Method for pre-treating specimens during analysis and specimen pre-treatment device

Abstract

To provide sample analysis pretreatment method capable of reducing the contamination of a reagent into a sample in a sample pretreatment, and to provide sample pretreatment devices capable of performing the analysis pretreatment method.SOLUTION: One aspect of the present embodiment is a sample analysis pretreatment method, the sample analysis pretreatment method comprising: step A of mixing a sample with a reagent A containing a carrier and a reducing agent immobilized on the carrier; step B of mixing the sample with a reagent B containing a carrier and an enzyme immobilized on the carrier; step X-A of separating the reagent A after step A and supernatant; and step X-B of separating the reagent B after step B and supernatant.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for pre-treatment of a sample for analysis and a sample pre-treatment device. [Background technology]

[0002] Analysis of peptides and proteins by LC-MS is important in various fields such as food, medicine, and diagnosis. When analyzing peptides and proteins by LC-MS, fragmentation using enzyme digestion is generally required. In conventional techniques, the sample is reduced and alkylated, and then fragmented by applying an enzyme solution, but this method can cause adverse effects on downstream processes by contaminating the sample with residues of the reagents used for reduction and alkylation, residues of the enzyme, and enzyme fragments.

[0003] In response to this, a method using a column in which an enzyme is immobilized has been proposed (see, for example, Patent Document 1). Also, a method using an immobilized enzyme or the like as a dispersion liquid has been proposed (see, for example, Non-Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2007-44043 A [Non-patent literature]

[0005] [Non-Patent Document 1] Analytical and Bioanalytical Chemistry volume 412, pages6833-6848(2020). Summary of the Invention [Problem to be solved by the invention]

[0006] In the invention described in Patent Document 1, there was a possibility of deterioration and cross-contamination due to repeated use of the column. In the invention described in Non-Patent Document 1, the dispersion liquid containing the immobilized enzyme could be used in a disposable system, but residues of the reagents used for reduction and alkylation could get mixed into the sample, adversely affecting the subsequent process.

[0007] Therefore, an object of the present disclosure is to provide a method for pre-analysis of a sample that can reduce contamination of the sample with a reagent during pre-treatment of the sample.

[0008] Another object of the present disclosure is to provide a specimen pretreatment device capable of executing an analysis pretreatment method capable of achieving the above object. [Means for solving the problem]

[0009] An example aspect of this embodiment is as follows.

[0010] A method for pre-analysis of a sample containing a peptide or protein, comprising the steps of: A step A of mixing a sample with a reagent A containing a carrier and a reducing agent immobilized on the carrier; A step B of mixing a sample with a reagent B containing a carrier and an enzyme immobilized on the carrier; A step XA of separating the reagent A from the supernatant after the step A; A step XB of separating the reagent B from the supernatant after the step B; An analytical pretreatment method comprising the steps of:

[0011] A specimen pretreatment device capable of performing the analysis pretreatment method, A specimen pretreatment device comprising a control mechanism, a sample container storage mechanism, a sample container heating and shaking mechanism, a reagent dispensing mechanism, and a supernatant recovery mechanism. Effect of the Invention

[0012] By carrying out the analysis pretreatment method of this embodiment, it is possible to reduce contamination of samples with reagents compared to conventional analysis pretreatment methods. In addition, since it is easy to construct a disposable system, cross-contamination can also be suppressed. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a process flow diagram showing one embodiment of the analysis pretreatment method of this embodiment. [Diagram 2] FIG. 2 is a process flow diagram showing one embodiment of the analysis pretreatment method of this embodiment. [Diagram 3] FIG. 3 is a process flow diagram showing one embodiment of the analysis pretreatment method of this embodiment. [Figure 4] FIG. 4 is a conceptual diagram showing one aspect of the specimen pretreatment device of this embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] One aspect of this embodiment is an analytical pretreatment method for a sample containing a peptide or protein, comprising step A of mixing the sample with reagent A containing a carrier and a reducing agent immobilized on the carrier, step B of mixing the sample with reagent B containing a carrier and an enzyme immobilized on the carrier, step XA of separating reagent A from the supernatant after step A, and step XB of separating reagent B from the supernatant after step B.

[0015] According to the above-mentioned embodiment, it is possible to reduce contamination of the specimen that has been subjected to the analytical pretreatment method with the reagents. This is because the reagents (reagent A, reagent B) contain a carrier and the reducing agent and enzyme are immobilized on the carrier, so that in step XA and step XB, it is easy to separate the reagents A and B after step A and step B from the supernatant containing the specimen. Furthermore, by constructing the analytical pretreatment method as a disposable system, it is possible to suppress cross-contamination that occurs, for example, due to repeated use of a column.

[0016] One aspect of the present embodiment is a specimen pretreatment device capable of carrying out the analysis pretreatment method, which is provided with, for example, a control mechanism, a sample container storage mechanism, a sample container heating and shaking mechanism, a reagent dispensing mechanism, and a supernatant recovery mechanism.

[0017] The present embodiment will be described in detail below. In the following description, the present embodiment will be described using the drawings as appropriate. The following description shows a specific example of the contents of the present embodiment, and the present embodiment is not limited to these descriptions. Various changes and modifications are possible by those skilled in the art within the scope of the technical ideas disclosed in this specification. In addition, in all the drawings for explaining the present embodiment, parts having the same functions are given the same reference numerals, and repeated explanations thereof may be omitted.

[0018] [Pre-analysis method] The analysis pretreatment method according to this embodiment is an analysis pretreatment method for a specimen containing a peptide or protein, and is characterized by including a step A of mixing the specimen with a reagent A containing a carrier and a reducing agent immobilized on the carrier, a step B of mixing the specimen with a reagent B containing a carrier and an enzyme immobilized on the carrier, a step XA of separating the reagent A from the supernatant after the step A, and a step XB of separating the reagent B from the supernatant after the step B. The analysis pretreatment method according to this embodiment uses a reagent in which a reducing agent or an enzyme is immobilized on a carrier, and therefore separation is easy.

[0019] The specimen to which the analysis pretreatment method is applied may contain a peptide or a protein, and is not particularly limited, but is usually a specimen containing at least one selected from a peptide and a protein. An example of at least one selected from a peptide and a protein is an antibody. In the analysis pretreatment method, denaturation and digestion of peptides and proteins are performed. Here, denaturation refers to reductively cleaving disulfide bonds in a molecule or changing a higher-order structure, and is performed for the purpose of increasing digestion efficiency. Digestion refers to breaking covalent bonds in a molecule and fragmenting peptides and proteins, and is performed for the purpose of increasing detection efficiency by LC-MS. In the analysis pretreatment method according to this embodiment, denaturation can be performed by reagent A, and digestion (fragmentation) can be performed by reagent B.

[0020] Although digested (fragmented) peptides and proteins have been analyzed in the past, the analytical pretreatment method according to the present embodiment can contribute to speeding up analysis because the time required for pretreatment is short. In addition, the reagents used for denaturation and digestion are solid-phased, making them easy to recover, and it is possible to prevent the reagents from being mixed into the specimen (sample) after digestion, which is expected to improve the accuracy of analysis.

[0021] The specimen used in the analytical pretreatment method is preferably in a liquid state from the viewpoint of efficient reaction with the reagent. The specimen may be a liquid sample, for example, a body fluid such as blood, urine, sweat, or tears, or may be a sample other than a body fluid, for example, a sample derived from a cell (for example, an animal cell, a bacterial cell, or a plant cell). Examples of samples derived from a cell include a cell extract, a secretion during culture, and a culture solution. The sample may be a reaction solution containing a cell-free protein synthesis, or a reaction solution in which a protein has been chemically modified. The specimen may also be a specimen whose concentration has been adjusted using a solvent as described below. The specimen may be a specimen derived from a human or a non-human. Examples of non-human specimens include specimens derived from mammals (excluding humans) and specimens derived from organisms other than mammals, for example, birds, fish, insects, plants, algae, and fungi. Examples of mammals (excluding humans) include mice, rats, horses, sheep, pigs, goats, and cows.

[0022] It is desirable that the specimen to which the pre-analysis method is applied is one that has been appropriately treated as necessary, such as by removing impurities, adsorption treatment, desalting, solvent replacement, dialysis, concentration, etc.

[0023] In the analysis pretreatment method, reagent A containing a carrier and a reducing agent immobilized on the carrier, and reagent B containing a carrier and an enzyme immobilized on the carrier are used. In addition, in the analysis pretreatment method, reagent C containing a carrier and an alkylating agent immobilized on the carrier may also be used. Note that "reagent A containing a carrier and a reducing agent immobilized on the carrier" is also referred to as "reagent A" or "immobilized reducing agent". "Reagent B containing a carrier and an enzyme immobilized on the carrier" is also referred to as "reagent B" or "immobilized enzyme". Furthermore, "reagent C containing a carrier and an alkylating agent immobilized on the carrier" is also referred to as "reagent C" or "immobilized alkylating agent".

[0024] In conventional denaturation, thiols such as dithiothreitol, cysteine, glutathione, and phosphines such as TCEP (tris(2-carboxyethyl)phosphine hydrochloride) are generally used as reducing agents for disulfide bonds, and the reducing agent contained in reagent A is not particularly limited and may include these reducing agents, but it is preferable that the reducing agent contained in reagent A contains a thiol or a phosphine as a partial structure. By adding reagent A to a sample and reacting it under appropriate conditions, it is possible to reductively cleave the disulfide bonds in the target molecule.

[0025] In the analysis pretreatment method according to this embodiment, alkylation may be performed. Alkylation can be performed for the purpose of suppressing the reductively cleaved disulfide bond from returning to a disulfide bond through a reverse reaction caused by oxygen oxidation or the like. As an alkylating agent used for alkylation, a nucleophilic reagent such as iodoacetamide or iodoacetic acid can generally be used, but in this embodiment, alkylation can be performed using reagent C. The alkylating agent contained in reagent C is not particularly limited, including these alkylating agents. However, as the alkylating agent, a nucleophilic reagent having selectivity for soft bases such as thiolate is preferable in order to suppress competition with the alkylation of amine residues in the target molecule.

[0026] The enzyme contained in the reagent B is not particularly limited as long as it can digest peptides and proteins in the sample. In one preferred embodiment, the enzyme is a protease. The enzyme preferably contains at least one enzyme selected from trypsin, chymotrypsin, pepsin, Lys-C, Lys-N, and Glu-C.

[0027] In the reagents A, B, and C, the method of immobilizing the reducing agent, enzyme, or alkylating agent is not particularly limited, and for example, existing techniques can be used. Here, immobilization means complexing the reducing agent, enzyme, or alkylating agent with a carrier while maintaining the activity of the reducing agent, enzyme, or alkylating agent within an acceptable range, and means such as chemical immobilization or physical adsorption can be used. In the immobilization, the selection of the carrier, the surface treatment of the carrier, the selection of the linker, the immobilization method of the linker, the quantification of the amount of immobilization, and the like can be appropriately performed using existing techniques.

[0028] The material of the carrier is not limited, but it is preferable that the carrier is easily mixed with the specimen, easily separated from the specimen in a subsequent process, and has low non-specific adsorption of the target molecules in the specimen for pretreatment. The carrier may be a single composition or a composite material made of multiple materials, and may have a surface coating. Examples of the surface coating include a polysaccharide coating, a hydrophilic polymer coating, and a zwitterionic polymer coating, which are used to reduce non-specific adsorption.

[0029] As the carrier, for example, a metal oxide such as silica or titania, an organic resin such as polystyrene or acrylic resin, a polysaccharide such as cross-linked agarose, a metal element such as metal nanoparticles, a semiconductor such as semiconductor nanoparticles, etc. can be used. In addition, a carrier to which magnetism such as superparamagnetism has been imparted may be used so as to facilitate separation in a later process. Means for imparting magnetism include the use of a magnetic material, and compounding with particles made of a magnetic material. Examples of magnetic materials include, but are not limited to, magnetic metal oxides such as iron oxide and cobalt oxide. It is preferable that the carrier contains at least one material selected from silica, polystyrene, acrylic resin, polysaccharides, a metal element, a semiconductor, a metal oxide, and a magnetic metal oxide.

[0030] The type, length, hydrophilicity, hydrophobicity, etc. of the linker are not limited, but it is desirable that the linker does not inhibit the activity of the reagent. Examples of the linker include oligoethylene glycol, polyethylene glycol, and alkyl groups.

[0031] The shape of the carrier is not limited, but since it is preferable to ensure the reproducibility of the analysis pretreatment, it is desirable to show a certain shape such as a sphere. The carrier may be porous or non-porous, but as the specific surface area increases, non-specific adsorption tends to increase, and the carrier can be selected according to the purpose. The size of the carrier is not limited, but as the particle diameter of the carrier increases, separation in the subsequent process tends to be easier, and the carrier can be selected according to the purpose. The average diameter of the carrier is preferably 0.5 μm to 500 μm, more preferably 1 μm to 100 μm, and even more preferably 2 μm to 30 μm. The narrower the size distribution of the carrier, the easier it is to ensure reproducibility, but it is not limited and can be selected according to the purpose.

[0032] The carriers contained in the reagents A, B, and C may be the same type of carrier or different types of carriers.

[0033] The analysis pretreatment method according to this embodiment includes a step A of mixing a specimen with a reagent A, a step B of mixing a specimen with a reagent B, a step XA of separating the reagent A from the supernatant after the step A, and a step XB of separating the reagent B from the supernatant after the step B. The analysis pretreatment method according to this embodiment may also include a step C of mixing a specimen with a reagent C, and a step XC of separating the reagent C from the supernatant after the step C. There is no particular restriction on the order in which the steps A and B are performed, and the steps A and B may be performed simultaneously, or one step may be performed after the other step, but preferably the steps A and B are performed simultaneously, or the step B is performed after the step A. When the step C is performed, it is preferable that the step C is performed simultaneously with the step A, after the step A and before the step B, or after the step A and simultaneously with the step B. That is, the order in which step A, step B and optionally step C are performed may include, for example, a mode in which step A and step B are performed simultaneously, a mode in which step A is performed followed by step B, a mode in which steps A and C are performed simultaneously and then step B is performed, a mode in which step A is performed followed by step C and then step B (a mode in which step C is performed between step A and step B), a mode in which step A is performed followed by steps B and C simultaneously, and a mode in which steps A, B and C are performed simultaneously.

[0034] Steps XA, XB, and XC are steps that are performed after steps A, B, and C, respectively, and there is no particular limitation on the timing of performing them. For example, step XC, which is performed when steps XA, XB, and C are performed after step A, step B, and optionally performed step C, may be performed simultaneously, or step XA may be performed after step A, step XC may be performed after step C, and step XB may be performed after step B.

[0035] An example of the embodiment of the analysis pretreatment method according to the present embodiment is shown below. For example, an embodiment in which step A is performed, then step B is performed, and then steps XA and XB are performed simultaneously (FIG. 1). An example including step C is an embodiment in which step A is performed, then step C is performed, then step B is performed, and then steps XA, XB, and XC are performed simultaneously (FIG. 2). An example in which steps A and B are performed simultaneously is an embodiment in which steps A and B are performed simultaneously, and then steps XA and XB are performed simultaneously (FIG. 3). Steps A and B may be performed simultaneously or separately as shown in FIG. 1 and FIG. 3.

[0036] When step B (digestion) is performed under conditions where the reverse reaction in which the disulfide bond cleaved by step A returns to a disulfide bond due to oxygen oxidation or the like can be ignored, step C is not necessarily required. Examples of cases where step C is not required include cases where the target peptide or protein of the pretreatment is structurally difficult to refold after being reduced and denatured, and cases where enzymatic digestion can be performed at a sufficiently high speed relative to the reverse reaction. In the analysis pretreatment method according to this embodiment, the use of an immobilized enzyme allows enzymatic digestion to be performed at a sufficiently high speed. When the alkylation step (step C) is not required, the pretreatment time can be shortened by reducing the number of steps.

[0037] In the pre-analysis method according to the present embodiment, by using immobilized reagents as the reducing agent and the optional alkylating agent, the post-treatment process can be made more efficient than when non-immobilized reagents are used. For example, in many cases, the alkylating agent reacts with the reducing agent. In addition, the activity of some enzymes is reduced by the reducing agent. For this reason, when a reducing agent solution is used, unreacted reducing agent may reduce the reaction efficiency in the post-treatment process.

[0038] In the analysis pretreatment method according to the present embodiment, by using a solid-phase reducing agent, it is possible to reduce the amount of unreacted reducing agent remaining in the system, and the efficiency of the subsequent steps can be improved. In addition, when an alkylating agent solution is used, it is common to add a quenching step because the unreacted alkylating agent may reduce the reaction efficiency in the subsequent steps, but by using a solid-phase alkylating agent, it is possible to reduce the amount of unreacted alkylating agent remaining in the system, and the efficiency of the subsequent steps can be improved.

[0039] In the analysis pretreatment method according to this embodiment, when mixing a specimen with reagents (reagent A, reagent B, reagent C), the specimen and the reagents may be mixed directly, or the reagents may be mixed in advance with a solvent described below and mixed with the specimen as a suspension or dispersion.

[0040] The analysis pretreatment method according to this embodiment may include a heating step, preferably a heating step within a range of 30° C. to 70° C. The heating step may be performed as a separate step from the above-mentioned steps, or may be performed simultaneously by performing the above-mentioned steps under heating conditions. The heating step is preferably performed simultaneously with at least one of the above-mentioned steps A, B, and optional step C, and more preferably performed by performing step A, B, and optional step C under heating conditions.

[0041] The method for carrying out the heating step is not particularly limited, and can be carried out using existing techniques such as, for example, heating by thermal conduction, microwave heating, or infrared heating. The temperature (heating temperature) for the heating step is selected within a range that does not adversely affect the specimen or the enzyme, and is, for example, 30 to 70°C, preferably 35 to 60°C, and more preferably 37 to 55°C. The heating step can be carried out, for example, under normal pressure conditions, and in this case, it is preferable to carry out the heating step at the above-mentioned temperature. The heating step can also be carried out under pressurized conditions, and when carried out under pressurized conditions, the temperature limit at which the enzyme activity is lost may be higher than that under normal pressure conditions, and in such cases, the heating range can be higher than the above-mentioned temperature.

[0042] The above-mentioned step A, step B, and optional step C can be carried out under shaking or non-shaking conditions. For shaking, existing techniques including physical shaking and ultrasonic shaking can be used. The time taken for the above-mentioned step A, step B, and optional step C is not limited. As long as the reaction progresses sufficiently to the subsequent step, the time can be kept short. The reaction time is, for example, 1 minute to 4 hours.

[0043] The equivalent amount of the reagent added in the above-mentioned step A, step B, and optional step C is not limited, but may be a large excess amount for rapid pretreatment. For example, the equivalent amount of the reducing agent contained in the reagent A is preferably 2 equivalents to 1,000,000 equivalents relative to the disulfide bond in the sample, more preferably 10 equivalents to 100,000 equivalents, and particularly preferably 100 equivalents to 10,000 equivalents. Note that equivalents refer to molar equivalents. The equivalent amount of the alkylating agent contained in the reagent C can be exemplified as being in the same range as the equivalent amount of the reducing agent contained in the reagent A. The content of the enzyme contained in the reagent B is preferably 1:1,000 to 100:1 in weight ratio to the peptide or protein in the sample, more preferably 1:200 to 50:1, and particularly preferably 1:50 to 10:1. In the analysis pretreatment method according to this embodiment, it is possible to reduce the contamination of the reagent with the sample after pretreatment, so that even if a large excess amount of the reagent is used, adverse effects on the subsequent steps are unlikely to occur. Therefore, compared to conventional sample pretreatment methods, it is possible to increase the amount of reagent (reagent equivalent) and speed up the reaction, thereby reducing the time required for pretreatment.

[0044] The steps (steps XA, XB, and XC) of separating the reagents (reagent A, reagent B, and reagent C) from the supernatant can be carried out by using, for example, existing techniques. For example, natural sedimentation, centrifugation, magnetic accumulation, sonic trapping, filtration, and the like. These techniques may be carried out by combining two or more methods. That is, it is preferable that each of the steps XA and XB includes at least one method selected from natural sedimentation, centrifugation, magnetic accumulation, sonic trapping, and filtration. It is also preferable that the step XC includes at least one method selected from natural sedimentation, centrifugation, magnetic accumulation, sonic trapping, and filtration. It is to be noted that the steps XA, XB, and XC may be carried out as separate steps or as the same step.

[0045] After step XA, step XB, or step XC, a step of washing the separated reagent A, reagent B, or reagent C, a step of mixing the washing liquid used to wash the reagent with the supernatant, etc. may be added. Furthermore, the supernatant thus recovered may be subjected to other additional steps before being subjected to LC-MS measurement, etc.

[0046] In the analysis pretreatment method according to the present embodiment, a solvent may be used. The solvent may be an aqueous buffer, an organic solvent, or a mixture of an aqueous buffer and an organic solvent. The solvent may contain a surfactant or a denaturant, but it is preferable to use less components that affect the analysis. When using an aqueous buffer or the like and a salt is used, the salt is preferably a volatile salt such as ammonium carbonate or ammonium formate, and when a non-volatile salt is used, it is preferable to desalt the salt before analysis. The organic solvent is preferably water-soluble, for example, acetonitrile or a lower alcohol.

[0047] A specimen treated using the analytical pretreatment method according to this embodiment can be subjected to LC-MS analysis or the like as is, or after appropriate treatment such as impurity removal, adsorption treatment, desalting, solvent replacement, dialysis, concentration, etc. as necessary. Furthermore, a specimen treated using the analytical pretreatment method according to this embodiment can also be subjected to analyses other than LC-MS, such as electrophoresis or ELISA, and the use is not limited.

[0048] The specimen pretreatment device according to this embodiment may be any device capable of carrying out the above-described analysis pretreatment method according to this embodiment. For example, the specimen pretreatment device may include a control mechanism 1, a sample container storage mechanism 5, a sample container heating and shaking mechanism 7, a reagent dispensing mechanism 9, and a supernatant recovery mechanism 11 (FIG. 4).

[0049] The sample container storage mechanism 5 stores the sample container 3. Usually, a specimen is added to the sample container 3 before or after the sample container 3 is stored in the sample container storage mechanism 5. The sample container heating and shaking mechanism 7 may be capable of heating and shaking the sample container 3. In FIG. 4, the top of the sample container heating and shaking mechanism 7 is open, but it may have, for example, an openable and closable lid. The reagent dispensing mechanism 9 and the supernatant recovery mechanism 11 may be provided in the sample container heating and shaking mechanism 7. The reagent dispensing mechanism 9 is configured to be able to add the reagent A, the reagent B, and the optionally used reagent C to the sample container 3, preferably in the form of a dispersion or suspension. The supernatant recovery mechanism 11 may be configured to separate the supernatant from the reagents (reagent A, reagent B, and reagent C) and recover the supernatant, and acts as a supernatant reagent separation mechanism. The sample container 3 that holds the specimen is not particularly limited. The technology used for heating and shaking is not particularly limited as described above. The mechanism for feeding the liquid in the dispensing mechanism and the supernatant recovery mechanism may be, but is not limited to, a pump, etc. As described above, the technique for separating the supernatant from the reagent is not particularly limited. EXAMPLES

[0050] The present embodiment will be described in detail below with reference to examples, although the present embodiment is not limited to the following examples.

[0051] [Example 1] In Example 1, a method for pre-analysis of a sample was carried out using reagent A containing a carrier and a reducing agent immobilized on the carrier, reagent B containing a carrier and an enzyme immobilized on the carrier, and reagent C containing a carrier and an alkylating agent immobilized on the carrier.

[0052] A suspension of a phosphine-based reducing agent immobilized on cross-linked agarose beads with an average diameter of 120 μm via an oligoethylene glycol-based linker (reducing agent amount: 0.04 mmol) (reagent A) was added to a solution of human IgG in ABC buffer (1 wt%, 1.0 mL, approximately 0.00016 mmol equivalent to disulfide bonds), and the mixture was heated with shaking at 55 ° C for 1 hour using a block bath shaker. The resulting supernatant was collected, and a suspension of an α-substituted acetamide-based alkylating agent physically adsorbed on cross-linked agarose beads with an average diameter of 120 μm (alkylating agent amount: 0.04 mmol) (reagent C) was added, and the mixture was heated with shaking at 55 ° C for 30 minutes. The resulting supernatant was collected, and 0.08 mL of a dispersion of trypsin (ResynBioscience) immobilized on magnetic beads (reagent B) was added, and the mixture was heated at 55 ° C for 1 hour using a block bath shaker. The resulting reaction mixture was centrifuged for 5 minutes and the supernatant was then removed to separate the carrier from the supernatant.

[0053] The amount of human IgG remaining in the supernatant was quantified, and the residual rate of undigested human IgG was calculated to be 25%. The entire process of the analytical pretreatment method took approximately 2.5 hours.

[0054] Example 1 suggests that human IgG can be rapidly pretreated by reduction, alkylation, and enzymatic digestion using a reagent (solid-phase reagent) that is easily separated from the supernatant and can be easily used in a disposable system.

[0055] [Example 2] In Example 2, a method for pre-analysis of a sample was carried out using Reagent A containing a carrier and a reducing agent immobilized on the carrier, and Reagent B containing a carrier and an enzyme immobilized on the carrier.

[0056] A suspension of a reducing agent immobilized on magnetic beads (reducing agent amount: 0.08 mmol) (reagent A) was added to a solution of human IgG in ABC buffer (1 wt%, 1.0 mL, approximately 0.00016 mmol equivalent to disulfide bonds), and heated with shaking at 55°C for 1 hour using a block bath shaker. The resulting reaction mixture was left to stand near a magnet for 1 minute, and the resulting supernatant was collected, and 0.08 mL of a dispersion of trypsin (ResynBioscience) immobilized on magnetic beads (reagent B) was added, and the mixture was heated at 55°C for 1 hour using a block bath shaker. The resulting reaction mixture was left to stand near a magnet for 1 minute, and the supernatant was removed to separate the carrier from the supernatant.

[0057] The amount of human IgG remaining in the supernatant was quantified, and the residual rate of undigested human IgG was calculated to be 19%. The entire process of the analytical pretreatment method took approximately 2 hours.

[0058] Example 2 suggests that human IgG can be pretreated rapidly by reduction and enzymatic digestion using a reagent (solid-phase reagent) that is easily separated from the supernatant and can be used in a disposable system, even without performing an alkylation step.

[0059] [Example 3] In Example 3, a method for pre-analysis of a sample was carried out by simultaneously using a reagent A containing a carrier and a reducing agent immobilized on the carrier, and a reagent B containing a carrier and an enzyme immobilized on the carrier.

[0060] A suspension of reducing agent immobilized on cross-linked agarose beads (reducing agent amount: 0.16 mmol) (reagent A) and a dispersion of trypsin (ResynBioscience) immobilized on magnetic beads (reagent B) (0.16 mL) were added to a solution of human IgG in ABC buffer (1 wt%, 1.0 mL, approximately 0.00016 mmol equivalent to disulfide bonds), and the mixture was heated at 55°C for 1 hour using a block bath shaker. The resulting reaction mixture was sucked up with a syringe, and the carrier and the supernatant were separated using a syringe filter.

[0061] The amount of human IgG remaining in the obtained supernatant was quantified, and the residual rate of undigested human IgG was calculated to be 40%. The entire process of the analytical pretreatment method took about 1 hour.

[0062] Example 3 suggests that human IgG can be pretreated rapidly in a process using reagent A and reagent B simultaneously.

[0063] [Comparative Example 1] In Comparative Example 1, a method for pre-analysis of a sample was carried out using a reducing agent solution, an alkylating agent solution, and a reagent B containing a carrier and an enzyme immobilized on the carrier.

[0064] A reducing agent (0.04 mmol) was added to a solution of human IgG in ABC buffer (1 wt%, 1.0 mL, approximately 0.00016 mmol as disulfide bond equivalent), and the mixture was heated with shaking at 55 ° C. for 1 hour using a block bath shaker. The resulting solution was collected, an alkylating agent (0.16 mmol) was added, and the mixture was heated with shaking at 55 ° C. for 1 hour. The resulting solution was collected, a reducing agent (0.08 mmol) was added, and the mixture was heated with shaking at 55 ° C. for 1 hour. The resulting supernatant was collected, and 0.08 mL of a dispersion of trypsin (ResynBioscience) immobilized on magnetic beads was added, and the mixture was heated at 55 ° C. for 1 hour using a block bath shaker. The resulting reaction mixture was centrifuged for 5 minutes, and the supernatant was removed to separate the carrier from the supernatant.

[0065] The amount of human IgG remaining in the resulting supernatant was quantified, and the residual rate of undigested human IgG was calculated to be 65%. In addition to the high residual rate due to reduced activity derived from unreacted substances and by-products of the reducing agent and alkylating agent, the decrease in mass spectrometry sensitivity due to their contamination of the sample was confirmed. The entire process of the analytical pretreatment method took approximately 4 hours.

[0066] Comparative Example 1 suggests that pretreatment using non-immobilized reagents (reducing agents, alkylating agents) tends to be less efficient, have more impurity contamination, and require longer treatment times than pretreatment using immobilized reagents.

[0067] The examples and comparative examples suggest that samples subjected to the analytical pretreatment method of this embodiment can reduce contamination of the sample with reagents compared to samples subjected to conventional analytical pretreatment methods, and that cross-contamination can also be suppressed because it is easy to construct a disposable system.

[0068] The upper limit and / or lower limit of the numerical ranges described in this specification can be arbitrarily combined to define a preferred range. For example, the upper limit and lower limit of the numerical range can be arbitrarily combined to define a preferred range, the upper limit of the numerical range can be arbitrarily combined to define a preferred range, and the lower limit of the numerical range can be arbitrarily combined to define a preferred range.

[0069] The claims following this written disclosure are expressly incorporated herein into this written disclosure, with each claim standing on its own as a separate embodiment. The present disclosure includes all instances in which an independent claim is replaced by its dependent claim. Furthermore, any additional embodiments derived from the independent claim and the subsequent dependent claims are also expressly incorporated into this written specification.

[0070] Those skilled in the art can use the above description to make the most of the present disclosure. The claims and embodiments disclosed herein are merely illustrative and exemplary and should not be construed as limiting the scope of the present disclosure in any way. With the aid of this disclosure, changes can be made to the details of the above embodiments without departing from the basic principles of the present disclosure. In other words, various modifications and improvements of the embodiments specifically disclosed in the above specification are within the scope of the present disclosure. The following additional notes are disclosed regarding the present invention.

[0071] (Additional Note) (Appendix 1) A method for pre-analysis of a sample containing a peptide or protein, comprising the steps of: A step A of mixing a sample with a reagent A containing a carrier and a reducing agent immobilized on the carrier; A step B of mixing a sample with a reagent B containing a carrier and an enzyme immobilized on the carrier; A step XA of separating the reagent A from the supernatant after the step A; A step XB of separating the reagent B from the supernatant after the step B; An analytical pretreatment method comprising the steps of: (Appendix 2) The analysis pretreatment method according to claim 1, A step C of mixing a sample with a reagent C containing a carrier and an alkylating agent immobilized on the carrier; and a step XC of separating the reagent C from the supernatant after the step C, The analysis pretreatment method is characterized in that the step C is carried out between the step A and the step B. (Appendix 3) The analysis pretreatment method according to claim 1 or 2, An analytical pretreatment method, characterized in that the step A and the step B are carried out simultaneously, or one of the steps is carried out after the other step is carried out. (Appendix 4) The analysis pretreatment method according to any one of Supplementary Note 1 to Supplementary Note 3, The reagent A contains a thiol or a phosphine as a partial structure of a reducing agent, The enzyme contained in the reagent B is a protease, An analytical pretreatment method, wherein the carrier has an average diameter of 0.5 μm to 500 μm. (Appendix 5) The analysis pretreatment method according to any one of Supplementary Note 1 to Supplementary Note 4, An analytical pretreatment method comprising a heating step within the range of 30°C to 70°C. (Appendix 6) The analysis pretreatment method according to any one of Supplementary Note 1 to Supplementary Note 5, The equivalent amount of the reducing agent contained in the reagent A is 2 to 1,000,000 equivalents relative to the disulfide bond in the sample, The enzyme comprises at least one enzyme selected from trypsin, chymotrypsin, pepsin, Lys-C, Lys-N, and Glu-C; 13. A method for pre-analysis, comprising the step of: providing a support that contains at least one material selected from the group consisting of silica, polystyrene, acrylic resin, polysaccharides, simple metals, semiconductors, metal oxides, and magnetic metal oxides. (Appendix 7) The analysis pretreatment method according to any one of Supplementary Note 1 to Supplementary Note 6, An analytical pretreatment method, wherein the step XA and the step XB each include at least one method selected from the group consisting of spontaneous precipitation, centrifugation, magnetic accumulation, sonic trapping, and filtration. (Appendix 8) A specimen pretreatment device capable of carrying out the analysis pretreatment method according to any one of Supplementary Notes 1 to 7. (Appendix 9) The specimen pretreatment device according to claim 8, A specimen pretreatment device comprising a control mechanism, a sample container storage mechanism, a sample container heating and shaking mechanism, a reagent dispensing mechanism, and a supernatant recovery mechanism. [Explanation of symbols]

[0072] 1: Control mechanism 3: Sample container 5: Sample container storage mechanism 7: Sample container heating and shaking mechanism 9: Reagent dispensing mechanism 11: Supernatant recovery mechanism

Claims

1. A method for pre-analysis of a sample containing a peptide or protein, comprising the steps of: A step A of mixing a sample with a reagent A containing a carrier and a reducing agent immobilized on the carrier; A step B of mixing a sample with a reagent B containing a carrier and an enzyme immobilized on the carrier; A step C of mixing a sample with a reagent C containing a carrier and an alkylating agent immobilized on the carrier; A step X-A of separating the reagent A from the supernatant after the step A; A step X-B of separating the reagent B from the supernatant after the step B; A step X-C of separating the reagent C from the supernatant after the step C; wherein step C is performed between step A and step B.

2. The analysis pre-processing method according to claim 1, 11. An analytical pretreatment method, comprising the steps A and B, each of which is performed after the other step is performed.

3. The analysis pre-processing method according to claim 1, The reagent A contains a thiol or a phosphine as a partial structure of a reducing agent, The enzyme contained in the reagent B is a protease, An analytical pretreatment method, characterized in that the average diameter of the carrier is 0.5 μm to 500 μm.

4. The analysis pre-processing method according to claim 1, An analytical pretreatment method comprising a heating step within the range of 30°C to 70°C.

5. The analysis pre-processing method according to claim 1, The equivalent amount of the reducing agent contained in the reagent A is 2 to 1,000,000 equivalents relative to the disulfide bond in the sample, The enzyme comprises at least one enzyme selected from trypsin, chymotrypsin, pepsin, Lys-C, Lys-N, and Glu-C; An analytical pretreatment method, wherein the carrier contains at least one material selected from the group consisting of silica, polystyrene, acrylic resin, polysaccharides, simple metals, semiconductors, metal oxides, and magnetic metal oxides.

6. The analysis pre-processing method according to claim 1, The analytical pretreatment method, wherein the step X-A and the step X-B each include at least one method selected from the group consisting of spontaneous precipitation, centrifugation, magnetic accumulation, sonic trapping, and filtration.

7. A specimen pretreatment device capable of carrying out the analysis pretreatment method according to claim 1, A specimen pretreatment device comprising a control mechanism, a sample container storage mechanism, a sample container heating and shaking mechanism, a reagent dispensing mechanism, and a supernatant recovery mechanism.

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