Method for producing glycopeptide sample

Abstract

Provided is a more efficient method for producing a glycopeptide sample with a higher ratio of glycopeptides to non-glycopeptides. The method for producing a glycopeptide sample includes: (3) a step of bringing particles that retain a protein on the surface thereof into contact 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). The particles are cellulose-coated particles. An ion pair agent is added together with the organic solvent in step (4).

Description

Method for producing glycopeptide samples

[0001] The present invention relates to a method for producing a glycopeptide sample.

[0002] In the field of bottom-up proteomics research, various pretreatment methods have been investigated to improve data quality (e.g., number of protein identifications, yield stability), streamline operations, and support automation. One such method is the single-pot, solid-phase-enhanced sample preparation (SP3) method, which uses magnetic particles with diameters ranging from several tens of nanometers to several micrometers to perform proteomics sample preparation in a single tube (Non-Patent Document 1). Proteins in the sample solution are retained on the magnetic particles by adding an organic solvent (e.g., ethanol or acetonitrile) to aggregate them, and after washing, they are subjected to the protein digestion process using digestive enzymes. Purification using magnetic particles is highly efficient, allowing the use and subsequent removal of strong denaturants, improving digestion efficiency. Peptides, the digestion products, can also be retained and purified on the magnetic particles using acetonitrile.

[0003] On the other hand, glycoproteomics, a branch of proteomics that focuses on glycosylation of proteins, is difficult to obtain comprehensive data by analyzing total peptides prepared from biological samples using conventional proteomics techniques, because the detection sensitivity of glycosylated peptides (glycopeptides) in mass spectrometry is generally lower than that of non-glycosylated peptides.

[0004] To improve the data quality of glycopeptide analysis, it is necessary to remove non-glycosylated peptides from the total peptide mixture generated by protein digestion and separate glycopeptides. The most commonly used purification method for selectively purifying glycopeptides is based on the principle of hydrophilic interaction liquid chromatography (HILIC), which utilizes the glycan moieties attached to glycopeptides for separation. Glycopeptides are retained on a hydrophilic stationary phase under high organic solvent conditions (mainly acetonitrile), and after washing, the glycopeptides are eluted with a solvent with a high water content. However, protein pretreatment and specific glycopeptide purification are performed independently, making this method unsuitable for efficient processing, especially for automated protocols for large-scale analysis.

[0005] Mol Syst Biol. 2014 Oct 30;10(10):757. doi: 10.15252 / msb.20145625. PMID: 25358341; PMCID: PMC4299378.

[0006] An object of the present invention is to provide a more efficient method for producing a glycopeptide sample having a higher ratio of glycopeptides to non-glycopeptides.

[0007] In view of the above-mentioned problems, the present inventors have conducted extensive research and have found that the above-mentioned problems can be solved by a method for producing a glycopeptide sample, comprising: (3) a step of contacting particles carrying proteins on their surfaces 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 agent is added together with the organic solvent in step (4). Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0008] Item 1. A method for producing a glycopeptide sample, comprising: (3) a step of contacting particles carrying proteins on their surfaces 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 hydrophilic polymer-coated particles, and an ion pairing agent is added together with the organic solvent in step (4).

[0009] Item 2. The method according to Item 1, wherein the hydrophilic polymer-coated particles are polysaccharide-coated particles.

[0010] Item 3. The method according to Item 2, wherein the polysaccharide-coated particles are cellulose-coated particles.

[0011] Item 4. The production method according to Item 1, further comprising the step of: (2) contacting the protein with particles in a solution containing an organic solvent before step (3), thereby retaining the protein on the particle surface.

[0012] Item 5. The manufacturing method according to Item 4, wherein step (2) comprises dispensing a pre-prepared particle suspension containing the particles and a thickener.

[0013] Item 6. The method according to Item 5, wherein the thickener is glycerol.

[0014] Item 7. The method according to Item 1, wherein the hydrophilic polymer-coated particles are carboxylic acid polymer-coated particles.

[0015] Item 8. The method according to Item 1, wherein the hydrophilic polymer-coated particles are magnetic particles.

[0016] Item 9. The method according to Item 1, wherein the ion pairing agent is an organic acid.

[0017] Item 10. The method according to Item 9, wherein the ion pairing agent comprises a halogenated short-chain fatty acid.

[0018] Item 11. The method according to Item 10, wherein the ion pairing agent is a mixture containing a halogenated short-chain fatty acid and a non-halogenated short-chain fatty acid.

[0019] Item 12. The production method according to Item 1, wherein the organic solvent is at least one selected from the group consisting of alcohol, acetonitrile, and acetone.

[0020] Item 13. The production method according to any one of Items 1 to 12, wherein all steps are carried out in the same vessel.

[0021] Item 14. The production method according to any one of Items 1 to 12, wherein the glycopeptide sample is a sample for mass spectrometry.

[0022] Item 15. A glycopeptide sample obtained by the production method according to any one of Items 1 to 12.

[0023] Item 16. A method for analyzing glycosylation of a protein, comprising a step of subjecting the glycopeptide sample according to Item 15 to mass spectrometry.

[0024] According to the present invention, a more efficient method for producing a glycopeptide sample having a higher ratio of glycopeptides to non-glycopeptides can be provided, and further, a method for analyzing protein glycan modifications using the glycopeptide sample can be provided.

[0025] This shows an outline of the experimental procedure for Test Example 1. This shows the results of a comparison of the number of peptides identified by Byonic search in Test Example 1. This shows the amount (total) of glycopeptides containing N-linked glycosylation detected in Test Example 1. This shows the amount (total) of non-glycopeptides not containing N-linked glycosylation detected in Test Example 1. This shows a representative example of the quantitative values ​​of peptides derived from major blood glycoproteins in Test Example 1. The amino acid sequences in the figure correspond to SEQ ID NOS: 1 to 6 in the Sequence Listing. This shows an outline of the experimental procedure for Test Example 2. This shows the amount (total) of non-glycopeptides not containing N-linked glycosylation detected in Test Example 2. This shows photographs depicting the state of glycopeptide purification by HILIC in Test Example 2. This shows micrographs of a carboxyl group-modified polymer magnetic particle suspension and a cellulose magnetic particle suspension, as well as photographs of the container appearance 1, 6, and 10 minutes after stirring in Test Example 3. This shows the composition of the HILIC wash solution and the number of identified N-linked glycopeptides prepared from untreated serum in Test Example 4. FIG. 1 shows the results of comparing the recovery amounts of N-linked glycopeptides prepared from serum (untreated, HSA- and Ig-removed) in Test Example 4. The amino acid sequences in the figure correspond to SEQ ID NOs: 7 to 8 in the Sequence Listing.

[0026] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0027] In one aspect, the present invention relates to a method for producing a glycopeptide sample (sometimes referred to herein as the "production method of the present invention"), which comprises: (3) a step of contacting particles carrying 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 agent is added together with the organic solvent in step (4).

[0028] The protein is not particularly limited as long as it is a protein for which analysis of fragmented peptides is desired. In a preferred embodiment, the protein is a protein derived from a biological sample of a subject. Examples of the biological species of the subject and the biological species from which the protein is derived include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, and preferably humans. The biological sample is not particularly limited as long as it contains a protein, and examples include body fluids, skin, mucous membranes, and internal tissues. Among these, from the viewpoints of ease of collection and minimal invasiveness, body fluids, skin, and mucous membranes are preferred, and body fluids are more preferred. Examples of body fluids include blood, follicular fluid, menstrual blood, saliva, cerebrospinal fluid, synovial fluid, urine, interstitial fluid, sweat, and tears. Examples of mucous membranes include oral mucosa and nasal mucosa. Biological samples are generally subjected to appropriate pretreatment depending on the sample, and treatments to remove some components and to concentrate and / or purify proteins are preferred. In one aspect of the present invention, from the viewpoint of efficiently processing multiple specimens, the biological sample is preferably a biological sample that has not been subjected to a process for removing abundant proteins (e.g., albumin (particularly serum albumin) and / or immunoglobulins (particularly immunoglobulin G)). Examples of such a removal process include processes that utilize chromatography, centrifugation, etc., taking advantage of differences in affinity, adsorptive property, solubility, etc. depending on the type of abundant protein. The removal process can be a process that removes, for example, 30% by mass or more, 40% by mass or more, 50% by mass or more, 60% by mass or more, 70% by mass or more, 80% by mass or more, 90% by mass or more, or 95% by mass or more of the abundant protein.

[0029] In one aspect of the present invention, the protein may be a protein contained in a blood sample or a sample obtained by removing some components contained in blood (e.g., albumin, antibodies such as IgG, etc.).

[0030] From the viewpoints of the efficiency of hydrolysis by proteases, suitability for mass spectrometry, etc., it is preferable that the protein has been subjected to a treatment such as denaturation, reduction, alkylation, etc. From this viewpoint, the production method of the present invention preferably includes the step of (1) subjecting the protein to at least one treatment selected from the group consisting of denaturation, reduction, and alkylation before step (3) (in a preferred embodiment, before step (2)).

[0031] The denaturation treatment is a treatment for unfolding the protein structure, and is not particularly limited as long as it is performed using a protein denaturant such as a surfactant. Examples of surfactants used in the denaturation treatment include anionic surfactants such as dodecyl sulfate, deoxycholate, and lauroyl sarcosinate. In the production method of the present invention, the protein is retained on the particle surface during treatment as in step (3), allowing for solution replacement. Therefore, components with strong denaturing properties that have a strong effect on protease activity can be used. Among the above components, alkyl sulfates such as dodecyl sulfate (e.g., sodium salt) are preferred.

[0032] The reduction treatment is a treatment for cleaving disulfide bonds within proteins and / or between proteins, and is not particularly limited as long as it is carried out using a reducing agent. Examples of reducing agents used in the reduction treatment include tris(2-carboxyethyl)phosphine hydrochloride, dithiothreitol, 2-mercaptoethanol, tributylphosphine, etc. Among these, tris(2-carboxyethyl)phosphine hydrochloride is preferred because of its strong denaturing effect.

[0033] The alkylation treatment is a treatment for irreversibly alkylating cysteine ​​residues in a protein, and is not particularly limited as long as it is carried out using an alkylating agent. Examples of alkylating agents used in the alkylation treatment include halogenated acetamides such as chloroacetamide and iodoacetamide. Among these, chloroacetamide is preferred.

[0034] More specifically, step (1) can be carried out by reacting the protein in a solution containing at least one selected from the group consisting of a surfactant, a reducing agent, and an alkylating agent. In one embodiment (particularly when a phosphine-based reducing agent is used as the reducing agent), a solution containing a reducing agent and an alkylating agent (and, if necessary, a surfactant) can be used. In another embodiment (particularly when a reducing agent based on the reaction of thiol groups (dithiothreitol, 2-mercaptoethanol, etc.) is used), a solution containing a reducing agent (and, if necessary, a surfactant) can be used before adding the alkylating agent.

[0035] When the solution in step (1) contains a surfactant, its concentration is, for example, 0.1 to 10 w / v%, preferably 0.2 to 5 w / v%, and more preferably 0.5 to 2 w / v%. When the solution in step (1) contains a reducing agent, its concentration is, for example, 1 to 100 mM, preferably 2 to 50 mM, and more preferably 5 to 20 mM. When the solution in step (1) contains an alkylating agent, its concentration is, for example, 5 to 200 mM, preferably 10 to 100 mM, and more preferably 20 to 60 mM. Water is usually used as the solvent.

[0036] The treatment temperature in step (1) is, for example, 15 to 100°C, preferably 40 to 100°C, more preferably 60 to 100°C, and even more preferably 80 to 98°C. The treatment time can be appropriately set depending on the treatment temperature, for example, 2 minutes to 3 hours. In the case of treatment at a high temperature (for example, 90°C or higher), the treatment time is preferably 2 minutes to 1 hour, more preferably 2 to 30 minutes, and preferably 5 to 15 minutes.

[0037] The particles are not particularly limited 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 metal particles such as gold, silver, copper, iron, aluminum, nickel, manganese, titanium, and oxides thereof; resin particles such as polystyrene and latex; and silica particles. The shape of the particles is not particularly limited, and examples include spheres, rectangular parallelepipeds, cubes, triangular pyramids, and shapes similar to these. The particles may be coated with other molecules or modified with functional groups (e.g., epoxy groups, amino groups, carboxy groups, azide groups, etc.). The average particle size of the particles is not particularly limited, and is, for example, 100 nm to 500 μm. In one embodiment, the average particle size of the particles is, for example, 100 nm to 10 μm, more preferably 300 nm to 5 μm, and even more preferably 500 nm to 3 μm. In one embodiment, the average particle size of the particles is, for example, 500 nm to 300 μm, preferably 1 μm to 200 μm, and more preferably 10 μm to 100 μm.

[0038] In the production method of the present invention, the particles used are preferably hydrophilic polymer-coated particles, more preferably polysaccharide-coated particles and / or carboxylic acid polymer-coated particles. The hydrophilic polymer is not particularly limited as long as it is a polymer having a hydrophilic functional group such as a carboxy group or a hydroxy group. The polysaccharide is not particularly limited, and examples thereof include cellulose, dextran, agarose, etc. The carboxylic acid polymer includes, for example, a styrene-acrylic acid copolymer, a glycidyl methacrylate polymer, etc.

[0039] In the glycopeptide purification process in step (4), the hydrophilic stationary phase, i.e., the hydroxyl and / or carboxyl groups constituting polysaccharide molecules, can be utilized as hydrophilic groups in HILIC, and therefore the presence or absence of glycans (difference in hydrophilic interactions) allows for more efficient extraction of glycopeptide samples. Polysaccharide-coated particles are preferred, with cellulose-coated particles being particularly preferred, from the viewpoint of more efficiently obtaining glycopeptide samples with a higher ratio of glycopeptides to non-glycopeptides and of achieving such effects without using highly corrosive reagents (e.g., TFA) as ion-pairing agents.

[0040] In the production method of the present invention, the particles are preferably magnetic particles. The magnetic particles may be electromagnet particles. This allows for simple and rapid replacement of the solution around the particles and makes the production method of the present invention more suitable for automated protocols for large-scale analysis.

[0041] The particles may be commercially available products as they are, or may be produced according to or in accordance with a known method.

[0042] Particles carrying proteins on their surfaces can be obtained according to or in accordance with known methods, for example, by adding an organic solvent to cause the proteins to aggregate on the particle surface. From this perspective, the production method of the present invention preferably includes (2) a step of contacting the proteins with the particles in a solution containing an organic solvent to carry out the protein retention on the particle surface prior to step (3).

[0043] In step (2), the protein and particles are dispensed into the same container, allowing them to come into contact within the container. During the course of research, the inventors focused on the problem of particles settling in the container containing the particles before dispensing, making the dispensing process inefficient. This problem is particularly pronounced when handling multiple samples (e.g., 10 or more, 20 or more, 50 or more, 70 or more, 90 or more) in an automated dispenser. Therefore, in order to prevent particle settling before dispensing, step (2) preferably includes dispensing a pre-prepared particle suspension containing the particles and a thickener. This prevents particle settling in the container containing the particles before dispensing, avoids or minimizes the need for stirring the particles to homogenize them before dispensing, and enables efficient dispensing.

[0044] The thickener is not particularly limited, but polymer-based thickeners (synthetic polymers, thickening polysaccharides, etc.) are preferably avoided from the viewpoint of minimizing their effect on glycopeptide preparation. From the viewpoint of suppressing the sedimentation rate of particles and facilitating removal, the thickener is preferably a relatively low molecular weight polyhydric alcohol, more preferably a polyhydric alcohol having a molecular weight of 50 to 300 (even more preferably 50 to 200, 60 to 150, 70 to 120, or 70 to 100). The polyhydric alcohol is, for example, a dihydric or trihydric alcohol. Examples of such polyhydric alcohols include preferably glycerol and propylene glycol, with glycerol being particularly preferred. The concentration (vol %) of the thickener in the particle suspension prepared in advance is not particularly limited as long as it can suppress the settling rate of the particles and gives the particle suspension a viscosity that allows it to be used in a dispensing operation. For example, in the case of glycerol, the concentration is preferably 5 to 90%, more preferably 10 to 90%, even more preferably 20 to 85%, still more preferably 30 to 85%, particularly preferably 40 to 80%, and especially preferably 50 to 80%.

[0045] The organic solvent used in step (2) is not particularly limited as long as it can retain the protein on the particle surface by protein aggregation, and examples thereof include C1-C4 alcohols such as ethanol (especially preferably ethanol and 2-propanol), acetonitrile, acetone, etc., and preferably C1-C4 alcohols, acetonitrile, etc. The concentration of the organic solvent in the solution in step (2) is, for example, 40 to 90 v / v%, preferably 70 to 90 v / v%, more preferably 75 to 85 v / v%, in the case of ethanol, and preferably 40 to 60%, more preferably 45 to 55 v / v%, in the case of acetonitrile.

[0046] The treatment temperature in step (2) is, for example, 10 to 40°C, preferably 15 to 30°C, and the treatment time is, for example, 2 to 30 minutes, preferably 5 to 15 minutes. During the treatment, the solution is preferably stirred, preferably shaken (for example, at 500 to 2000 rpm).

[0047] After step (2), it is preferable to wash the particles carrying the protein on their surface before step (3). The solution used in step (2) can be used as the washing solution. It is preferable to wash the particles multiple times (for example, 2 to 5 times, preferably 2 to 3 times).

[0048] After step (2) or washing, it is preferable to remove the solvent by drying or the like.

[0049] The protease used in step (3) is not particularly limited as long as it is capable of fragmenting proteins, and examples thereof include endoproteases such as trypsin, Asp-N, Lys-C, and Glu-C, and exopeptidases such as aminopeptidase and carboxypeptidase.

[0050] Step (3) is usually carried out in solution.

[0051] The protease concentration in the solution used in 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. When trypsin is used, the ratio of protease to protein is, for example, 1:1000 to 1:10 (v / v), preferably 1:200 to 1:50. The solution preferably contains a surfactant, for example, 2 to 30 mM, preferably 5 to 20 mM, to promote digestion and to facilitate resolubilization of the sample after drying in step (2) or after washing. Preferred surfactants are anionic surfactants with a carboxyl group (e.g., deoxycholate, lauroyl sarcosinate, etc.), which have relatively weak protein denaturing properties. The solution may also contain other components, such as a buffer and metal salts necessary for protease activity.

[0052] The treatment temperature in step (3) is, for example, 20 to 40°C, preferably 30 to 40°C, more preferably 35 to 39°C, and particularly preferably 36 to 38°C, and the treatment time is, for example, 30 minutes to 48 hours, preferably 8 to 24 hours. During the treatment, the solution is preferably stirred, preferably shaken (for example, at 500 to 2000 rpm).

[0053] In step (3), the particulate protein is fragmented to generate peptides (glycopeptides and non-glycopeptides).

[0054] In step (4), an organic solvent is added after step (3).

[0055] As the organic solvent used in step (4), acetonitrile is mainly used among those shown in step (2).

[0056] The amount and concentration of the organic solvent added in step (4) are not particularly limited, as long as the organic solvent concentration in the container is sufficient to retain the peptide on the particles (e.g., 70 to 100 v / v%, preferably 75 to 90 v / v%).

[0057] In step (4), it is preferable to add an ion-pairing agent together with the organic solvent in order to obtain a glycopeptide sample with a higher ratio of glycopeptides to non-glycopeptides more efficiently. The ion-pairing agent is not particularly limited as long as it is one used in peptide analysis. A wide variety of organic acids can be used as the ion-pairing agent. Fatty acids are suitable as organic acids, with short-chain fatty acids (fatty acids with 5 or fewer carbon atoms) being particularly suitable. Examples of short-chain fatty acids include formic acid, acetic acid, propionic acid, isobutyric acid, and butyric acid, with formic acid being particularly preferred. From the above perspective and in terms of ease of removal, halogenated short-chain fatty acids are preferred. Halogenated short-chain fatty acids are particularly preferred, with fluoroacetic acid (especially trifluoroacetic acid) being particularly preferred. Conventional (non-halogenated) short-chain fatty acids (e.g., formic acid, acetic acid) can also be used. The combined use of halogenated and non-halogenated short-chain fatty acids can improve the recovery rate of hydrophobic glycopeptides, especially when using biological samples that have not been treated to remove abundant proteins. In this case, the ratio of non-halogenated short-chain fatty acids to halogenated short-chain fatty acids (non-halogenated short-chain fatty acid mass / halogenated short-chain fatty acid mass) is, for example, 1 to 500, preferably 2 to 300, more preferably 5 to 200, even more preferably 10 to 100, more preferably 20 to 80, even more preferably 30 to 70, and even more preferably 40 to 60. When an ion pairing agent is added, it can be added so that the concentration of the ion pairing agent in the container becomes, for example, 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, the ion pairing agent is added so that the concentration becomes 1-5% v / v, and in the case of halogenated short-chain fatty acids such as trifluoroacetic acid, the ion pairing agent is added so that the concentration becomes 0.1-1% v / v.

[0058] The treatment temperature in step (4) is, for example, 10 to 40°C, preferably 15 to 30°C, and the treatment time is, for example, 5 minutes to 2 hours, preferably 15 to 45 minutes. During the treatment, the solution is preferably stirred, preferably shaken (for example, at 500 to 2000 rpm).

[0059] In step (5), the particles obtained in step (4) are washed. The solution used in step (2) can be used as the washing solution. Washing is preferably performed multiple times (e.g., 2 to 5 times, preferably 2 to 3 times). In the final stage of washing, washing is preferably performed with acetonitrile of a high concentration (preferably 90 v / v% or more, particularly preferably 95 v / v% or more).

[0060] After step (5), the peptides are preferably eluted from the particles. The elution solution may be a solution containing the above-mentioned organic solvent at a relatively low concentration (e.g., 50 v / v% or less, preferably 40 v / v% or less). Elution under neutral conditions, rather than acidic conditions, can suppress hydrolysis of sialic acid. The elution solution preferably contains ammonium formate, for example, at 3 to 30 mM, preferably 5 to 15 mM. The elution temperature is, for example, 10 to 40°C, preferably 15 to 30°C, and the elution time is, for example, 2 minutes to 1 hour, preferably 5 to 30 minutes. During the elution, the solution is preferably stirred, preferably shaken (e.g., at 500 to 2000 rpm).

[0061] After step (5) or the elution treatment, it is preferable to remove the solvent by drying or the like.

[0062] In the production method of the present invention, all steps can be carried out in the same vessel, making it particularly suitable for automated protocols for large-scale analysis.

[0063] The glycopeptide sample obtained by the production method of the present invention has a higher ratio of glycopeptides to non-glycopeptides. Therefore, the glycopeptide sample obtained by the production method of the present invention is suitable as a sample for use in analytical methods, particularly mass spectrometry, which generally have lower detection sensitivity for glycopeptides than for non-glycopeptides. Therefore, in one embodiment of the present invention, the glycopeptide sample obtained by the production method of the present invention can be subjected to mass spectrometry.

[0064] Mass spectrometry can be performed according to or in accordance with known methods. Liquid chromatography-mass spectrometry is preferred because it is suitable for analyzing glycopeptides.

[0065] The ionization method in mass spectrometry is not particularly limited, and examples of ionization methods for peptides include laser desorption ionization (LDI) and electrospray ionization (ESI).

[0066] The method for measuring the mass-to-charge ratio in mass spectrometry is not particularly limited, and for example, time-of-flight, magnetic deflection, quadrupole, ion trap, Fourier transform ion cyclotron resonance, tandem, etc. can be used.

[0067] The technology of the present invention can be used, for example, to search for glycopeptide biomarkers in biological samples (e.g., plasma, urine, cerebrospinal fluid, saliva, autopsy tissue, etc.). Furthermore, glycoproteomics is now an essential technology for the quality control of biopharmaceuticals, such as antibody drugs. The technology of the present invention also enables high-throughput evaluation of antibody drugs.

[0068] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0069] Test Example 1. Comparison of conditions for protein pretreatment and glycopeptide purification using magnetic particles <1-1. Method> Digestion, HILIC purification, and type of magnetic particle were compared. The starting sample used was 10 μL of plasma that had been treated to remove albumin and IgG and then centrifuged and dried. Three samples were used in parallel for each condition. An overview of each condition is shown in Table 1, and an outline of the experimental procedure is shown in Figure 1.

[0070]

[0071] 1-1-1. Protein Denaturation, Reduction, and Alkylation. The starting sample was 10 μL of albumin- and IgG-removed, centrifuge-dried plasma. The procedure was performed in a 1.5-mL low-protein-binding polypropylene tube (ProteinLobind, Eppendorf). A solution for denaturing the sample protein, reducing disulfide bonds, and alkylating it (1% (w / v) sodium dodecyl sulfate (SDS), 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), 40 mM chloroacetamide (CAA)) was added. The sample was redissolved by shaking in a thermomixer at 1,000 rpm at 25°C for 1 min, followed by heating at 95°C for 10 min for the denaturation, reduction, and alkylation reactions.

[0072] 1-1-2. Protein Washing with Magnetic Particles After cooling the sample solution to room temperature, 30 μL or 50 μg / μL magnetic particle mixture was added. The magnetic particles varied depending on the condition: Sera-Mag Speedbeads, Carboxylate-modified, Hydrophilic [E7] / Hydrophobic [E3] = 1:1 (25 μg / μL each, equivalent to 1.5 mg of magnetic particles; Cytiva) were used for conditions A and C, while ReliaPrep Resin (equivalent to 1.5 mg of magnetic particles; Promega) was used for conditions E and G. The solution was mixed by shaking at 1,000 rpm at 25°C for 1 min in a thermomixer. Subsequently, 520 μL of ethanol (final concentration 80% v / v) was added to aggregate the protein onto the beads, and the mixture was shaken at 1,000 rpm at 25°C for 10 min in a thermomixer. The tube containing the sample solution was placed on a magnetic stand and allowed to stand for 2 minutes. The magnetic particles were then collected on the tube wall by the magnet, and the supernatant was removed with a pipette. To wash the tube, 800 μL of 80% (v / v) ethanol was added and allowed to stand for 2 minutes, after which the supernatant was removed. This washing procedure was repeated twice. For the next wash, 800 μL of acetonitrile was added and allowed to stand for 2 minutes, after which the supernatant was removed. The tube was then left on an aluminum block heater at 45°C for 15 minutes with the lid open to evaporate the acetonitrile and dry the beads.

[0073] <1-1-3. Digestion of Proteins on Beads> Next, 50 μL of a 2x concentrated detergent buffer (4.8 mM SDC, 4.8 mM SLS, 200 mM Tris-HCl pH 8.5) was added to the sample for reconstitution and digestion. The sample was resuspended by shaking in a thermomixer at 2,000 rpm at 25°C for 5 min (in most cases, protein-bead aggregates are not completely dispersed at this stage). 50 μL of enzyme solution for protein digestion (0.02 μg / μL Trypsin / Lys-C mix, 4 mM CaCl2, 2 mM acetic acid (acetic acid comes from the enzyme reconstitution solution)) was added, and the digestion reaction was carried out in a thermomixer at 1,000 rpm at 37°C for 16 h (stirring after addition of the digestive enzyme almost completely disperses the protein-bead aggregates).

[0074] <1-1-4. Purification of glycopeptides> *The magnetic particles added in the previous step were also used for purifying glycopeptides.

[0075] First, droplets were dropped down by flash centrifugation (tabletop mini centrifuge).

[0076] The digestion reaction was stopped by adding 200 μL of glycopeptide loading solution to retain glycopeptides on the beads. The glycopeptide loading solution (varied depending on the condition): acetonitrile (ACN) containing 1% (v / v) trifluoroacetic acid (TFA) for conditions A and E, and acetonitrile containing 3% (v / v) formic acid (FA) for conditions C and G.

[0077] To this was added 10 μL of a standard glycopeptide solution (1 μM sialylglycopeptide, 1 μM G2-peptide; Tokyo Chemical Industry Co., Ltd.), and the mixture was stirred in a thermomixer at 1,000 rpm, 25°C, and for 30 seconds.

[0078] Glycopeptide Loading Solution was then added according to the conditions. The amounts added were: Condition A: 900 μL, Condition C: 550 μL, Condition E: 400 μL, and Condition G: 240 μL. Glycopeptides were captured by shaking in a thermomixer at 1,500 rpm at 25°C for 30 min. Droplets were removed by flash centrifugation, and the tube containing the sample solution was placed on a magnetic stand and left to stand for 2 min before the supernatant was removed.

[0079] The tubes were washed twice with wash solution, shaken at 1,500 rpm at 25°C for 30 min, and then centrifuged to remove droplets. The tubes were then placed on a magnetic stand and allowed to stand for 2 min before the supernatant was removed. The wash solution was 800 μL and had different compositions depending on the condition: Condition A: 90% acetonitrile containing 1% TFA; Condition C: 85% acetonitrile containing 3% formic acid; Condition E: 82.5% acetonitrile containing 1% TFA; and Condition G: 77.5% acetonitrile containing 3% formic acid (concentrations by volume (v / v)).

[0080] Next, while the tube containing the sample and beads was still placed on the magnetic stand, 800 μL of TFA Rinse Sol. (98% (v / v) ACN) was added to remove the TFA surrounding the beads. After 2 min of incubation, the supernatant was removed. 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 in a thermomixer at 1,000 rpm at 25°C for 10 min. After 5 min of incubation on the magnetic stand, the supernatant separated from the beads was collected and transferred to a new 1.5-mL low-binding polypropylene tube. The solution was then dried in a centrifugal dryer.

[0081] 1-1-5. Reconstitution of Glycopeptides and LC / MS Measurement (Common to All Conditions) The dried glycopeptide sample was reconstituted in 60 μL of 0.5% (v / v) aqueous acetic acid, transferred to a filter (Millex LH 45 μm, 4 mm, Merck Millipore) attached to a low-binding 0.5-mL PP tube, and filtered by centrifugation at 2,000 × g, 25°C, for 2 min. The filter was then rinsed and diluted with 40 μL of 0.1% (v / v) aqueous acetic acid. The sample solution was mixed by vortexing to prepare a 1 / 10-diluted plasma glycopeptide solution (0.1 μL plasma / μL).

[0082] For the measurement, the 1 / 10 diluted plasma glycopeptide solution was transferred to a low-adsorption TPX resin HPLC vial (0.3 mL Proteosave-coated, AMR), and further diluted with four times the volume of 0.1% (v / v) formic acid aqueous solution to obtain a 1 / 50 diluted plasma glycopeptide solution, which was then placed in the autosampler of the high-performance liquid chromatography (HPLC).

[0083] Glycopeptide samples were analyzed using a liquid chromatography-mass spectrometry-based technique using a nanoflow 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).

[0084] The mobile phases used for HPLC were ultrapure water containing 0.1% (v / v) formic acid (A) and acetonitrile containing 0.1% (v / v) formic acid (B). A nano-HPLC capillary column (C18, 0.075 × 150 mm, 3 μm; Nikkyo Technos) with an integrated emitter was used. The flow rate during sample separation was 0.30 μL / min, and the column temperature was set at 40°C. The proportion of mobile phase B was fixed at 0% from 0 to 2 min, increased linearly to 1% from 2 to 2.5%, 40% from 2.5 to 35 min, and 95% from 35 to 40 min.

[0085] In the mass spectrometer, the ion source was set to a spray voltage of 1800 V and an ion transfer tube temperature of 275°C.

[0086] Full MS scan was performed with Orbitrap Resolution: 120,000, Scan Range: 500-1800 m / z, RF Lens: 70%, Normalized AGC Target: 300%, Maximum Injection Time: 50 ms, Microscans: 1, and Positive Polarity. Data-dependent MS 2 The filter settings were: MIPS: Peptide, Intensity Threshold: 1.0e4, Charge state: 2-8, Dynamic Exclusion duration: 20 s, Dynamic Exclusion Mass Tolerance: High-10, Low-10 ppm. The data-dependent MS2 (ddMS2) acquisition settings were: Data Dependent mode: Cycle time (1.5 s), Isolation window: 1.6 m / z, Collision Energy Type: Normalized, HCD collision energy: 20, 30, 40 (%), Orbitrap resolution: 15,000, Scan range: 150-2000 m / z, Normalized AGC target: 200%, Maximum injection time: 50 ms.

[0087] Measurements were performed using Full MS and data-dependent MS 2 The sample injection volume was set to 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).

[0088] The raw files of the obtained LC / MS data were searched using Byonic (Protein Metrics), a peptide analysis software compatible with glycopeptides, to identify glycopeptides, and analyzed using a combination of data alignment and Byonic search using Progenesis QI for Proteomics (Waters) to identify glycopeptides and perform quantitative comparison.

[0089] <1-2. Results> <1-2-1. Comparison of the number of peptides identified by Byonic search> Figure 2 shows the results of a comparison of the number of peptides identified by Byonic search. In Figure 2, the number of peptides identified based on data-dependent MS2 measurements under each condition is shown for glycopeptides (peptides with N-linked glycan modifications; blue bars) and non-glycopeptides (peptides without N-linked glycan modifications; red bars).

[0090] Overall, the number of glycopeptides detected was slightly higher when cellulose magnetic particles were used (E, G) than when carboxyl group-modified magnetic particles were used (A, C).

[0091] Furthermore, the number of identified non-glycopeptides (peptides without N-linked glycans), which are interfering substances in glycopeptide detection, was lower in the samples using cellulose magnetic particles (E, G), indicating that a higher degree of glycopeptide purification was achieved.

[0092] In HILIC purification using carboxyl group-modified magnetic particles, the addition of TFA (A) resulted in a higher number of identified glycopeptides and a lower number of identified non-glycopeptides than the addition of formic acid (C), demonstrating that a higher degree of glycopeptide purification was achieved.

[0093] In HILIC purification using cellulose magnetic particles, the number of identified glycopeptides was slightly higher when TFA was added (E) than when formic acid was added (G), but no dramatic difference was observed.

[0094] The acetonitrile (ACN) concentration used differed depending on the magnetic particles (carboxyl group-modified polymer magnetic particles or cellulose magnetic particles) and the acid added (1% TFA or 3% FA). The acetonitrile concentration was set to (presumably close to) the optimum to achieve higher glycopeptide recovery rates and higher non-glycopeptide removal rates.

[0095] Washing with a lower acetonitrile concentration increases the efficiency of removing non-glycopeptides, thereby increasing the degree of glycopeptide purification.

[0096] On the other hand, when the acetonitrile concentration is low, the retention of glycopeptides tends to weaken.

[0097] It is likely that cellulose magnetic particles have a larger difference between the acetonitrile concentration at which non-glycopeptides are no longer retained and the acetonitrile concentration at which glycopeptides are sufficiently retained, compared to carboxyl group-modified magnetic particles, making it easier to increase the degree of purification of glycopeptides.

[0098] <1-2-2. Comparison of peptide detection amounts> Data-dependent analysis and Full MS-only measurement data under each condition were aligned using Progenesis QI for proteomics, and peptides containing N-linked glycan modifications were searched for using Byonic search to identify and quantify peptides from Full MS-only measurement data. Quantitative comparisons between conditions were made based on these quantitative values.

[0099] Figure 3 shows the detected amounts (total values) of glycopeptides containing N-linked glycosylation.

[0100] No dramatic differences were observed in the combined detection signals of glycopeptides.

[0101] Overall, the detection values ​​were slightly higher when using cellulose magnetic particles (E, G) than when using carboxyl group-modified magnetic particles (A, C), indicating that higher glycopeptide recovery was achieved.

[0102] Figure 4 shows the detected amount (total value) of non-glycopeptides not containing N-linked glycosylation. This is the total detection signal of non-glycopeptides that act as interfering substances in glycopeptide detection.

[0103] There were significant differences depending on the conditions.

[0104] Overall, the detection values ​​were lower when using cellulose magnetic particles (E, G) than when using carboxyl group-modified magnetic particles (A, C), indicating that a higher degree of glycopeptide purification was achieved (it is thought that the higher degree of purification leads to an increase in the number of glycopeptide identifications in data-dependent MS2 measurements).

[0105] Under conditions where TFA-HILIC purification was performed after on-bead digestion, it was shown that cellulose magnetic particles (E) achieved a higher degree of purification than carboxyl group-modified magnetic particles (A).

[0106] When cellulose magnetic particles were used, the amount of non-glycopeptides detected tended to be lower, particularly under conditions (E) in which on-bead digestion was followed by TFA-HILIC purification, which was slightly lower than under HILIC conditions (G) using FA (formic acid).

[0107] Figure 5 shows representative examples of quantitative values ​​for peptides derived from major blood glycoproteins.

[0108] As shown in Figure 5, when on-bead digestion and TFA-based HILIC purification were performed using carboxyl-modified magnetic particles and cellulose magnetic particles (conditions A and E), there was no dramatic difference in the overall glycopeptide yield, and even when examining individual glycopeptides derived from major blood glycoproteins, the difference was small (ac). On the other hand, the yield was low under carboxyl-modified magnetic particle conditions A and C, and some glycopeptides were detected in higher amounts under cellulose magnetic particle conditions E and G (df). Furthermore, the non-glycopeptide portion derived from glycoproteins (fibrinogen alpha chain) was found to be higher under carboxyl-modified magnetic particle conditions (A and C) and lower under cellulose magnetic particle conditions E and G, with the amount detected being particularly low under TFA condition E (g).

[0109] Carboxylic acid-modified magnetic particles require higher concentrations of acetonitrile to retain (glyco)peptide molecules. However, it has been reported that high acetonitrile concentrations tend to aggregate glycan molecules, resulting in their loss during HILIC purification. This is particularly true for glycopeptides with large glycans and peptides with many hydrophilic residues. Therefore, some may not be retained on the magnetic particles and may form fine aggregates that are removed during washing. Because the peptide solubilization ability is greater with TFA than with formic acid, more hydrophilic glycans may be lost in formic acid (d: Oligomannose, f: Triantennary). Furthermore, cellulose magnetic particles are operated at lower acetonitrile concentrations, which may reduce the likelihood of such aggregation.

[0110] Test Example 2. Comparison of Cellulose Magnetic Particles, On-Bead Digestion and In-Solution Digestion <2-1. Method> Digestion methods were compared. Experimental procedures were carried out in parallel for three samples under each condition. An overview of each condition is shown in Table 2, and an overview of the experimental procedures is shown in Figure 6.

[0111]

[0112] On-Beads protein digestion and glycopeptide purification under condition E were carried out in the same manner as in Test Example 1. Redissolution of glycopeptides and LC / MS measurement were carried out in the same manner as in Test Example 1.

[0113] Condition F In Solution protein digestion and glycopeptide purification were performed as follows.

[0114] 2-1-1. Protein Denaturation, Reduction, and Alkylation. The starting sample was 10 μL of albumin- and IgG-removed, centrifuge-dried plasma. The procedure was performed in a 1.5-mL low-protein-binding polypropylene tube (ProteinLobind, Eppendorf). 50 μL of a 2x-concentrated detergent buffer (4.8 mM sodium deoxycholate (SDC), 4.8 mM sodium lauroyl sarcosinate (SLS), 200 mM Tris-HCl pH 8.5) was added to the tube for sample reconstitution and digestion. 10 μL of 100 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) for disulfide bond reduction and 10 μL of 400 mM chloroacetamide (CAA) for alkylation were added, followed by 28 μL of ultrapure water for dilution. The sample was redissolved by shaking in a thermomixer at 1,000 rpm at 25°C for 1 minute, and then subjected to denaturation, reduction, and alkylation reactions by heat treatment (95°C, 10 minutes).

[0115] <2-1-2. Digestion of proteins in solution> After the sample solution was cooled to approximately room temperature, 10 μL of 20 mM CaCl2 to stabilize the digestive enzyme and 2 μL of digestive enzyme solution (0.5 μg / μL Trypsin / Lys-C mix, 50 mM acetic acid) were added, and the sample was stirred by shaking in a thermomixer at 1,000 rpm at 25°C for 1 minute, and then the enzymatic digestion reaction was carried out in a stationary state at 37°C for 16 hours.

[0116] 2-1-3. HILIC Purification of Glycopeptides First, a droplet was dropped down by flash centrifugation (tabletop mini centrifuge). 20 μL of a 50 μg / μL magnetic particle mixture solution was added. ReliaPrep Resin (equivalent to 1 mg of magnetic particles; manufactured by Promega) was used as the magnetic particles.

[0117] To the mixture, 200 μL of Glycopeptide Loading Solution (1% (v / v) trifluoroacetic acid (TFA) in acetonitrile (ACN)) was added to quench the digestion reaction. Then, 10 μL of a standard glycopeptide solution (1 μM sialylglycopeptide, 1 μM G2-glycopeptide; Tokyo Chemical Industry Co., Ltd.) was added, and the mixture was stirred in a thermomixer at 1,000 rpm at 25°C for 30 seconds.

[0118] An additional 510 μL of Glycopeptide Loading Solution was added. The mixture was shaken in a thermomixer at 1,500 rpm at 25°C for 30 min to capture glycopeptides. The droplets were removed by flash centrifugation, and the tube containing the sample solution was placed on a magnetic stand and allowed to stand for 2 min before the supernatant was removed.

[0119] Further washing was performed by adding 800 μL of wash solution (82.5% (v / v) acetonitrile containing 1% TFA), shaking at 1,500 rpm at 25°C for 30 min, and then flash centrifugation to remove droplets. The tube containing the sample solution was then placed on a magnetic stand and allowed to stand for 2 min before removing the supernatant. This procedure was repeated twice.

[0120] Next, while the tube containing the sample and beads was still placed on the magnetic stand, 800 μL of TFA Rinse Sol. (98% (v / v) ACN) was added to remove the TFA surrounding the beads. After 2 min of incubation, the supernatant was removed. 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 in a thermomixer at 1,000 rpm at 25°C for 10 min. After 5 min of incubation on the magnetic stand, the supernatant separated from the beads was collected and transferred to a new 1.5-mL low-binding polypropylene tube. The solution was then dried in a centrifugal dryer.

[0121] 2-2. Results FIG. 7 shows the detected amounts (total values) of non-glycopeptides not containing N-linked glycosylation.

[0122] The total detection signal value of non-glycopeptides, which are interfering substances in glycopeptide detection, was lower in on-bead digestion (condition E) than in in-solution digestion (condition F).

[0123] FIG. 8 shows a photograph showing the state of glycopeptide purification by HILIC.

[0124] Protein digestion was performed in deep-well plates (Protein LoBind® Plates, Eppendorf), followed by the addition of 200 μL of Glycopeptide Loading Solution (acetonitrile (ACN) containing 1% (v / v) trifluoroacetic acid (TFA)) to terminate the digestion reaction. 10 μL of standard glycopeptide solution was then added, followed by 310 μL of Glycopeptide Loading Solution, and the mixture was shaken at 1,500 rpm in a thermomixer. The solution in the deep wells did not overflow during shaking at 1,500 rpm.

[0125] Test Example 3: Particle Sedimentation Test. Carboxyl-modified polymer magnetic particle suspensions and cellulose magnetic particle suspensions were dropped into plastic Petri dishes and observed under an inverted microscope. A magnified microscopic image is shown in Figure 9. Compared to carboxyl-modified polymer magnetic particles with a particle diameter of approximately 1 μM, the cellulose magnetic particles formed larger particle clumps. A container containing the cellulose magnetic particle suspension (Table 1 in Test Example 1) was placed on a magnetic stand, and the supernatant (original storage solution: water, containing <0.1% sodium azide) was separated and removed. After removing the container from the magnetic stand, various concentrations of glycerol aqueous solutions were added and stirred. Figure 9 shows photographs of the container's appearance 1, 6, and 10 minutes after stirring. The addition of glycerol suppressed particle sedimentation. Furthermore, the effect of suppressing particle sedimentation improved with increasing glycerol concentration.

[0126] Test Example 4: Examination of the effect of combined use of ion-pairing agents Starting samples used were 2.5 μL of untreated serum (commercially available, purchased from Cosmo Bio) and the serum treated with serum albumin and immunoglobulin depletion resin (High-Select HSA / Immunoglobulin Depletion Resin; purchased from Thermo Fisher) (HSA&Ig-dep serum, equivalent to 10 μL).

[0127] [Protein Denaturation, Reduction, and Alkylation] Before preparation, samples were transferred to low-protein-binding 1.5-mL polypropylene tubes (ProteinLobind, Eppendorf) and centrifuged to dry. A solution for denaturing the sample proteins, reducing disulfide bonds, and alkylating them (1% (w / v) sodium dodecyl sulfate (SDS), 10 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), and 40 mM chloroacetamide (CAA)) was added. The samples were redissolved by shaking at 1,000 rpm at 25°C for 1 min in a thermomixer, and then heated at 95°C for 10 min to carry out the denaturation, reduction, and alkylation reactions.

[0128] [Washing of Proteins with Magnetic Particles] After cooling the sample solution to room temperature, a 50 μg / μL cellulose magnetic particle suspension (ReliaPrep Resin; Promega) was added. The amount of magnetic particles added was 40 μL (equivalent to 2 mg) for untreated serum and 30 μL (equivalent to 1.5 mg) for HSA&Ig-dep serum, so that the weight of the magnetic particles was approximately 10 times the weight of the sample protein. The solution was mixed by shaking at 1,000 rpm and 25°C for 1 min in a thermomixer. Subsequently, to aggregate the protein onto the beads, ethanol (final concentration 80% v / v; 560 μL for untreated serum and 520 μL for HSA&Ig-dep serum) was added, and the mixture was shaken at 1,000 rpm and 25°C for 10 min in a thermomixer. The tube containing the sample solution was placed on a magnetic stand and allowed to stand for 2 min. After the magnetic beads were allowed to accumulate on the tube wall using the magnet, the supernatant was removed with a pipette. To wash the plate, 800 μL of 80% (v / v) ethanol was added and left to stand for 2 minutes, after which the supernatant was removed. This washing procedure was repeated three times.

[0129] [Digestion of proteins on magnetic particles] Next, 50 μL of a 2x concentrated detergent buffer (4.8 mM SDC, 4.8 mM SLS, 200 mM Tris-HCl pH 8.5) was added to the sample for reconstitution and digestion. The sample was resuspended by shaking in a thermomixer at 2,000 rpm at 25°C for 5 min. To this was added 50 μL of enzyme solution for protein digestion (0.02 μg / μL Trypsin / Lys-C mix, 4 mM CaCl2, 2 mM acetic acid (acetic acid comes from the enzyme reconstitution solution)), and the digestion reaction was carried out in a thermomixer at 1,000 rpm at 37°C for 16 h.

[0130] [Purification of glycopeptides] The compositions of the glycopeptide loading solution used to retain glycopeptides on the beads and the wash solution used for washing differ depending on the ion pairing agent added thereto.

[0131] Table 3 shows the HILIC purification solutions used to compare the addition of ion-pairing agents to the wash solution between untreated serum samples. (1) 1% TFA added. (2) 5% formic acid added. (3) 5% formic acid and 0.1% TFA added (only formic acid added to the Glycopeptide Loading Solution).

[0132]

[0133] Table 4 shows the HILIC purification solutions for comparison of the addition of wash solvents and ion-pairing agents in untreated serum and HSA & Ig-depleted serum. (1) 1% TFA added. (2) 5% formic acid and 0.1% TFA added.

[0134]

[0135] First, droplets were dropped down by flash centrifugation (tabletop mini centrifuge).

[0136] To terminate the digestion reaction, 100 μL of Glycopeptide Loading Solution was added to each sample to retain glycopeptides on the beads. Then, 10 μL of a standard glycopeptide (Sialylglycopeptide; Tokyo Chemical Industry) solution (untreated serum: 1 μM, HSA & Ig-dep serum: 2 μM) was added, and the mixture was stirred in a thermomixer at 1,000 rpm at 25°C for 30 seconds.

[0137] Additionally, glycopeptide loading solution was added according to the conditions. The total amount added is shown in Tables 3 and 4. Glycopeptides were captured by shaking in a thermomixer at 1,500 rpm at 25°C for 30 min. Droplets were removed by flash centrifugation, and the tube containing the sample solution was placed on a magnetic stand and left to stand for 2 min before the supernatant was removed.

[0138] Further, for washing, wash solution was added, and the mixture was shaken at 1,500 rpm at 25°C for 30 minutes, followed by flash centrifugation to remove droplets. The tube containing the sample solution was then placed on a magnetic stand and allowed to stand for 2 minutes before the supernatant was removed. This procedure was repeated twice.

[0139] Next, while the tube containing the sample and beads was still placed on the magnetic stand, 800 μL of TFA Rinse Sol. (98% (v / v) ACN) was added to remove the TFA around the beads. After 2 min, the supernatant was removed. 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 in a thermomixer at 1,000 rpm at 25°C for 10 min. After 5 min on the magnetic stand, the supernatant separated from the beads was collected and transferred to a new 1.5-mL low-binding polypropylene tube. The solution was then dried in a centrifugal dryer.

[0140] [Reconstitution of glycopeptides and LC / MS measurement (common to all conditions)] The dried glycopeptide sample was reconstituted in 50 μL of 1% (v / v) acetic acid, 2% (v / v) acetonitrile aqueous solution, transferred to the inside of a filter (Millex LH 45 μm, 4 mm, Merck Millipore) attached to a low-binding 0.5-mL PP tube, and filtered by centrifugation at 2,000 × g, 25°C, for 2 min. For measurement, the glycopeptide solution was transferred to a 96-well plate (Sarstedt) for low-protein binding PCR and diluted with 0.1% (v / v) formic acid aqueous solution to prepare a 1 / 50-diluted plasma glycopeptide solution. This solution was then loaded onto the autosampler of the high-performance liquid chromatography (HPLC). Analysis of the glycopeptide sample was performed using a liquid chromatography-mass spectrometry method. The equipment used was a nanoflow high-performance liquid chromatography (HPLC) system (Vanquish-Neo UHPLC system, Thermo Fisher Scientific) and a quadrupole-Orbitrap mass spectrometer (Orbitrap Exploris 240, Thermo Fisher Scientific).

[0141] The mobile phases used for HPLC were: A: ultrapure water containing 0.1% (v / v) formic acid; B: acetonitrile containing 0.1% (v / v) formic acid. A nano-HPLC capillary column (C18, 0.075 × 150 mm, 3 μm; Nikkyo Technos) with an integrated emitter was used. The flow rate during sample separation was 0.30 μL / min, and the column temperature was set at 40°C. The proportion of mobile phase B was fixed at 0% from 0 to 2 min, 1% from 2 to 2.5%, 44.5% from 2.5 to 60.5 min, and linearly increased to 95% from 60.5 to 65.5 min.

[0142] In the mass spectrometer, the ion source was set to a spray voltage of 1800 V and an ion transfer tube temperature of 275°C.

[0143] The full MS scan was performed with the following settings: Orbitrap Resolution: 120,000, Scan Range: 500-1800 m / z, RF Lens: 70%, Normalized AGC Target: 300%, Maximum Injection Time: 50 ms, Microscans: 1, and Positive Polarity. The filter settings for data-dependent MS2 were: MIPS: Peptide, Intensity Threshold: 1.0e4, Charge state: 2-8, Dynamic Exclusion duration: 20 s, and Dynamic Exclusion Mass Tolerance: High-10, Low-10 ppm. The data-dependent MS2 (ddMS2) acquisition settings were: Data Dependent mode: Cycle time (1.5 s), Isolation window: 1.6 m / z, Collision Energy Type: Normalized, HCD collision energy: 20, 30, 40 (%), Orbitrap resolution: 30,000, Scan range: 150–2000 m / z, Normalized AGC target: 200%, Maximum injection time: 75 ms. The raw LC / MS data were searched using Byonic (Protein Metrics), a peptide analysis software compatible with glycopeptides, for glycopeptide identification. Furthermore, Proteome discoverer (Thermo Fisher) combined data alignment and Byonic search for glycopeptide identification and quantitative comparison.

[0144] [Results] ・The number of glycopeptide identifications was highest in the wash solution containing only TFA (1%) as an ion-pairing agent, lower in the wash solution containing only formic acid (5%), and higher in the wash solution containing a mixture of formic acid and TFA (5% and 0.1%) than in the wash solution containing only formic acid (Figure 10). ・In the abundant protein (serum albumin and immunoglobulin)-depleted (HSA&Ig-dep) sample, the type of ion-pairing agent had little effect on the HILIC wash solvent (Figures 11(a) and (b), HSA&Ig-dep). ・In untreated serum, glycopeptides with highly hydrophilic peptide chains were less affected by the ion-pairing agent in the wash solvent (e.g., Complement C3 [74-94] Hex2HexNAc6; Figure 11(a)). On the other hand, glycopeptides containing many hydrophobic residues showed low recovery when using a wash solvent containing only TFA, whereas the recovery rate was significantly improved by using a wash solvent containing a mixture of formic acid and TFA (e.g., Alpha-1-antitrypsin [94-125] Hex5HexNAc4NeuAc2; Figure 11(b)). These results are summarized as follows: (1) Using an acetonitrile-based HILIC wash solvent containing a mixture of formic acid and TFA improved the recovery rate of glycopeptides prepared from untreated serum / plasma compared with a wash solvent containing only TFA as an ion pairing agent. (2) For the first time, we observed a synergistic effect of combining TFA and formic acid in the analysis of untreated (non-depleted) serum. (3) Although abundant protein-removed serum / plasma has the advantage of easily increasing the depth of analysis, it has the drawback of requiring additional preparation procedures and high costs. (4) Analysis of untreated serum and plasma can proceed almost directly to the glycopeptide preparation process from the specimen, which is high-throughput and low-cost, making it suitable for large-scale analysis. (5) The investigation of ion-pairing agents has improved the reliability of sample preparation for analysis of untreated serum and plasma.

Claims

1. Step (3) of contacting a particle that holds a protein on its surface with a protease; (4) step of adding an organic solvent after step (3); and (5) step of washing the particle obtained in step (4), the method for producing a glycopeptide sample, comprising adding an ion pair agent together with the organic solvent in step (4), wherein the particle is a hydrophilic polymer-coated particle.

2. The production method according to claim 1, wherein the hydrophilic polymer-coated particle is a polysaccharide-coated particle.

3. The production method according to claim 2, wherein the polysaccharide-coated particle is a cellulose-coated particle.

4. The production method according to claim 1, comprising (2) a step of contacting a protein and a particle in a solution containing an organic solvent before step (3) to hold the protein on the particle surface.

5. The production method according to claim 4, wherein in step (2), the method includes a step of dispensing a previously prepared particle suspension containing the particle and a thickening agent.

6. The production method according to claim 5, wherein the thickening agent is glycerol.

7. The production method according to claim 1, wherein the hydrophilic polymer-coated particle is a carboxylic acid polymer-coated particle.

8. The production method according to claim 1, wherein the hydrophilic polymer-coated particle is a magnetic particle.

9. The production method according to claim 1, wherein the ion pair agent is an organic acid.

10. The production method according to claim 9, wherein the ion pair agent contains a halogenated short-chain fatty acid.

11. The production method according to claim 10, wherein the ion pair agent is a mixture containing a halogenated short-chain fatty acid and a non-halogenated short-chain fatty acid.

12. The production method according to claim 1, wherein the organic solvent is at least one selected from the group consisting of alcohol, acetonitrile, and acetone.

13. The production method according to any one of claims 1 to 12, wherein all steps are carried out in the same container.

14. The production method according to any one of claims 1 to 12, wherein the glycopeptide sample is a sample for mass spectrometry.

15. A glycopeptide sample obtained by the production method according to any one of claims 1 to 12.

16. A method for analyzing the sugar chain modification of a protein, comprising the step of mass-analyzing the glycopeptide sample according to claim 15.

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