are polysaccharide-coated particles.

3. The manufacturing method as described in claim 2, characterized in that: The polysaccharide-coated particles are cellulose-coated particles.

4. The manufacturing method as described in claim 1, characterized in that, include: (2) A process of contacting the protein with the particles in a solution containing an organic solvent before step (3) to keep the protein on the surface of the particles.

5. The manufacturing method as described in claim 4, characterized in that: In step (2), there is a step of dispensing a pre-prepared particle suspension containing the particles and a thickener.

6. The manufacturing method as described in claim 5, characterized in that: The thickener is glycerin.

7. The manufacturing method as described in claim 1, characterized in that: The hydrophilic polymer-coated particles are carboxylic acid polymer-coated particles.

8. The manufacturing method as described in claim 1, characterized in that: The hydrophilic polymer-coated particles are magnetic particles.

9. The manufacturing method as described in claim 1, characterized in that: The ion-pairing reagent is an organic acid.

10. The manufacturing method as described in claim 9, characterized in that: The ion-pairing reagent contains halogenated short-chain fatty acids.

11. The manufacturing method as described in claim 10, characterized in that: The ion-pairing reagent is a mixture containing halogenated short-chain fatty acids and non-halogenated short-chain fatty acids.

12. The manufacturing method as described in claim 1, characterized in that: The organic solvent is selected from at least one of alcohols, acetonitrile, and acetone.

13. The manufacturing method according to any one of claims 1 to 12, characterized in that: All processes are carried out within the same container.

14. The manufacturing method according to any one of claims 1 to 12, characterized in that: The glycopeptide sample is a sample used for mass spectrometry analysis.

15. A glycopeptide sample, characterized in that: It is obtained by the manufacturing method according to any one of claims 1 to 12.

16. A method for analyzing glycan modifications of proteins, characterized in that, include: The procedure for performing mass spectrometry analysis on the glycopeptide sample according to claim 15.

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