Method for preparing peptide-containing samples for mass spectrometry, and method for separating non-magnetic solid-phase supports.

Abstract

The present invention addresses the problem of providing a method for preparing a peptide-containing sample, whereby it becomes possible to identify a greater number of trace proteins and treat a greater number of samples automatically in a proteome analysis of a biological sample such as dried paper filter spots. A fibrillar assembly having a biological sample adsorbed thereon is washed with a water-based solvent to remove a hydrophilic protein contained in the biological sample from the fibrillar assembly. Remaining proteins bound to the fibrillar assembly are digested with a protein degrading enzyme to remove the fibrillar assembly from a digestion reaction mixture, thereby producing a peptide mixture. Magnetic particles such as an iron powder are adhered to the fibrillar assembly, and the fibrillar assembly is magnetically adsorbed. In this manner, an operation such as the collection, washing and transfer of the fibrillar assembly having the biological sample adsorbed thereon is performed.　The present invention also addresses the problem of providing a technology for easily and reliably separating a non-magnetic solid carrier dispersed / floating in a liquid from the liquid. A liquid containing magnetic particles and a non-magnetic solid carrier is stirred to cause the magnetic particles to be adhered to the non-magnetic solid carrier. The non-magnetic solid carrier having the magnetic particles adhered thereto is magnetically adsorbed, thereby separating the non-magnetic solid carrier from the liquid. When a non-magnetic solid carrier having a porous structure is used, the magnetic particles penetrate into voids in the porous structure by the stirring, and therefore the non-magnetic solid carrier can be magnetically adsorbed more reliably. When a thickening agent is added to the liquid, the sedimentation rates of the non-magnetic solid carrier and the magnetic particles are reduced. As a result, both of the non-magnetic solid carrier and the magnetic particles can be mixed in the liquid more satisfactorily, the magnetic particles are incorporated in the non-magnetic solid carrier uniformly, and the adsorption to a magnet can be ensured more reliably.

Description

Technical Field

[0001] The present invention relates to a method for preparing a peptide-containing sample for identifying proteins contained in a biological sample by mass spectrometry. The present invention also relates to a method for separating a non-magnetic solid-phase carrier dispersed and suspended in a liquid from the liquid.

Background Art

[0002] Early detection of genetic diseases is important for preventing onset and reducing symptoms. Newborn screening is widely performed to detect diseases in newborns, and dried blood spots (DBS), which are minimally invasive, excellent in storage stability and transportability, are widely used therein. Conventional newborn screening targeting specific proteins such as metabolites and enzymes in DBS has different detection methods for each protein, and thus has the problem that labor, cost and time increase as the number of test items increases. On the other hand, non-targeted DBS proteome analysis can simultaneously and collectively detect a large number of disease-causing proteins in a single analysis, and it is considered that rapid and inexpensive newborn screening can be realized. In addition, in recent years, screening by genome-wide sequencing has also attracted attention, but it is still expensive, and DNA sequence information easily enables individual identification, which also involves ethical problems. On the other hand, newborn screening based on comprehensive protein detection has the potential to realize low-cost, rapid and comprehensive disease screening. Furthermore, proteome information is not directly linked to individual identification unlike genome information, so it is considered that ethical problems are also minor.

[0003] In DBS proteome analysis, proteins are typically extracted directly from DBS, subjected to reductive alkylation treatment as needed, and then analyzed by liquid chromatography-mass spectrometry (LC-MS) after enzymatic digestion. However, hydrophilic proteins such as hemoglobin, albumin, and globulin, which are present in large quantities in DBS, interfere with the detection of trace proteins. Therefore, it is necessary to remove high-abundance proteins as much as possible during the pretreatment stage to maximize the content ratio of trace proteins. However, there is a dilemma in that removing high-abundance proteins may inadvertently remove coexisting trace proteins. Normally, these high-abundance proteins are specifically adsorbed and removed using antibody columns conjugated with antibodies against them. However, commercially available antibody columns are very expensive, costing 6,000 to 7,000 yen each, making them unsuitable for neonatal screening where lower costs are required. Using the present invention, it is possible to easily remove high-abundance proteins and detect trace proteins using inexpensive general reagents.

[0004] To date, many groups have attempted to improve the number of proteins and peptides identified in non-targeted proteomic analysis of DBS. Non-patent document 1 describes how proteins in DBS were extracted by treating the surface of DBS with a 50 mM aqueous ammonium bicarbonate solution, and how 120 proteins were identified by tryingpsinizing the extract and performing LC-MS / MS analysis using data-dependent acquisition (DDA).

[0005] Non-patent document 2 describes how proteins were extracted from DBS using 25 mM ammonium bicarbonate, 1% sodium deoxycholate, and 5 mM tris(2-carboxyethyl)phosphine hydrochloride. The resulting protein extract was alkylated with iodoacetamide, triedpsinized, and then analyzed by LC-MS / MS with DDA to identify 253 proteins.

[0006] In Non-Patent Literature 3, proteins were extracted from DBS using 50 mM ammonium bicarbonate buffer (ABC buffer), the extract was treated with trypsin, and LC-MS / MS analysis was performed using DDA combined with Field Asymmetric Ion Mobility Spectrometry (FAIMS), identifying approximately 350 proteins.

[0007] Non-patent document 4 describes how solid-phase-liquid extraction was performed from DBS disks using 50 mM ammonium bicarbonate buffer and 2% sodium deoxycholate, the liquid phase was recovered, alkylated with iodoacetamide, treated with trypsin, and analyzed by LC-MS / MS with DDA to identify 295 proteins.

[0008] Meanwhile, Nieman et al. treated DBS samples with 6M urea, 50 mM ammonium bicarbonate, and 0.1 mM dithiothreitol to redissolve the proteins, alkylated them with 0.1 mM iodoacetamide, and then triedpsinized them. They then performed LC-MS / MS analysis using Data Independent Acquisition (DIA) and successfully identified 712 proteins (Non-Patent Literature 5).

[0009] To overcome the effects of hematocrit values ​​and sample heterogeneity, van den Broek et al. utilized volumetric absorbent microsampling (VAMS) instead of DBS. They extracted proteins from samples using 2% w / v octyl-beta-glucopyranoside, 8.3 mM Tris(2-carboxyethyl)phosphine, and 73.3 mM Tris, 2.9 mM calcium chloride buffer (pH 8.5). The extracts were alkylated with 200 mmol / L MMTS, trypsinized, and analyzed by LC-MS / MS using DIA, identifying 423 proteins (Non-Patent Literature 6).

[0010] The inventors have successfully increased the number of identified proteins in a single LC-MS analysis to over 2000 proteins, including 500 disease-causing proteins, by using the sodium carbonate precipitation method (SCP) to easily concentrate hydrophobic proteins from DBS and introducing DIA-MS (Non-Patent Literature 7). In this method, DBS is crushed in a 100 mM sodium carbonate aqueous solution, the proteins adsorbed onto filter paper are extracted into a sodium carbonate aqueous solution, and the insoluble filter paper is precipitated by centrifugation at 3000 xg, the supernatant is collected, and the filter paper is removed. Next, the obtained supernatant is centrifuged at 17,400 xg to precipitate poorly soluble proteins. This precipitate is washed with a 100 mM sodium carbonate aqueous solution, centrifuged again at 17,400 xg to precipitate again, and the supernatant is removed to isolate the SCP. This SCP is subjected to reductive alkylation treatment, Trypsin digestion, and LC-MS / MS analysis. However, this method has drawbacks: the extracted proteins are difficult to visualize during the extraction process, and there is a high risk of losing the small, slippery hydrophobic protein precipitate. Therefore, it requires a well-trained operator to perform the entire procedure with meticulous care. Consequently, it is not well-suited for throughput processing and is difficult to apply to neonatal screening where rapid processing of large quantities of samples is required.

[0011] On the other hand, when washing a solid support containing an analytical sample to remove unwanted substances such as dirt, or when removing a solid support that is no longer needed after extracting analytes such as proteins and metabolites from it, it is generally necessary to add the desired solution to the solid support, stir it, centrifuge it to settle the solid support, and then remove or recover the supernatant. However, when using centrifugation to recover the solid support, there is a risk of aspirating the settled solid support when removing or recovering the supernatant, or the settled solid support may float back up due to vibrations during the recovery operation, so the procedure must be carried out carefully. As a result, the operation becomes complicated when processing large quantities of samples, and automated processing is difficult. It is also possible to directly pick up the solid support in the container with tweezers, but there is a risk of sample contamination by the tweezers, and the operation is complicated, making it unsuitable for processing large quantities of samples.

[0012] When magnetic beads are used as the solid phase support, operations such as recovery, washing, and separation of the solid phase support can be automated by combining them with automated equipment equipped with magnetic rods, such as the Maelstrom 8 Autostage (MS8) (manufactured by Taiwan Advanced Nanotech). However, the types of samples that can be analyzed using commercially available magnetic beads are limited, and when using non-magnetic solid phase supports such as filter paper, gel pieces, or cotton, the process remains cumbersome.

[0013] Patent documents 1 and 2 disclose a method for removing non-magnetic suspensions (colloidal substances) in a liquid by magnetic coagulation together with magnetic suspensions. In this method, a magnetic field is formed by arranging the magnetic poles of multiple magnets in a counter-pole structure on two rotating plates attached to a magnetic coagulation treatment tank, and a liquid containing impurities is passed through it. As a result, the impurities magnetically coagulate with the magnetic suspensions as nuclei, while embracing the non-magnetic suspensions, and these magnetic aggregates are attracted to magnets. This technology is used in electrical discharge machining (EDM) machines to remove fine impurities (colloidal substances) such as magnetic suspensions like chips and metal powders, and non-magnetic suspensions like carbon, mud, and oil, from oily or aqueous machining fluids used during electrical discharge machining. [Prior art documents] Patent Literature

[0014] Patent Literature 1 Japanese Unexamined Patent Publication No. Hei 6-71195 Patent Literature 2 Japanese Unexamined Patent Publication No. Hei 9-248483 Non-Patent Literature

[0015] Non-Patent Literature 1 Martin et al., Journal of the American Society for Mass Spectrometry, 24(8), 1242-1249, 2013 Non-Patent Literature 2 Chambers et al., Journal of the American Society for Mass Spectrometry, 24(9), 1338-1345, 2013 Non-Patent Literature 3 Rosting et al., Journal of Proteome Research, 17(6), 1997-2004, 2018 Non-Patent Literature 4 Eshghi et al., Molecular and Cellular Proteomics, 19(3), 540-553, 2020 Non-Patent Literature 5 Nieman et al., Proteomes, 8(1), 4, 2020 Non-Patent Literature 6 Van Den Broek et al., Clinical Mass Spectrometry, 4-5, 25-33, 2017 Non-Patent Literature 7 Nakajima et al., Journal of Proteome Research, 19(7), 2821-2827 Summary of the Invention Problems to be Solved by the Invention

[0016] An object of the present invention is to provide a method for preparing a peptide-containing sample, which enables identification of a larger number of trace proteins and automatic processing of a large number of samples by inexpensively and rapidly removing high-abundance proteins in blood in proteome analysis. Another object of the present invention is to provide a technique for easily and reliably separating non-magnetic solid-phase carriers dispersed and suspended in a liquid from the liquid. Means for Solving the Problems

[0017] The inventors of the present invention have conducted intensive studies to solve the above problems, and found that in all conventional DBS proteome analysis, proteins adsorbed on filter paper are extracted into an aqueous solution, the obtained proteins in the extract are treated with an enzyme or the like, and analyzed by LC-MS / MS, and the filter paper is discarded (Non-Patent Documents 1 to 7). The inventors of the present invention focused on the filter paper residue that had been discarded after protein extraction, and analyzed the proteins remaining therein. Surprisingly, it was found that even after the protein extraction operation, many proteins including trace proteins still remain adsorbed on the filter paper residue. After washing this filter paper residue, the proteins adsorbed on the filter paper were trypsinized while remaining adsorbed, and the obtained peptide fragments were analyzed by LC-MS / MS. As a result, more than 4000 types of proteins were successfully identified. This greatly exceeded the number of identified proteins obtained by the conventional method of analyzing the protein extract from filter paper by LC-MS. The washing operation of the filter paper appropriately washed away hydrophilic proteins such as albumin contained in large amounts in DBS, which had hindered the detection of trace proteins, while most of the trace proteins remained adsorbed on the filter paper, leading to a high number of identified proteins.

[0018] Furthermore, when magnetic particles such as iron powder were suspended and stirred in a liquid containing a non-magnetic solid support, and then a magnet was brought near, the magnetic particles adhered to the non-magnetic solid support. The non-magnetic solid support with the attached magnetic particles was then attracted to the magnet, successfully separating the non-magnetic solid support from the liquid in a magnetic way. As a result, it became possible to wash, recover, and remove the non-magnetic solid support without using centrifugation or other methods. When a non-magnetic solid support with a porous structure was used, stirring caused the magnetic particles to enter the voids within the porous structure, allowing the non-magnetic solid support to firmly embrace the magnetic particles, resulting in more reliable attraction and recovery of the non-magnetic solid support to the magnet. In particular, when a fibrous aggregate such as filter paper was used as the non-magnetic solid support, stirring loosened the fibers, widening the voids between the fibers, allowing a large amount of magnetic particles to be incorporated into the non-magnetic solid support, thereby strengthening its attraction to the magnet. Adding a thickener to the liquid reduced the settling velocities of both the non-magnetic solid support and the magnetic particles to a similarly low level. This allowed for thorough mixing of the two in the liquid, resulting in uniform incorporation of the magnetic particles into the non-magnetic solid support and more reliable magnetization. As a result, it became possible to automatically process large quantities of non-magnetic solid support samples while minimizing the risk of errors in recovering the non-magnetic solid support during the process.

[0019] Accordingly, in order to apply this method for separating non-magnetic solid-phase carriers using magnetic particles to the above-mentioned extraction method for filter paper-binding proteins, and to adapt this method to disease screening that requires processing multiple samples as quickly and accurately as possible, the present inventors aimed to establish an automatic extraction method for filter paper-binding proteins using an automatic nucleic acid extraction apparatus. By adding iron powder when crushing filter paper in a solution, the iron powder enters between the fibers of the swollen, pulp-like filter paper to form a "complex" of filter paper and iron powder, and this complex can be easily recovered using a magnet. By this method, the automatic processing of DBS crushing, complex formation, recovery, washing, and buffer replacement was successfully achieved. The finally obtained filter paper was suspended in a buffer for trypsin digestion, and the protein bound to the filter paper was digested with trypsin to form peptides, which were then subjected to proteomic analysis by DIA-MS. As a result, 5817 proteins, a number far exceeding that obtained by conventional methods, could be identified. Furthermore, as a result of comparison with the OMIM database, it was found that as many as 1895 disease-related proteins were included, indicating that this method has the potential to be used for far more disease screening than conventional methods. Furthermore, as a result of examining the reproducibility between days and between analyzers, a high Pearson correlation coefficient r of 0.96 or higher was obtained, demonstrating that the method can be sufficiently applied to the inspection of samples submitted daily. Based on these findings, the present inventors conducted further studies and completed the present invention.

[0020] That is, the present invention relates to the following. [1] A method for preparing a peptide-containing sample for identifying a protein contained in a biological sample by mass spectrometry from a fiber aggregate to which a biological sample containing a protein is adsorbed, comprising the following steps: 1) washing a fiber aggregate to which a biological sample containing a protein is adsorbed with an aqueous solvent to remove hydrophilic proteins contained in the biological sample from the fiber aggregate, and recovering the fiber aggregate to which residual proteins are bound; 2) treating the recovered fiber aggregate to which residual proteins are bound with a proteolytic enzyme, thereby digesting the residual proteins bound to the fiber aggregate with the proteolytic enzyme; and 3) Recover the peptide mixture as a digestion product by removing fibrous aggregates from the digestion reaction mixture. [2] A method for preparing [1], wherein the fiber aggregate is a cellulose fiber aggregate. [3] A method for preparing [2], wherein the cellulose fiber aggregate is filter paper. [4] A method of preparation of any of [1] to [3], wherein a fibrous aggregate to which a protein-containing biological sample has been adsorbed is a dried blood spot. [5] A preparation method according to any of [1] to [4], further comprising subjecting the fibrous aggregate to which residual protein is bound to a reductive alkylation treatment. [6] A method for separating a non-magnetic solid support from a liquid containing a non-magnetic solid support, comprising the following steps: 1) To provide a liquid containing magnetic particles and a non-magnetic solid support; 2) Stir the liquid containing magnetic particles and a non-magnetic solid support to cause the magnetic particles to adhere to the non-magnetic solid support; 3) Bringing a magnet close to a liquid containing magnetic particles and a non-magnetic solid support, and magnetically adsorbing the non-magnetic solid support to which the magnetic particles have adhered; and 4) Separating a magnetically adsorbed non-magnetic solid support from a liquid. [7] The method of [6] wherein the non-magnetic solid support has a porous structure, and magnetic particles enter the voids in the porous structure by stirring. [8] The method of [7], wherein the nonmagnetic solid-phase support comprises a fibrous aggregate. [9] Any of the methods [6] to [8] wherein the nonmagnetic solid support is in the form of a sheet.

[10] Any method of [6] to [9], wherein the nonmagnetic solid support is selected from the group consisting of paper, gel, cloth, resin, cotton and swab.

[11] A method in which the liquid contains a thickening agent, one of the methods in [6] to

[10] .

[12] Any method of [6] to

[11] wherein the magnetic particles are ferromagnetic metal particles or ferromagnetic metal oxide particles.

[13] The method of

[12] wherein the magnetic particles are iron powder.

[14] Any of the methods [6] to

[13] , wherein the average particle diameter of the magnetic particles is 150 μm or less.

[15] Disperse 0.3 mg or more of magnetic particles in 1 ml of liquid using any of the methods [6] to

[14] .

[16] Any of the methods [6] to

[15] , wherein 0.2 mg or more of magnetic particles are dispersed in a liquid per 1 mg of non-magnetic solid support.

[17] A method for preparing [1], further comprising stirring a fibrous aggregate on which a biological sample containing magnetic particles and protein has been adsorbed in an aqueous solvent to cause the magnetic particles to adhere to the fibrous aggregate.

[18] The preparation method of

[17] , wherein the aqueous solvent used in step 1 contains magnetic particles, and step 1 is performed by stirring the fibrous aggregate on which a biological sample containing protein has been adsorbed with the aqueous solvent containing magnetic particles to wash the fibrous aggregate with the aqueous solvent, thereby removing the hydrophilic protein contained in the biological sample from the fibrous aggregate, and attaching the magnetic particles to the fibrous aggregate, and recovering the fibrous aggregate to which the magnetic particles have been attached and to which residual protein has been bound.

[19] A method for preparing

[17] or

[18] , wherein the fibrous aggregate to which residual protein in step 1 is bound is recovered by magnetic adsorption.

[20] A preparation method of any of

[17] to

[19] , wherein the fibrous aggregates in step 3 are removed by magnetic adsorption.

[21] A method of preparation of any of

[17] to

[20] , wherein the magnetic particles are iron powder.

[22] A method for preparing any of the following,

[17] to

[21] , wherein the aqueous solvent contains a thickening agent.

[23] A method for preparing

[22] , wherein the thickener is glycerol. [Effects of the Invention]

[0021] The present invention makes it possible to easily prepare a peptide-containing sample from a fibrous aggregate to which a biological sample has been adsorbed, in which the content of peptides derived from hydrophilic proteins such as albumin, which are present in large quantities in the biological sample, is suppressed, and peptides derived from trace proteins are abundant. By subjecting the peptide-containing sample obtained by the preparation method of the present invention to LC-MS analysis, it becomes possible to identify numerous proteins, including trace proteins.

[0022] Furthermore, the present invention makes it possible to separate non-magnetic solid-phase supports from liquids in a magnetic-dependent manner, thus enabling the washing, recovery, and removal of non-magnetic solid-phase supports without the use of centrifugation or the like. Conventionally, when continuously washing, extracting, or otherwise processing non-magnetic solid-phase supports with liquid, the non-magnetic solid-phase supports are left in the container and precipitated by centrifugation before the washing and extraction liquid is added and recovered. As a result, when removing or recovering the liquid from the container, the precipitated non-magnetic solid-phase supports may be sucked in or float to the surface, leading to loss of the non-magnetic solid-phase supports. In the method of the present invention, the non-magnetic solid-phase supports are removed from the container while the washing and extraction liquid remains in the container, thus minimizing the loss of non-magnetic solid-phase supports while continuously washing and extracting them with liquid. Since automated devices for handling magnetic solid-phase supports such as magnetic beads are already widely available, combining this method with them enables large-scale automated processing of non-magnetic solid-phase supports, which was previously difficult.

[0023] Furthermore, by applying this method of separating non-magnetic solid-phase supports using magnetic particles to the preparation of peptide-containing samples from fiber aggregates to which biological samples have been adsorbed, and by attaching magnetic particles to the fiber aggregates, operations such as washing, recovery, and buffer replacement of fiber aggregates to which biological samples have been adsorbed by magnetic adsorption can be easily performed. In addition, by automating the process using an automated nucleic acid extraction device, it becomes possible to process a large number of samples in a short time, and the reproducibility of the results is also improved, making it possible to apply this to mass screening of neonatal congenital diseases using dried blood spots. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows flowcharts for the preparation of insoluble proteins from sodium carbonate precipitate (A) and filter paper-bound proteins from DBS (B). [Figure 2]Figure 2 shows the optimization of the m / z range for DIA-MS for FPBP proteome analysis. The bar graphs show the number of proteins (left) and peptides (right) detected in each m / z range. The left y-axis represents the number of proteins, and the right y-axis represents the number of peptides. [Figure 3] Figure 3 shows a Venn diagram of the number of proteins isolated by the SCP and FPBP methods. "SCP" stands for sodium carbonate precipitate, and "FPBP" stands for filter paper-bound protein. [Figure 4] A schematic diagram of the automated isolation process for FPBP using the Maelstrom 8 Autostage / Maelstrom 9610 is shown. [Figure 5A] Figure 5A shows a Venn diagram of the number of proteins identified using two different preparation methods. "FPBP" refers to the manual protein preparation method using filter paper-bound proteins, and "Auto-FPBP" refers to the automated protein preparation method using filter paper-bound proteins. [Figure 5B] Figure 5B is a bar graph showing the total intensity of high-abundance proteins in blood prepared by manual or automated FPBP methods. The high-abundance proteins were HBB, HBA2, IGGH1, APOA1, and ALB. [Figure 5C] Figure 5C is a violin plot showing the coefficient of variation of protein quantification values ​​for FPBP and Auto-FPBP. The Y-axis represents CV(%). [Figure 6A] Figure 6A is a graph comparing the number of proteins identified using three different preparation methods. "SCP" refers to the protein preparation method using sodium carbonate precipitation, "FPBP" refers to the manual protein preparation method using filter paper-bound proteins, and "Auto-FPBP" refers to the automated protein preparation method using filter paper-bound proteins. From top to bottom, the graph shows the number of plasma-derived proteins, blood cell-derived proteins, and other proteins. [Figure 6B] Figure 6B is a graph showing the number of membrane proteins identified using the three different preparation methods. [Figure 6C] Figure 6C is a graph showing the number of OMIM-hit proteins in the proteins identified using the three different preparation methods. [Figure 7]Figure 7 is a graph showing the evaluation of inter-instrument and inter-day reproducibility in automated protein preparation using filter paper-bound proteins. Protein intensities of samples 1-12, 13-24, and 25-36, prepared by the Auto-FPBP method on days 0, 32, and 60, respectively, were measured using LC-MS instruments A and B, and Pearson correlation analysis was performed. [Figure 8] Figure 8 is a schematic diagram comparing the pretreatment flow of proteins attached to filter paper for proteome analysis using the conventional method and the separation method (improved method) of the present invention. [Figure 9] Figure 9 shows how the filter paper and gel pieces in the TBST are magnetically attracted by the magnetic force between the iron powder suspended in the TBST and the magnet. [Figure 10] Figure 10 shows DBS and iron powder being added to TBST and stirred with MS8. [Figure 11] Figure 11 shows how DBS in the liquid phase is recovered by the magnetic force between the iron powder suspended in the liquid phase and the magnet provided in the MS8. [Modes for carrying out the invention]

[0025] 1. Method for preparing peptide-containing samples The present invention provides a method (hereinafter referred to as the preparation method of the present invention) for preparing a peptide-containing sample for identifying proteins contained in a biological sample by mass spectrometry, from a fibrous aggregate on which a protein-containing biological sample has been adsorbed, comprising the following steps. 1) Washing the fibrous aggregate to which a protein-containing biological sample has adsorbed with an aqueous solvent removes the hydrophilic protein contained in the biological sample from the fibrous aggregate, and recovers the fibrous aggregate to which residual protein is bound; 2) Digesting the residual proteins bound to the fiber aggregates with proteolytic enzymes by treating the recovered residual proteins bound to the fiber aggregates with proteolytic enzymes; and 3) Recover the digested product as a peptide mixture by removing fibrous aggregates from the digested mixture.

[0026] In conventional preparation methods, proteins adsorbed to fiber aggregates, including a large amount of hydrophilic proteins, are extracted into an aqueous solution. The proteins in the resulting extract are then treated with enzymes and analyzed by LC-MS / MS, while the fiber aggregates themselves are discarded (Non-Patent Documents 1-7). The inventors focused on the discarded fiber aggregate residue and analyzed the proteins remaining in it. Surprisingly, they found that despite the protein extraction procedure, a large amount of proteins, including trace amounts, remained adsorbed to the fiber aggregate residue. This invention was completed based on these findings.

[0027] Examples of biological samples used in the present invention include, but are not limited to, body fluids such as blood (whole blood, serum, plasma), saliva, urine, sweat, lymph, sputum, tears, nasal mucus, semen, cerebrospinal fluid, pleural fluid, ascites, synovial fluid, pericardial fluid, and interstitial fluid, cells, tissues or parts thereof, cell or tissue lysates, cell or tissue extracts, biopsy materials, swab samples, feces, cell cultures, bacteria, viruses, and fungi. Biological samples may also be extracts or concentrates of specific biological substances. Biological samples typically contain proteins.

[0028] A fiber aggregate is a structure in which fibers are layered in three dimensions, and the fibers may or may not be intertwined. Examples of fiber aggregates include, but are not limited to, cotton-like, paper-like, cloth-like, or felt-like structures. Examples of fibers that make up a fiber aggregate include, but are not limited to, natural fibers such as cellulose, keratin, silk, chitin, and hemicellulose, and synthetic fibers such as nylon, polyester, acrylic, and rayon. The fibers that make up a fiber aggregate are preferably cellulose. That is, the fiber aggregate is preferably a cellulose fiber aggregate. The cellulose may or may not be modified. Examples of modifications include, but are not limited to, nitration, esterification (nitrate esterification, sulfate esterification, etc.), amination, acetylation, and methoxylation. The cellulose is preferably unmodified cellulose. Cellulose becomes hydrophobic and insoluble when hydrophilic β-glucose units are polymerized linearly by β-1,4 glucosidic bonds. While not bound by theory, cellulose fibers, possessing both hydrophilic and hydrophobic portions, can adsorb proteins of diverse properties. Examples of cellulose fiber aggregates include, but are not limited to, paper (filter paper, etc.), cotton, cotton yarn, and cotton cloth. Filter paper is preferred as the cellulose fiber aggregate.

[0029] The fiber aggregates may be contained in the aqueous solvent in a suspended, dispersed, or floating state. The size (maximum diameter) of the fiber aggregates is not particularly limited, as long as the fiber aggregates can be suspended, dispersed, or floated in the aqueous solvent within the container in which the preparation method of the present invention is carried out. Preferably, the size (maximum diameter) of the fiber aggregates is less than the inner diameter of the opening of the container (well) in which the preparation method of the present invention is carried out, so as to facilitate the separation of the fiber aggregates from the aqueous solvent. The size (maximum diameter) of the fiber aggregates is usually 100 μm or more (e.g., 500 μm or more, 1 mm or more, 3 mm or more, 5 mm or more).

[0030] In one embodiment, the fibrous aggregate on which the biological sample is adsorbed is a dried blood spot (DBS). A dried blood spot is made by soaking collected blood into filter paper and drying it, and is used in newborn screening and doping tests for athletes. DBS is usually made by dropping 1 to 2 drops (about 20 to 40 microliters) of human blood onto filter paper in a circle with a diameter of about 1 cm, and then allowing it to air dry at room temperature. In one embodiment, a disc with a diameter of 3.2 mm (equivalent to about 3 microliters of blood) punched out from the DBS is used in the preparation method of the present invention. In this specification, a dried blood spot may be referred to as dried filter paper blood.

[0031] In the preparation method of the present invention, first, the fibrous aggregate to which the biological sample has been adsorbed is washed with an aqueous solvent. This washing operation removes hydrophilic proteins contained in the biological sample from the fibrous aggregate. Regardless of the washing operation, various proteins from the biological sample, including trace amounts of proteins, remain bound to the fibrous aggregate. Therefore, the fibrous aggregate to which the residual proteins are bound is recovered and used for sample preparation for mass spectrometry. Many biological samples contain large amounts of hydrophilic proteins such as albumin, γ-globulin, β-globin, hemoglobin A2, immunoglobulin, and apolipoprotein A. When these biological samples are directly analyzed by LC-MS, the signals of trace proteins are masked by the signals of these large amounts of hydrophilic proteins, making it difficult to identify the trace proteins. In the preparation method of the present invention, hydrophilic proteins present in large quantities in the biological sample are eluted into the aqueous solvent and removed by the washing operation. By subjecting the residual proteins bound to the fiber aggregates to LC-MS analysis, the masking of trace protein signals by hydrophilic proteins is suppressed, making it possible to detect and identify trace proteins with high sensitivity.

[0032] It is preferable to add an appropriate buffer to the aqueous solvent used for washing in order to maintain the pH. Suitable buffers include, but are not limited to, Tris-HCl, phosphates, citrates, acetates, HEPES, borates, and tartrates. Inorganic salts may also be added to the aqueous solvent used for washing to suppress the influence of salt concentration in the biological sample. Examples of inorganic salts include, but are not limited to, sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and ammonium sulfate. Surfactants may also be added to the aqueous solvent used for washing to promote the dissolution of hydrophilic proteins. Examples of surfactants include, but are not limited to, nonionic surfactants such as Tween-20, Tween-80, Triton X-100, and Nonidet P-40, ionic surfactants such as sodium dodecyl sulfate and sodium deoxycholate, and amphoteric surfactants such as CHAPS. Chelating agents may also be added to the aqueous solvent used for washing. Examples of chelating agents include, but are not limited to, EDTA, EGTA, DTPA, and HEDTA. The buffer, inorganic salt, surfactant, and chelating agent may be used individually or in combination of multiple types. The pH of the aqueous solvent used for washing is usually 5.5 to 9.5, preferably 6.5 to 9.0, and more preferably 7.0 to 8.5. Suitable aqueous solvents for washing include, but are not limited to, physiological saline with a buffer and a nonionic surfactant added, such as TBS-Tween (physiological saline with Tris-HCl and Tween-20 added) at pH 8.0.

[0033] Washing of fibrous aggregates to which biological samples have been adsorbed with an aqueous solvent can be performed, for example, by placing the fibrous aggregates to which the biological sample has been adsorbed and the aqueous solvent in a suitable tube, stirring, and centrifuging to remove the supernatant containing dissolved hydrophilic proteins from the biological sample, and then recovering the precipitate of fibrous aggregates to which residual proteins have been bound. The washing operation may be performed multiple times, and multiple types of aqueous solvents may be used for the washing operation.

[0034] To cleave intermolecular and intramolecular disulfide bonds between cysteine ​​molecules of the protein and prevent thissulfide formation, the fibrous aggregate to which residual protein is bound may be subjected to a reductive alkylation treatment. The reduction of residual protein can be carried out by heating the fibrous aggregate to which residual protein is bound with a reducing agent such as 1,4-dithiothreitol (DTT), 2-mercaptoethanol, or tris(2-carboxyethyl)phosphine. Alkylation of residual protein can be carried out by reacting the fibrous aggregate to which the reduced residual protein is bound with an alkylating reagent such as iodoacetamide, iodoacetic acid, acrylamide, or chloroacetamide. To promote reductive alkylation, a chaotropic agent may be added during the reduction treatment and / or the alkylation treatment. Examples of chaotropic agents include, but are not limited to, urea, thiourea, guanidine hydrochloride, thiocyanates, and sarcosine. After the reductive alkylation reaction, the fiber aggregate to which the reductive alkylated residual protein is bound may be washed with an aqueous solvent to remove unreacted reducing and alkylating agents. The aqueous solvents listed for removing hydrophilic proteins from the fiber aggregate can be used. Note that the reductive alkylation treatment is an optional step, and it may be omitted if speed and labor saving are prioritized.

[0035] Next, the fibrous aggregate to which residual protein (including reduced alkylated residual protein) is bound is treated with a protease to digest the residual protein bound to the fibrous aggregate. Specifically, the fibrous aggregate to which residual protein is bound is incubated in an aqueous solvent containing the protease. Examples of proteases that can be used include, but are not limited to, endoproteases such as trypsin, Glu-C, Lys-N, Lys-C, Asp-N, and chymotrypsin. Multiple types of proteases may be used in combination. As the aqueous solvent, those listed as aqueous solvents used to remove hydrophilic proteins from the fibrous aggregate can be used. Suitable aqueous solvents include aqueous buffer solutions, such as Tris-HCl buffer pH 8.0, but are not limited to these. When the residual protein bound to the fibrous aggregate is digested by the protease treatment, the digestion product, peptides, are released from the fibrous aggregate into the aqueous solvent.

[0036] Then, by removing the fibrous aggregates from the digestion reaction mixture, the peptide mixture is recovered as the digestion product, and this is used as a peptide-containing sample for identifying proteins contained in the biological sample by mass spectrometry. Specifically, for example, the aqueous solvent containing the peptide mixture, which is the digestion product, and the digestion reaction mixture containing the fibrous aggregates are centrifuged to precipitate the fibrous aggregates, and the aqueous solvent containing the peptide mixture, which is the digestion product, is recovered from the supernatant.

[0037] To analyze the recovered peptide mixture in aqueous solvent by LC-MS, it is usually subjected to a desalting treatment to remove low molecular weight substances such as salts, buffers, and chaotropic agents, thereby purifying the peptide mixture. Desalting can be performed by placing the aqueous solvent containing the peptide mixture on a reversed-phase column. Examples of reversed-phase columns include, but are not limited to, columns in which hydrophobic groups such as octadecyl (C18), octyl (C8), butyl (C3), phenyl, and cyanopropyl groups are bonded to a solid support (e.g., silica gel), and styrene-divinylbenzene (SDB) copolymer columns. Preferably, the reversed-phase column is a column in which octadecyl (C18) groups are bonded to a solid support (e.g., silica gel) (referred to as a C18 column) or an SDB copolymer column. Commercially available reversed-phase columns can be used, and commercially available kits such as EVOSEP ONE or SDB-STAGE tips may also be used.

[0038] When an aqueous solvent containing a peptide mixture is applied to a reversed-phase column, the peptide mixture is adsorbed onto the column, while low-molecular-weight substances such as salts, buffers, and chaotropic agents are eluted and removed without binding to the column. The peptide mixture adsorbed onto the reversed-phase column can then be eluted to obtain a purified peptide mixture. The solvent used for peptide elution is typically a mixed solvent of water and an organic solvent such as acetonitrile, methanol, tetrahydrofuran, isopropanol, or acetone. To promote protonation, it is preferable to add an acid such as trifluoroacetic acid, formic acid, acetic acid, or hydrochloric acid to the water.

[0039] The eluted peptide mixture is dried to remove the solvent. Drying of the peptide mixture can be done, for example, using a centrifugal evaporator.

[0040] The resulting dried peptide mixture is redissolved in water. To promote protonation, an acid such as formic acid, trifluoroacetic acid, acetic acid, or hydrochloric acid may be added to the water. Alternatively, an organic solvent such as acetonitrile, methanol, tetrahydrofuran, isopropanol, or acetone may be added to the water. For example, the dried peptide mixture is dissolved in water containing 0.1% (v / v) formic acid to obtain an aqueous solution of the peptide mixture.

[0041] The resulting aqueous solution of the peptide mixture is then subjected to mass spectrometry. Based on the mass spectrometry results, the peptides contained in the peptide mixture are identified, and based on the information of the identified peptides, the proteins in the biological sample from which they originate are identified. Mass spectrometry of the peptide mixture is usually performed using a liquid chromatography-mass spectrometer (LC-MS), preferably a tandem mass spectrometer (LC-MS / MS) connected online to a liquid chromatograph. The measurement mode can be appropriately selected according to the purpose of measurement, but when comprehensively identifying proteins contained in a biological sample by non-targeted proteomics, measurement modes such as data-dependent acquisition (DDA) or data-independent acquisition (DIA) are selected, and when identifying or quantifying specific proteins by targeted proteomics, measurement modes such as S / MRM (Selected or Multiple Reaction Monitoring) may be selected.

[0042] DDA automatically selects multiple precursor ions with strong signal intensity from the top and acquires product ion spectra. From the obtained data, a file (peak list) is created that links the product ion spectra to peaks on the MS spectrum, and a database search is performed. In the database search, the peptide sequences obtained when all protein sequences registered in the arbitrary sequence database are enzymatically digested are theoretically predicted (for example, cleavage at the C-terminus of K / R in trypsin), and candidate peptide sequences with masses matching the precursor ions are narrowed down from these. Next, the m / z values ​​of the N-terminal ion (β-ion) and C-terminal ion (y-ion) generated when a peptide bond in the candidate peptide sequence is cleaved at one site are calculated for all peptide bonds, and the theoretical product ion spectra obtained are compared with the measured product ion spectra to identify specific peptide sequences.

[0043] In DIA, without selecting a precursor ion, the mixed product ion spectrum of all precursor ions that fit within a set m / z width Q1 window is repeatedly acquired while shifting the window. In DIA, a chromatogram of fragment ions derived from the peptide sequence to be investigated is extracted using an MS / MS information library created from separately acquired DDA data, and the chromatographic peak of the target peptide is identified from the co-elution pattern of the fragment ions.

[0044] In S / MRM, the combination of the m / z of the precursor ion of the target peptide identified by DDA and the m / z of the fragment generated by CID is specified in the measurement method, and the ions of the target peptide in the sample (fragment ion signal) are selectively monitored to obtain a chromatogram.

[0045] In DDA, if multiple analytes elute simultaneously and their abundances differ significantly, there is a relatively high risk that analytes with low abundances may not be detected in the initial MS spectrum, or that the mass spectrometer's speed may not keep up with acquiring MS / MS spectra for all peaks detected in MS mode (too slow for the complexity of the sample). In contrast, DIA collects the full MS / MS spectra of all detectable analytes passing through each Q1 window, and the entire mass range is analyzed within the time of LC analysis. Therefore, it is possible to obtain complete MS and MS / MS spectra for virtually all detectable peaks in the sample, which is advantageous for the detection and identification of analytes with low abundances. In the preparation method of the present invention, hydrophilic proteins such as albumin, which are present in large quantities in the biological sample, are removed during the pretreatment step while maintaining trace amounts of protein. Therefore, it is possible to prepare a peptide-containing sample with a high content of peptides derived from trace proteins. Accordingly, by subjecting the peptide-containing sample prepared by the preparation method of the present invention to LC-MS / MS analysis in DIA measurement mode, it becomes possible to detect and identify trace amounts of protein and peptides derived therefrom in the biological sample with higher sensitivity, thereby achieving more sensitive and in-depth proteomic analysis.

[0046] Therefore, the preparation method of the present invention can also be considered as a method for identifying proteins contained in a biological sample by mass spectrometry, comprising the following steps: 0) To provide a fibrous aggregate on which a biological sample containing protein has been adsorbed; 1) Washing the fibrous aggregate to which a protein-containing biological sample has adsorbed with an aqueous solvent removes the hydrophilic protein contained in the biological sample from the fibrous aggregate, and recovers the fibrous aggregate to which residual protein is bound; 2) Digesting the residual proteins bound to the fibrous aggregates with proteolytic enzymes; 3) Recovering the peptide mixture as a digestion product by removing fibrous aggregates from the digestion reaction mixture; 4) The recovered peptide mixture is subjected to a desalting treatment to obtain a purified peptide mixture; 5) Subject the purified peptide mixture to mass spectrometry and identify the peptides contained in the peptide mixture based on the obtained mass spectrometry results; and 6) Identify the protein in the biological sample from which the identified peptide originates, based on the information obtained from the peptide.

[0047] As detailed in the examples, by mass spectrometry of a peptide-containing sample prepared from DBS (preferably neonatal DBS) using the preparation method of the present invention, proteins listed in Tables 1-1 to 1-20 that could not be detected by the conventional SCP method were identified. These included OMIM hit proteins listed in Tables 2-1 to 2-7. Therefore, in one embodiment, by mass spectrometry of a peptide-containing sample prepared from DBS (preferably neonatal DBS) using the preparation method of the present invention, at least one protein selected from the proteins listed in Tables 1-1 to 1-20 in the DBS, for example, at least 5, 10, 20, 50, 100, 200, 300, 400, 500, or 1000 proteins, can be identified in the DBS. Furthermore, in one embodiment, a peptide-containing sample prepared from DBS (preferably neonatal DBS) by the preparation method of the present invention is subjected to mass spectrometry to identify at least one protein selected from the OMIM hit proteins listed in Tables 2-1 to 2-7, for example, at least 5, 10, 20, 50, 100, 200, 300, or 400 proteins in the DBS.

[0048] Table 3 shows some of the proteins that can be identified by mass spectrometry of peptide-containing samples prepared from DBS (preferably neonatal DBS) using the preparation method of the present invention, and which are expected to be applicable to neonatal screening. Accordingly, in one embodiment, by mass spectrometry of peptide-containing samples prepared from DBS (preferably neonatal DBS) using the preparation method of the present invention, at least one protein, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, selected from the proteins listed in Table 3, is identified in the DBS. Of the proteins listed in Table 3, Coagulation factor VIII and Collagen alpha-1(I) chain were not detected by SCP and were detected from DBS for the first time using the preparation method of the present invention. Accordingly, in one embodiment, by mass spectrometry of peptide-containing samples prepared from DBS (preferably neonatal DBS) using the preparation method of the present invention, at least one, preferably both, selected from Coagulation factor VIII and Collagen alpha-1(I) chain, is identified in the DBS.

[0049] 2. Method for separating non-magnetic solid-phase supports The present invention provides a method for separating a non-magnetic solid support from a liquid containing a non-magnetic solid support (hereinafter referred to as "the separation method of the present invention"), comprising the following steps: 1) To provide a liquid containing magnetic particles and a non-magnetic solid support; 2) Stir the liquid containing magnetic particles and a non-magnetic solid support to cause the magnetic particles to adhere to the non-magnetic solid support; 3) Bringing a magnet close to a liquid containing magnetic particles and a non-magnetic solid support, and magnetically adsorbing the non-magnetic solid support to which the magnetic particles have adhered; and 4) Separating a magnetically adsorbed non-magnetic solid support from a liquid.

[0050] In the separation method of the present invention, first, a liquid containing magnetic particles and a non-magnetic solid support is provided. Specifically, a liquid containing magnetic particles and a non-magnetic solid support is obtained by adding magnetic particles to a liquid containing a non-magnetic solid support, adding a non-magnetic solid support to a liquid containing magnetic particles, or adding a non-magnetic solid support and magnetic particles to a liquid.

[0051] In this specification, "carrier" means a substance that serves as a base for adsorbing or fixing other substances. The carrier used in the separation method of the present invention is a non-magnetic solid-phase carrier. In this specification, "solid phase" also includes gels. In one embodiment, the non-magnetic solid-phase carrier used in the separation method of the present invention contains a sample for analysis (e.g., a biological sample), and the separation method of the present invention is carried out for purposes such as removing unwanted substances from the non-magnetic solid-phase carrier, extracting necessary substances from the non-magnetic solid-phase carrier, or modifying the sample contained in the non-magnetic solid-phase carrier, as a pretreatment for the intended analysis. The non-magnetic solid-phase carrier used in the separation method of the present invention preferably has a porous structure and has voids inside. These voids are large enough for magnetic particles to enter. In the separation method of the present invention, when a non-magnetic solid-phase carrier having a porous structure is used, the magnetic particles enter the voids in the porous structure by stirring, and the magnetic particles adhere firmly to the non-magnetic solid-phase carrier and become difficult to detach from the non-magnetic solid-phase carrier, so more reliable magnetic adsorption can be expected when a magnet is brought close. Porous structures include fibrous aggregates, foams, and network structures. A fibrous aggregate is a structure in which fibers are layered in three dimensions, and the fibers may or may not be intertwined. Examples of fibrous aggregates include, but are not limited to, cotton-like, paper-like, cloth-like, or felt-like structures. A foam is a structure in which many air bubbles are dispersed in a matrix, and examples include sponge-like structures. A network structure is a structure in which polymers are bonded together in a three-dimensional network.

[0052] The materials constituting the non-magnetic solid support used in the separation method of the present invention include, but are not limited to, paper (filter paper, etc.), gel (agarose gel, polyacrylamide gel, etc.), cloth (nonwoven fabric, etc.), resin, cotton, swab, glass, non-magnetic metal, ceramics, etc. In one embodiment, the non-magnetic solid support used in the separation method of the present invention includes a fibrous aggregate composed of materials such as paper (filter paper, etc.), cloth (nonwoven fabric, etc.), resin, cotton, swab, glass, non-magnetic metal, and ceramics.

[0053] In one embodiment, the non-magnetic solid support used in the separation method of the present invention is in the form of a sheet. "Sheet-like" means having a two-dimensional surface and two opposing surfaces (front and back) separated by a distance equal to its thickness. Its thickness is not particularly limited, but is, for example, 5 mm or less (e.g., 1 nm to 5 mm), preferably 1 mm or less (e.g., 1 nm to 1 mm). When a sheet-like non-magnetic solid support is used, when a magnet is brought near, magnetic particles spread onto the surface of the sheet opposite the magnet and adhere to the sheet, attracting the non-magnetic solid support in the direction of the magnet (magnetic force direction). Furthermore, because it is in the form of a sheet, the distance between the magnetic particles spread on the surface opposite the magnet and the magnet becomes shorter, thus enabling more reliable magnetic adsorption.

[0054] In a preferred embodiment, the non-magnetic solid support used in the separation method of the present invention has a porous structure (e.g., a fibrous aggregate) and is in the form of a sheet. When a sheet-shaped non-magnetic solid support having a porous structure is used in the separation method of the present invention, magnetic particles enter the voids in the porous structure, causing the magnetic particles to adhere firmly to the non-magnetic solid support. Furthermore, the magnetic particles spread out on the surface of the sheet opposite to the magnet and adhere to the sheet, attracting the non-magnetic solid support in the direction of the magnet (magnetic force direction). Also, because the distance between the magnetic particles spread out on the surface opposite to the magnet and the magnet becomes smaller, reliable magnetic adsorption can be expected. Examples of non-magnetic solid supports having a porous structure and being in the form of a sheet include paper (filter paper, etc.) and cloth (nonwoven fabric, etc.).

[0055] The non-magnetic solid phase support may be contained in the liquid in a suspended, dispersed, or floating state. The size (maximum diameter) of the non-magnetic solid phase support is not particularly limited, as long as the non-magnetic solid phase support can be suspended, dispersed, or floated in the liquid within the container in which the separation method of the present invention is performed. To facilitate the separation of the non-magnetic solid phase support from the liquid, the size (maximum diameter) of the non-magnetic solid phase support is preferably less than the inner diameter of the opening of the container (well) in which the separation method of the present invention is performed. The size (maximum diameter) of the non-magnetic solid phase support is usually 100 μm or more (e.g., 500 μm or more, 1 mm or more, 3 mm or more, 5 mm or more).

[0056] The amount of non-magnetic solid support used is not particularly limited, as long as the non-magnetic solid support to which the magnetic particles are attached can be magnetically attracted when a magnet is brought close to the liquid containing dispersed magnetic particles and the non-magnetic solid support. The specific amount of non-magnetic solid support added is adjusted as appropriate, taking into consideration the volume of the liquid, etc. If the amount of non-magnetic solid support contained in the liquid is too large, it becomes difficult to mix the magnetic particles and the non-magnetic solid support, so the volume of non-magnetic solid support per 1 ml of liquid is preferably 0.6 ml or less, more preferably 0.4 ml or less, and even more preferably 0.2 ml or less.

[0057] If the specific gravity of the non-magnetic solid phase support is too high, the non-magnetic solid phase support will quickly settle in the liquid, making it difficult for magnetic particles to adhere to it through magnetic adsorption. Therefore, the apparent specific gravity of the non-magnetic solid phase support when immersed in the liquid used (hereinafter simply referred to as "apparent specific gravity of the non-magnetic solid phase support") is preferably 3.0 or less, more preferably 2.0 or less, even more preferably 1.5 or less, and even more preferably 1.2 or less. Even if the apparent specific gravity of the non-magnetic solid phase support is lower than the specific gravity of the liquid, it is still possible to carry out the separation method of the present invention with the non-magnetic solid phase support floating on the surface of the liquid; however, it is preferable that the apparent specific gravity of the non-magnetic solid phase support is less than or equal to the specific gravity of the liquid used.

[0058] The magnetic particles used in the separation method of the present invention are not particularly limited as long as they contain a metal that is attracted to a magnet and have the property of being attracted to a magnet, and may include single metal particles, alloy particles, metal oxide particles, particles coated with a metal that is attracted to a magnet, etc. Preferably, the magnetic particles are ferromagnetic metal particles such as iron, nickel, and cobalt, or ferromagnetic metal oxide particles such as iron oxide and chromium oxide, more preferably iron particles (iron powder) or iron oxide particles, and even more preferably iron particles. The purity of iron in the iron particles is usually 90% (w / w) or higher, preferably 95% (w / w) or higher. The magnetic particles may or may not be coated with a polymer or the like to prevent deformation (oxidation, etc.) in the liquid.

[0059] If the particle size of the magnetic particles is too large, the settling velocity will be too fast, causing them to accumulate at the bottom of the container. Even if the liquid containing the magnetic particles and the non-magnetic solid support is stirred, the magnetic particles will not adhere well to the non-magnetic solid support. Therefore, the average particle size of the magnetic particles is preferably 150 μm or less, more preferably 45 μm or less, and even more preferably 5 μm or less. The average particle size of the magnetic particles is usually 1 μm or more. In this specification, the average particle size of the magnetic particles refers to the average particle size based on volume. The average particle size of the magnetic particles can be measured using laser scattering analysis.

[0060] The amount of magnetic particles added is sufficient to magnetically adsorb the non-magnetic solid support to which the magnetic particles are attached when a magnet is brought near the liquid containing the magnetic particles and the non-magnetic solid support. The specific amount of magnetic particles to add is adjusted as appropriate, taking into account the volume of the liquid, the weight of the non-magnetic solid support, etc. If the amount of magnetic particles per unit volume of liquid is too small, it will be difficult to magnetically adsorb the non-magnetic solid support to which the magnetic particles are attached even when a magnet is brought near. Therefore, preferably 0.3 mg or more, more preferably 0.6 mg or more, even more preferably 1.0 mg or more, even more preferably 3.0 mg or more, and even more preferably 5.0 mg or more of magnetic particles are added per 1 ml of liquid. Also, if the volume of magnetic particles per unit volume of liquid is too large, the magnetic adsorption of the non-magnetic solid support to which the magnetic particles are attached will be hindered by the excess magnetic particles when a magnet is brought near. Therefore, the volume of magnetic particles added per 1 ml of liquid is preferably 0.5 ml or less, more preferably 0.25 ml or less, and even more preferably 0.1 ml or less. Furthermore, if the amount of magnetic particles per unit weight of the non-magnetic solid support is too small, magnetic adsorption of the non-magnetic solid support to which the magnetic particles are attached becomes difficult. Therefore, for every 1 mg of non-magnetic solid support, preferably 0.2 mg or more, more preferably 0.4 mg or more, even more preferably 0.66 mg or more, even more preferably 0.83 mg or more, even more preferably 1.66 mg or more, even more preferably 2.0 mg or more, and even more preferably 3.3 mg or more of magnetic particles are added.

[0061] The specific gravity of the magnetic particles (if the magnetic particles have voids, this refers to the apparent specific gravity when immersed in the liquid in which they are dispersed) is preferably greater than the specific gravity of the liquid in which they are dispersed.

[0062] If the specific gravity of the magnetic particles differs significantly from the apparent specific gravity of the non-magnetic solid support, when the two are mixed, one will settle faster, making it difficult to uniformly adhere the magnetic particles to the non-magnetic solid support, which may reduce the recovery rate by magnetic adsorption. Therefore, it is preferable to keep the difference between the specific gravity of the magnetic particles and the apparent specific gravity of the non-magnetic solid support small so that the settling rates of the non-magnetic solid support and the magnetic particles are as similar as possible. For example, if the smaller of the specific gravity of the magnetic particles and the apparent specific gravity of the non-magnetic solid support is set to 1, then the specific gravity of the other should be 10.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.0 or less, 3.0 or less, 2.0 or less, 1.5 or less, or 1.2 or less.

[0063] The liquid used in the separation method of the present invention may be aqueous or oil-based, but is preferably aqueous. The type of liquid is not particularly limited, but examples include a washing solution for removing a desired substance from a non-magnetic solid support, an extraction solution for extracting a desired substance from a non-magnetic solid support, and a reaction solution for modifying a sample contained in a non-magnetic solid support.

[0064] In one embodiment, the liquid used in the separation method of the present invention contains a thickening agent. When magnetic particles having a specific gravity greater than that of the liquid are used, their sedimentation makes accurate dispensing difficult. However, by adding a thickening agent to the liquid, the sedimentation rate is reduced, enabling accurate dispensing. Examples of thickening agents include, but are not limited to, polyhydric alcohols having 3 to 6 carbon atoms such as glycerol, propylene glycol, and diglycerol; natural polysaccharides such as carrageenan, xanthan gum, dextrin, and hyaluronic acid; semi-synthetic polysaccharides such as methylcellulose, ethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; and synthetic polymers such as polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, carboxyvinyl polymer, and acrylic acid / alkyl acrylate (10 to 30 carbon atoms) copolymer. The thickening agent is added so that the viscosity of the liquid exceeds that of pure water (1 mPa.s) (for example, 2 mPa.s or more, 4 mPa.s or more, or 6 mPa.s or more). If the viscosity is too high, while the settling of magnetic particles can be suppressed, the excessive viscosity can make dispensing operations difficult. Therefore, the viscosity of the liquid should normally be 50 mPa.s or less (e.g., 20 mPa.s or less, 10 mPa.s or less).

[0065] Next, the liquid containing the magnetic particles and the non-magnetic solid support is stirred to cause the magnetic particles to adhere to the non-magnetic solid support. The stirring operation can be carried out by well-known methods such as a vortex mixer, plate mixer, or spin tip provided in a MaelStrom 8, etc. The magnetic particles may adhere to the outside of the non-magnetic solid support or they may penetrate into the inside of the non-magnetic solid support and adhere thereto. If a porous structure is used as the non-magnetic solid support, stirring causes the magnetic particles to enter the voids in the porous structure, resulting in strong adhesion of the magnetic particles to the non-magnetic solid support. In particular, if a fibrous aggregate such as filter paper is used as the non-magnetic solid support, stirring loosens the fibers and widens the voids between the fibers, allowing a large amount of magnetic particles to be incorporated into the non-magnetic solid support. Furthermore, adding a thickener to the liquid reduces the settling velocities of the non-magnetic solid support and the magnetic particles to roughly the same degree, allowing them to mix well in the liquid and enabling uniform incorporation of the magnetic particles into the non-magnetic solid support.

[0066] Once magnetic particles adhere to a non-magnetic solid support, a magnet is brought close to the liquid containing the magnetic particles and the non-magnetic solid support. As the magnetic particles are attracted to the approaching magnet, the non-magnetic solid support to which the magnetic particles are attached is also attracted to the magnet along with the magnetic particles and magnetically adsorbed to the magnet or the side of the container. The magnetic force (surface magnetic flux density) of the magnet used is strong enough to magnetically adsorb the non-magnetic solid support to which the magnetic particles are attached, and is usually 1000 gauss or more, preferably 2000 gauss or more, and more preferably 3000 gauss or more. The magnet may be a permanent magnet or an electromagnet. Electromagnets offer superior operability because their magnetic field can be electrically switched on and off. To prevent the magnetic particles and non-magnetic solid support from being directly adsorbed to the magnet, the magnet may be covered with a resin cap or the like.

[0067] Then, the magnetically adsorbed non-magnetic solid support is separated from the liquid. The non-magnetic solid support may be separated from the liquid by moving the magnet, or the liquid in the container may be removed while the non-magnetic solid support is magnetically adsorbed, thereby separating the non-magnetic solid support from the liquid.

[0068] In one embodiment, a magnet is brought close to the side of a container containing a liquid with magnetic particles and a non-magnetic solid support, causing the non-magnetic solid support, to which the magnetic particles have adhered, to magnetically attract to the inner wall surface of the container near the magnet. For example, a suitable magnetic separator (magnetic stand) corresponding to the shape of the container used is used as the magnet. Then, while holding the non-magnetic solid support to the inner wall surface of the container, the liquid is removed from the container, or the magnet is moved, separating the non-magnetic solid support magnetically attracted to the wall surface from the liquid along the wall surface.

[0069] In another embodiment, a magnet is inserted along the direction of gravity into a liquid containing magnetic particles and a non-magnetic solid support, and the non-magnetic solid support to which the magnetic particles are attached is magnetically attracted to the magnet. Preferably, the magnet is a rod-shaped magnet (magnetic rod) having a magnetic pole at its tip, and by inserting its magnetic pole along the direction of gravity into a liquid containing magnetic particles and a non-magnetic solid support, the non-magnetic solid support to which the magnetic particles are attached is magnetically attracted to the magnetic pole together with the magnetic particles. Preferably, magnetic particles having a specific gravity greater than the specific gravity of the liquid are used. When the magnet is inserted along the direction of gravity, the magnetic particles that are abundantly attached to the surface of the non-magnetic solid support that is more in the direction of gravity are pulled up in the opposite direction to gravity together with the non-magnetic solid support, so that they can be attracted to the magnet more reliably. Therefore, the risk of losing the non-magnetic solid support or the non-magnetic solid support falling from the magnet during the operation is suppressed. In particular, when using a sheet-like non-magnetic solid support such as paper or cloth, magnetic particles that spread and adhere to the underside of the side of the sheet-like non-magnetic solid support that is more in the direction of gravity (the bottom surface) pull the non-magnetic solid support upward in the opposite direction of gravity. Furthermore, because it is in sheet form, the distance between the magnet and the magnetic particles becomes as close as the thickness of the sheet, allowing the non-magnetic solid support to be more reliably maintained in an attached state to the magnet.

[0070] The separation method of the present invention makes it possible to separate a non-magnetic solid support from a liquid in a magnetic-dependent manner. Therefore, by combining it with an automated device for handling magnetic solid supports such as magnetic beads (for example, the Maelstrom 8 Autostage (MS8) (manufactured by Taiwan Advanced Nanotech)), it becomes possible to automatically process large quantities of non-magnetic solid supports. For example, a non-magnetic solid support, magnetic particles, and liquid 1 are added to the first well of a container provided with a plurality of arranged wells, such as a 96-well plate, and liquid 2 is added to the second well. By stirring the mixture of the non-magnetic solid support, magnetic particles, and liquid 1 in the first well, the non-magnetic solid support is treated with liquid 1, and the magnetic particles adhere to the non-magnetic solid support. Next, a rod-shaped magnet (magnetic rod) having a magnetic pole at its tip is inserted into the liquid 1 containing the non-magnetic solid support to which the magnetic particles have adhered, along the direction of gravity, and the non-magnetic solid support to which the magnetic particles have adhered is attracted to the magnet. The magnet is preferably an electromagnet that can switch the magnetism ON / OFF. The magnet may be fitted with a cover made of resin or the like to prevent the liquid or magnetic particles from directly contacting the magnet. For example, a tubular cover may be attached to a rod-shaped magnet, and with the cover still attached, the rod-shaped magnet is inserted into the liquid 1 containing a non-magnetic solid-phase support to which magnetic particles are attached, along the direction of gravity. Alternatively, the tubular cover is first inserted into the liquid 1 containing the non-magnetic solid-phase support to which magnetic particles are attached, and then the rod-shaped magnet is inserted through the tube of the tubular cover along the direction of gravity. By pulling the magnet out of the liquid 1 in the opposite direction to gravity, the non-magnetic solid-phase support to which magnetic particles attached that have been attracted to the magnet can be separated from the liquid 1. The separated non-magnetic solid-phase support to which magnetic particles are attached, while still attracted to the magnet, is then moved into the liquid 2 of the second well. The non-magnetic solid-phase support to which magnetic particles are attached is then released into the liquid 2 by turning off the electromagnet switch or by pulling the rod-shaped magnet out of the tubular cover. Then, by stirring the second well, the non-magnetic solid support is treated with liquid 2, and a non-magnetic solid support with magnetic particles dispersed in liquid 2 attached is obtained. By performing this series of operations continuously, the non-magnetic solid support can be continuously treated with various types of liquids.Furthermore, by using multiple rod-shaped magnets arranged in accordance with the well arrangement (for example, 8- or 12-row magnetic rods corresponding to a 96-well plate), and automating operations such as magnet insertion, movement, and well agitation, it becomes possible to automatically and continuously process a large number of non-magnetic solid-phase supports with various types of liquids.

[0071] Conventionally, when continuously washing, extracting, or performing other treatments with a liquid on a non-magnetic solid phase support, the non-magnetic solid phase support is left in the container, precipitated by centrifugation, and then the washing / extraction liquid is added and recovered. This process carries the risk of losing the non-magnetic solid phase support if the precipitated non-magnetic solid phase support is sucked in or floats to the surface when the liquid is removed or recovered from the container. In contrast, the separation method of the present invention removes the non-magnetic solid phase support from the container while leaving the washing / extraction liquid in the container, thus minimizing the loss of the non-magnetic solid phase support while continuously washing, extracting, or performing other treatments with a liquid on the non-magnetic solid phase support. Therefore, for example, the separation method of the present invention is useful when continuously washing a non-magnetic solid phase support with a liquid while maintaining an intended substance (e.g., an analyte) within the non-magnetic solid phase support, or when treating the substance maintained within the non-magnetic solid phase support in a stepwise manner with a reaction solution.

[0072] 3. Preparation of peptide-containing samples using magnetic particles In one embodiment, the preparation method of the present invention, detailed in Chapter 1, is modified to apply the separation method of the present invention, detailed in Chapter 2, and magnetic particles are used. Magnetic particles are attached to a fiber aggregate, and the fiber aggregate to which the magnetic particles are attached is magnetically adsorbed to perform operations such as recovery and removal of the fiber aggregate. By performing processing operations on the fiber aggregate by magnetic adsorption, operations such as washing, recovery, and buffer replacement of the fiber aggregate to which the biological sample has been adsorbed can be easily performed. Furthermore, automation using an automated nucleic acid extraction device becomes possible, enabling the processing of a dramatically larger number of samples and improving the reproducibility of results.

[0073] In one embodiment, the preparation method of the present invention further comprises stirring a fibrous aggregate on which a biological sample containing magnetic particles and protein has been adsorbed in an aqueous solvent to cause the magnetic particles to adhere to the fibrous aggregate.

[0074] As magnetic particles, those described in Chapter 2 above as usable in the separation method of the present invention can be used. The magnetic particles are preferably iron particles (iron powder) or iron oxide particles, and more preferably iron particles. The magnetic particles may or may not be coated with a polymer or the like to prevent deformation (oxidation, etc.) in the liquid.

[0075] The particle size of the magnetic particles can be appropriately set in accordance with the description in Chapter 2 above.

[0076] The amount of magnetic particles added is sufficient to magnetically adsorb the fiber aggregate to which the magnetic particles are attached when a magnet is brought close to the aqueous solvent containing the magnetic particles and fiber aggregate, and can be set as described in Chapter 2 above.

[0077] The specific gravity of the magnetic particles (if the magnetic particles have voids, this refers to the apparent specific gravity when immersed in the aqueous solvent in which they are dispersed) is preferably greater than the specific gravity of the aqueous solvent in which they are dispersed, and can be appropriately set in accordance with the description in Chapter 2 above.

[0078] The structure of the fiber aggregate may include, but is not limited to, cotton-like, paper-like, cloth-like, or felt-like structures. However, when magnetic particles are used in this embodiment, the fiber aggregate used in the preparation method of the present invention preferably has voids inside (i.e., between fibers) and these voids are large enough for magnetic particles to enter. When a fiber aggregate to which a biological sample containing magnetic particles and protein has been adsorbed is stirred in an aqueous solvent, the magnetic particles enter the voids within the fiber aggregate due to the stirring, and the magnetic particles adhere firmly to the fiber aggregate and become difficult to detach from it, so more reliable magnetic adsorption can be expected when a magnet is brought close. When the fiber aggregate is stirred in an aqueous solvent, it is preferable that the fibers do not disperse randomly in the aqueous solvent, but rather intertwine with each other to maintain the shape of the aggregate and hold magnetic particles between the fibers. When a cellulose fiber aggregate such as paper (filter paper, etc.), cotton, cotton yarn, or cotton cloth is used as the fiber aggregate, it may be selected to produce voids large enough for magnetic particles to enter between the fibers when stirred in an aqueous solvent. For example, the paper is preferably one that, when stirred in an aqueous solvent, swells and becomes pulp-like, creating voids large enough for magnetic particles to enter between the fibers. Pulp-like refers to a state in which filament units or fibers close to filament units are disorderly intertwined. Filter paper, when stirred with magnetic particles in an aqueous solvent, swells and becomes pulp-like, and firmly holds the magnetic particles that have entered between the fibers, so it is suitably used in combination with magnetic particles in the preparation method of the present invention.

[0079] The fiber aggregate used in this embodiment may be in the form of a sheet. Its thickness is not particularly limited, but is, for example, 5 mm or less (e.g., 1 nm to 5 mm), preferably 1 mm or less (e.g., 1 nm to 1 mm). Examples of fiber aggregates that are in the form of a sheet include paper (filter paper, etc.) and cloth (nonwoven fabric, etc.).

[0080] The amount and specific gravity of the fiber aggregate used can be appropriately determined in accordance with the description in Chapter 2 above.

[0081] As the aqueous solvent used when stirring the fibrous aggregate on which the biological sample containing magnetic particles and proteins has been adsorbed, one of the aqueous solvents listed in step 1 of the preparation method of the present invention for removing hydrophilic proteins from the fibrous aggregate can be used.

[0082] A thickening agent may be added to the aqueous solvent. When magnetic particles with a specific gravity greater than that of the aqueous solvent are used, their sedimentation makes accurate dispensing difficult. However, by adding a thickening agent to the aqueous solvent, the sedimentation rate is reduced, enabling accurate dispensing. The thickening agents described in Chapter 2 above can be used. A thickening agent may be added so that the viscosity of the aqueous solvent exceeds that of pure water (1 mPa.s) (for example, 2 mPa.s or more, 4 mPa.s or more, 6 mPa.s or more). If the viscosity is too high, although the sedimentation of the magnetic carrier can be suppressed, the excessive viscosity will make dispensing difficult. Therefore, the viscosity of the aqueous solvent should normally be 50 mPa.s or less (e.g., 20 mPa.s or less, 10 mPa.s or less).

[0083] The stirring operation can be carried out in accordance with the description in Chapter 2. By stirring, the magnetic particles enter the voids between the fibers constituting the fiber aggregate, and the magnetic particles can adhere firmly to the fiber aggregate. In particular, when filter paper is used as the fiber aggregate, stirring loosens the fibers and widens the voids between the fibers, so a large amount of magnetic particles can be incorporated into the non-magnetic solid support. Furthermore, by adding a thickener to the aqueous solvent, the settling velocities of the fiber aggregate and the magnetic particles become similarly small, allowing them to mix well in the liquid and enabling the magnetic particles to be uniformly incorporated into the fiber aggregate.

[0084] The resulting fibrous aggregate, on which a biological sample containing magnetic particles and protein is adsorbed, can be subjected to operations from step 1 onward.

[0085] The attachment of magnetic particles to the fiber aggregate may be performed before step 1 or simultaneously during step 1. If the magnetic particle attachment step is performed before step 1, the resulting fiber aggregate to which the protein-containing biological sample has been adsorbed, to which the magnetic particles have been attached, is subjected to operations from step 1 onward. On the other hand, if the attachment of magnetic particles to the fiber aggregate is performed simultaneously during step 1, the magnetic particles are included in the aqueous solvent used to wash the fiber aggregate to which the protein-containing biological sample has been adsorbed. The fiber aggregate to which the protein-containing biological sample has been adsorbed is then stirred with the aqueous solvent containing the magnetic particles to wash the fiber aggregate, remove the hydrophilic protein contained in the biological sample from the fiber aggregate, and attach the magnetic particles to the fiber aggregate, thereby recovering the fiber aggregate to which the magnetic particles have been attached and to which residual protein has been bound.

[0086] Operations such as the recovery of fiber aggregates after magnetic particles have been attached to them can be carried out by magnetic adsorption of the fiber aggregates to which the magnetic particles have been attached. For example, at least one, preferably both, of the recovery of fiber aggregates to which residual protein has been bound in step 1 and the removal of fiber aggregates from the digestion reaction mixture in step 3 can be carried out by magnetic adsorption of the fiber aggregates to which the magnetic particles have been attached. Furthermore, in the case of reductive alkylation, the recovery and washing of fiber aggregates to which reduced residual protein has been bound, the recovery and washing of fiber aggregates to which reduced alkylated residual protein has been bound, and the proteolytic enzyme treatment of fiber aggregates to which residual protein has been bound in step 2 (addition of fiber aggregates to which residual protein has been bound to an aqueous solvent containing proteolytic enzymes) can also be carried out by magnetic adsorption of the fiber aggregates to which the magnetic particles have been attached.

[0087] Magnetic adsorption of a fiber assembly to which magnetic particles are attached can be carried out by bringing a magnet close to an aqueous solvent containing the fiber assembly to which magnetic particles are attached, as described in Chapter 2. When the magnetic particles are attracted to the approaching magnet, the fiber assembly to which the magnetic particles are attached is attracted to the magnet together with the magnetic particles and is magnetically adsorbed to the magnet or the side of the container. The magnetic force (surface magnetic flux density) of the magnet used is strong enough to magnetically adsorb the fiber assembly to which the magnetic carrier is attached, and is usually 1000 gauss or more, preferably 2000 gauss or more, and more preferably 3000 gauss or more. The magnet may be a permanent magnet or an electromagnet. Electromagnets offer excellent operability because the magnetism can be electrically switched on and off. To prevent the magnetic particles and fiber assembly from being directly adsorbed to the magnet, the magnet may be covered with a resin cap or the like.

[0088] Then, in accordance with the description in Chapter 2, the magnetically adsorbed fiber aggregate is separated from the aqueous solvent. The fiber aggregate may be separated from the aqueous solvent by moving the magnetically adsorbed fiber aggregate out of the aqueous solvent by moving the magnet, or the fiber aggregate may be separated from the aqueous solvent by removing the aqueous solvent from the container while the fiber aggregate is magnetically adsorbed.

[0089] In one embodiment, a magnet is brought close to the side of a container containing an aqueous solvent with a fibrous aggregate to which magnetic particles are attached, causing the fibrous aggregate with magnetic particles attached to the inner wall of the container near the magnet to be magnetically attracted. For example, a suitable magnetic separator (magnetic stand) corresponding to the shape of the container used is used as the magnet. Then, while holding the fibrous aggregate to the inner wall of the container, the aqueous solvent is removed from the container, or the magnet is moved, separating the fibrous aggregate magnetically attracted to the wall from the aqueous solvent along the wall.

[0090] In another embodiment, a magnet is inserted along the direction of gravity into an aqueous solvent containing a fiber aggregate to which magnetic particles are attached, and the fiber aggregate to which the magnetic particles are attached is magnetically attracted to the magnet. Preferably, the magnet is a rod-shaped magnet (magnetic rod) having a magnetic pole at its tip, and by inserting its magnetic pole along the direction of gravity into the aqueous solvent containing the fiber aggregate to which magnetic particles are attached, the fiber aggregate to which the magnetic particles are attached is magnetically attracted to the magnetic pole together with the magnetic particles. Preferably, magnetic particles having a specific gravity greater than the specific gravity of the aqueous solvent are used. When the magnet is inserted along the direction of gravity, the magnetic particles that are more abundantly attached to the surface and interior of the fiber aggregate in the direction of gravity are pulled up in the opposite direction of gravity together with the non-fiber aggregate, so that the fiber aggregate can be attracted to the magnet more reliably. Therefore, the risk of losing the fiber aggregate or the fiber aggregate falling from the magnet during the operation is suppressed. In particular, when using sheet-like fiber aggregates such as paper or cloth, magnetic particles that spread and adhere to the underside of the side of the sheet-like fiber aggregate that is more in the direction of gravity (the bottom surface) pull the fiber aggregate in the opposite direction of gravity. Also, because it is in sheet form, the distance between the magnet and the magnetic particles becomes as close as the thickness of the sheet, so the fiber aggregate can be more reliably maintained in an attached state to the magnet.

[0091] By using magnetic particles, it becomes possible to separate the fiber aggregate from the aqueous solvent in a magnetic-dependent manner. Therefore, by combining this with an automated device for handling magnetic solid-phase supports such as magnetic beads (for example, the Maelstrom 8 Autostage (MS8) (manufactured by Taiwan Advanced Nanotech)), it becomes possible to prepare a peptide-containing sample for identifying the protein contained in a biological sample by mass spectrometry from a fiber aggregate to which a protein-containing biological sample has been adsorbed, through automated processing. For example, an aqueous solvent for processing the fiber aggregate to which a protein-containing biological sample has been adsorbed in each step of the preparation method of the present invention is added to each well of a container provided with multiple wells arranged in a sequence, such as a 96-well plate, and the containers are arranged in the order of the steps. Then, the preparation method of the present invention is carried out by continuously adding the fiber aggregate to which the biological sample has been adsorbed to the wells containing the aqueous solvent for processing in each step, in the order of the steps. Here, the movement of the fiber aggregate to which the biological sample has been adsorbed between wells is performed by magnetic adsorption of the fiber aggregate to which magnetic particles have been attached.

[0092] Specifically, in a container having multiple wells arranged in a row, such as a 96-well plate, a fibrous aggregate to which a biological sample has been adsorbed, magnetic particles, and the aqueous cleaning solvent used in step 1 are added to the first well, and the aqueous solvent containing the proteolytic enzyme used in step 2 (aqueous solution of proteolytic enzyme) is added to the second well. By stirring the mixture of the fibrous aggregate to which the biological sample has been adsorbed, magnetic particles, and the aqueous cleaning solvent in the first well, hydrophilic proteins contained in the biological sample are removed from the fibrous aggregate into the aqueous cleaning solvent, and the magnetic particles adhere to the fibrous aggregate, resulting in a fibrous aggregate to which magnetic particles have been attached and residual proteins have been bound. Next, a rod-shaped magnet (magnetic rod) with a magnetic pole at its tip is inserted into the first well containing the fibrous aggregate to which magnetic particles have been attached and residual proteins have been bound, along the direction of gravity, and the fibrous aggregate to which magnetic particles have been attached and residual proteins have been bound is attracted to the magnet. Preferably, the magnet is an electromagnet that can switch the magnetism ON / OFF. The magnet may be fitted with a cover made of resin or the like to prevent it from coming into direct contact with the aqueous cleaning solvent or magnetic particles. For example, a tubular cover may be attached to a rod-shaped magnet, and with the cover still attached, the rod-shaped magnet is inserted along the direction of gravity into a first well containing a fibrous aggregate to which magnetic particles are attached and residual proteins are bound. Alternatively, a tubular cover may be first inserted into the first well containing the fibrous aggregate to which magnetic particles are attached and residual proteins are bound, and then a rod-shaped magnet may be inserted through the tube of the tubular cover along the direction of gravity. By pulling the magnet out of the first well in the opposite direction of gravity, the fibrous aggregate to which magnetic particles are attached and residual proteins are bound, which has been adsorbed onto the magnet, can be separated from the aqueous cleaning solvent in the first well. The separated fibrous aggregate to which magnetic particles are attached and residual proteins are bound moves into the proteolytic enzyme aqueous solution in the second well while still adsorbed onto the magnet. Then, by switching off the electromagnet or pulling the rod-shaped magnet out of the tubular cover, the magnetic particles that have attached to the fibrous aggregate to which residual proteins have bound are released into the proteolytic enzyme aqueous solution.Then, the second well is stirred as needed, and the fibrous aggregates to which magnetic particles have adhered and to which residual proteins have bound are treated with a proteolytic enzyme, thereby digesting the residual proteins bound to the fibrous aggregates with the proteolytic enzyme. The peptide mixture, which is the digestion product, is released from the fibrous aggregates into the proteolytic enzyme aqueous solution. A rod-shaped magnet is inserted into the second well containing the digestion reaction mixture, along the direction of gravity. Alternatively, a tubular cover is first inserted into the second well, and then a rod-shaped magnet is inserted through the tube of the tubular cover, along the direction of gravity. Then, by pulling the magnet out of the second well in the opposite direction of gravity, the fibrous aggregates to which magnetic particles adsorbed to the magnet have adhered are removed from the digestion reaction mixture, and the peptide mixture, which is the digestion product, is recovered in the second well.

[0093] In the example above, the aqueous cleaning solvent used in step 1 was added to the first well and the proteolytic enzyme aqueous solution used in step 2 was added to the second well. However, the method is not limited to this, and other wells containing treatment solutions according to the desired treatment may be provided, and the fibrous aggregates to which magnetic particles have adhered and residual proteins bound may be treated in these wells. By moving the fibrous aggregates between wells by inserting, moving, and removing magnets as described above, and performing a series of operations continuously, the fibrous aggregates to which magnetic particles have adhered and residual proteins bound can be continuously treated with various types of liquids. For example, by providing multiple wells containing aqueous cleaning solvents and continuously adding the fibrous aggregates to which magnetic particles have adhered and residual proteins bound to these wells, the cleaning in step 1 may be performed multiple times. Alternatively, a well containing 1) a reducing agent, 2) an alkylating agent, and 3) an aqueous washing solvent may be provided, and the fibrous aggregate to which the magnetic particles obtained in step 1 are attached and to which residual protein is bound may be added sequentially in the order of 1) the well containing the reducing agent → 2) the well containing the alkylating agent → 3) the well containing the aqueous washing solvent to perform a protein reduction and alkylation treatment, thereby obtaining a fibrous aggregate to which magnetic particles are attached and to which reduced alkylated residual protein is bound. The obtained fibrous aggregate to which magnetic particles are attached and to which reduced alkylated residual protein is bound may then be added to a well containing an aqueous solution of proteolytic enzyme and treated with the proteolytic enzyme to carry out step 2.

[0094] By using multiple rod-shaped magnets arranged in accordance with the well arrangement (for example, 8- or 12-row magnetic rods for a 96-well plate), and automating operations such as magnet insertion, movement, detachment, and well agitation, it becomes possible to automatically and continuously prepare peptide-containing samples for identifying proteins contained in biological samples by mass spectrometry from fibrous aggregates to which numerous proteins have been adsorbed.

[0095] Unless otherwise specified, the definitions of terms used in this chapter shall follow those given in Chapters 1 and 2.

[0096] All references cited herein, including publications and patent documents, are incorporated herein by reference to the same extent as they are individually and specifically referred to and their entire contents are specifically described.

[0097] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. [Examples]

[0098] [Example 1] I. Materials and Methods 1. Reagents Dried filter paper blood was prepared by soaking blood collected from the fingertips of healthy volunteers into blood collection filter paper (ADVANTEC (Tokyo, Japan)) and then drying it. Water, acetonitrile, 0.1% (v / v) formic acid-water, and 0.1% (v / v) formic acid-acetonitrile for mass spectrometry were purchased from Thermo Fisher Scientific (Waltham, MA). Trypsin / Lys-C Mix for mass spectrometry was obtained from Promega (Madison, WI). Iron powder (particle size 3-5 μm, 99.9%) was purchased from Kojun Kagaku Kenkyusho (Saitama, Japan). 5 mm zirconia beads (Tommy Seikou Co., Ltd., Tokyo, Japan), TBST: Tris-buffered saline containing Tween 20 (10x), and liquid were purchased from Nacalai Tesque Co., Ltd. (Kyoto, Japan). Sodium dodecanoate, 1M Tris-HCl (pH 8.0): Trizma® hydrochloride solution, pH 8.0, 1 molar was purchased from Sigma-Aldrich Co. LLC (Saint Louis, MO). n-dodecyl-β-D-maltoside (DDM) was purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). BCA, Pierce TM The BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA). The GL-Tip SDB was purchased from GL Sciences Co., Ltd. (Tokyo, Japan). Other reagents were purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan).

[0099] 2. Equipment The following equipment was used in this test. Stirring centrifuge: NSD-12 (Nisshin Rika, Tokyo, Japan) Automatic extraction device: Maelstrom 8 Autostage, Maelstrom 9610 (Taiwan Advanced Nanotech Inc., Taoyuan City 333, Taiwan (ROC)) Ultrasound device: BIORUPTOR, UCW-201 (Sonic Bio Co., Ltd., Kanagawa, Japan) Tissue Lyser (Qiagen, Hildenm, Germany)

[0100] 3. Extraction of hydrophobic proteins by sodium carbonate precipitation (SCP) Figure 1A shows an overview. Step 1: Two 3.2 mm diameter DBS discs punched from DBS and one zirconia bead were added to 900 μL of 100 mM sodium carbonate aqueous solution, and the DBS was crushed by vigorously stirring with a tissue lyserator at 25 Hz for 5 minutes. Then, 900 μL of 100 mM sodium carbonate aqueous solution was added, followed by sonication (BIORUPTOR, UCW-201, "High", 5 minutes). Step 2: After centrifugation (3,000g, 4°C, 3 minutes), the supernatant was transferred to a new 1.5 mL Eppendorf tube, and the precipitated filter paper residue was removed. Step 3: After centrifugation (17,400g, 4°C, 15 minutes), the supernatant was completely removed and the precipitate was isolated. Step 4: After adding 1.5 mL of 100 mM sodium carbonate aqueous solution, the mixture was sonicated (BIORUPTOR, UCW-201, "High", 5 minutes). The precipitate was washed by centrifugation (17,400 g, 4°C, 15 minutes) followed by complete removal of the supernatant. Step 5: 20 μL of 100 mM Tris-Cl pH 8.5, 0.5% sodium dodecanoate was added to the precipitate, and the mixture was dissolved by vigorous stirring (centrifuge, max, 1 min) and sonication (BIORUPTOR, UCW-201, "High", 5 min). The protein was reduced and alkylated, and then digested with trypsin. The resulting peptide was desalted using an SDB tip, dried, dissolved in 3% ACN, 0.1% TFA, and then analyzed by LC-MS. Details are described in Nakajima et al, Journal of Proteome Research, 2020, 19, 2821-2827.

[0101] 4. Extraction of filter paper-bound proteins (FPBP method) Figure 1B shows an overview. Steps 1 and 2: A 3.2 mm diameter disc was added to 500 μL of TBST, and the mixture was vigorously stirred (stirring centrifuge, max, 30 minutes). The mixture was then centrifuged (15,000 g, rt, 5 minutes), and the supernatant was removed to isolate the pulped DBS. Step 3: 500 μL of TBST was added, the mixture was vigorously stirred (stirring centrifuge, max, 10 minutes), centrifuged (15,000 g, rt, 5 minutes), and the supernatant was removed to wash the pulped DBS. Step 4: The pulped DBS was washed by adding 500 μL of 50 mM Tris-Cl pH 8.0, stirring vigorously (stirring centrifuge, max, 10 min), centrifuging (15,000 g, rt, 2 min), removing the supernatant, adding another 500 μL of 50 mM Tris-Cl pH 8.0, stirring vigorously (ballerina, max, 2 min), centrifuging (15,000 g, rt, 5 min), and removing the supernatant. Step 5: 200 μL of 50 mM Tris-Cl pH 8.0 was added, and the pulped DBS was suspended by vigorous stirring (centrifuge, max, 3 minutes). 4 μL of 500 ng / μL Trypsin / Lys-C Mix was added to the DBS suspension, and the protein was digested overnight at 37°C. The filter paper and iron powder composite was attached to a magnet, and the supernatant was collected in a new tube. 50 μL of 5% TFA was added to acidify the solution, and then desalting was performed using a GL-Tip SDB. A GL-Tip SDB was used for desalting. After equilibration with 25 μL of 80% ACN, 0.1% TFA and 50 μL of 3% ACN, 0.1% TFA, the acidified sample solution was added. After washing with 80 μL of 3% ACN and 0.1% TFA, the peptide was eluted with 30 μL of 50% ACN and 0.1% TFA. After drying, it was dissolved in 12.5 μL of 0.02% DDM aqueous solution, and the concentration was measured using the BCA method to adjust it to 200 ng / μL. 500 ng was then analyzed by LC-MS. While reductive alkylation was performed in the SCP method, in the FPBP method, preliminary studies showed that the number of detected disease-causing proteins did not decrease significantly even when reductive alkylation was omitted. Therefore, prioritizing speed and labor savings, reductive alkylation was not performed in this study.

[0102] 5. Automated extraction method for filter paper-bound proteins (FPBPs) (Figure 3) (1) Preparation of plates for MaelStrom 8 Autostage / MaelStrom 9610 processing 500 μL of 1 x TBST, 5 mg of iron powder solution (particle size 3-5 μm, 5 mg / 25 μL in 50% glycerol), and one 3.2 mm diameter disc punched from DBS were added to the first row / first 96-well plate. 500 μL of 1 x TBST was added to the second row / second 96-well plate. 500 μL of 50 mM Tris-Cl pH 8.0 was added to the third row / third 96-well plate. 500 μL of 50 mM Tris-Cl pH 8.0 was added to the fourth row / fourth 96-well plate. 200 μL of 50 mM Tris-Cl pH 8.0 was added to the fifth row / fifth 96-well plate.

[0103] (2) Details of automated extraction process using MaelStrom 8 Autostage / MaelStrom 9610 (2-1) Crushing of filter paper in TBST A spin tip was inserted into the first row / first plate, and the DBS was crushed by stirring in a TBST at 3,500 rpm for 30 minutes, inverting every 10 seconds. During this time, a composite of pulp-like DBS and iron powder was formed. A magnetic rod was inserted into the spin tip, and the composite was attracted to and collected by the spin tip over 60 seconds, and then transferred to the second row / second plate.

[0104] (2-2) Washing of the complex with TBST and 50 mM Tris-Cl pH 8.0 After removing the magnetic rod from the spin tip in TBST, the complex was washed by stirring at 3,500 rpm for 10 minutes, inverting every 10 seconds. The magnetic rod was inserted into the spin tip, and the complex was adsorbed and collected onto the spin tip over 60 seconds, then transferred to the 3rd row / 3rd plate. After removing the magnetic rod from the spin tip in 50 mM Tris-Cl pH 8.0, the complex was washed by stirring at 3,500 rpm for 2 minutes, inverting every 10 seconds. The magnetic rod was inserted into the spin tip, and the complex was adsorbed and collected over 60 seconds, then transferred to the 4th row / 4th plate. After removing the magnetic rod from the spin tip in 50 mM Tris-Cl pH 8.0, the complex was washed by stirring at 3,500 rpm for 2 minutes, inverting every 10 seconds. The spin tip was inserted into the 4th row / 4th plate, and the complex was washed by stirring at 3,500 rpm for 2 minutes, inverting every 10 seconds. A spin tip magnetic rod was inserted, and the composite material was attracted and collected over 60 seconds before being moved to the 5th row / 5th plate.

[0105] (2-3) Washing of the complex with TBST and 50 mM Tris-Cl pH 8.0 After removing the magnetic rod from the spin tip, the complex was released into the solution and suspended in 50 mM Tris-Cl pH 8.0 by stirring at 3,500 rpm for 2 minutes, inverting every 10 seconds. The same procedure as described above for manual extraction of filter paper-bound proteins was then followed up to LC-MS analysis.

[0106] II. Results and Discussion Figure 1 shows the protein extraction schemes using the SCP method and the filter paper-binding protein preparation method. As detailed in Nakajima et al, Journal of Proteome Research, 2020, 19, 2821-2827, the SCP method efficiently removes hydrophilic proteins such as hemoglobin, albumin, and immunoglobulins, which make up the majority of blood, by precipitating hydrophobic proteins in a 100 mM sodium carbonate aqueous solution, and has succeeded in significantly increasing the number of identified proteins. This procedure involves the precipitation of hydrophobic proteins, but the precipitate is small and difficult to see, and is slippery, so there is a risk of accidentally sucking up and discarding the precipitate when removing the supernatant. Therefore, it is necessary for well-trained operators to work carefully and with the utmost caution (Figure 1A). Therefore, in this study, we aimed to establish a simpler protein extraction method from DBS and attempted to extract filter paper-binding proteins. The filter paper-binding protein could be isolated by washing pulp-like filter paper in solution using TBST or Tris buffer, which are commonly used in biochemical experiments, through stirring and centrifugation (Figure 1B). The precipitate from the pulped filter paper was large and easily visible, minimizing the risk of accidentally suctioning and discarding it, and allowing even inexperienced operators to successfully process it.

[0107] For DIA-MS analysis, the window width was optimized for FPBP identification. As a result, the number of identified proteins and peptides was highest in the m / z range of 500-700 (Figure 2). DIA-MS analysis was performed under these conditions in the following experiments.

[0108] Figure 3 shows a comparison of the number of proteins identified in proteomic analysis using the SCP method and the filter paper-bound protein extraction (FPBP) method. The number of identified proteins was 3352 with the SCP method and 4782 with the FPBP method, showing a significant increase of 1.4 times with the FPBP method compared to the SCP method. Table 1 summarizes the proteins that were not detected by the SCP method but were detected by the FPBP method (either manual, automated, or both). The number of identified proteins in proteomic analysis without pre-fractionation of DBS by other groups remained below 1000 (Eshghi et al., Molecular and Cellular Proteomics, 19(3), 540-553, 2020; Nieman et al., Proteomes, 8(1), 4 2020), confirming the superiority of the present invention's method. 3091 proteins were common to both the SCP method and the filter paper-bound protein extraction method, while 1691 were common to the FPBP method alone. 161 proteins were also detected by the SCP method alone. However, the FPBP method was considered superior as a simple method for detecting as many different types of proteins as possible.

[0109] Manual preparation is time-consuming and difficult to process in large quantities, and variations in operator performance can make it challenging to achieve consistent results. Therefore, to further stabilize and accelerate the FPBP method and enable handling of multiple samples, we attempted to automate the process by applying an automated nucleic acid extraction system. Figure 4 shows the scheme of the automated extraction method. By adding iron powder when crushing DBS in TBST, a composite of iron powder and filter paper was generated during the crushing of DBS by the rotational motion of the spin tip. By inserting a magnetic rod into the spin tip, we confirmed that this composite could be magnetically bound to the spin tip and easily and completely recovered. The recovered composite was transferred to a new solution, and the suspension and recovery washing operation was repeated until the reddish-brown color exhibited by hemoglobin became colorless and transparent. Furthermore, in the latter half of the washing step, by replacing the buffer with 50 mM Tris-Cl pH 8.0, which is trypsin-digestible, the final product could be directly trypsin-digested. Once the reagents were placed in the 96-well plate, the automated extraction system could perform a series of processes—pulping of DBS, formation of a complex between the pulped DBS and iron powder, and washing and isolation of the complex—in approximately 30 minutes. Using the MaelStrom 8 Autostage, 8 samples could be processed at once, while the MaelStrom 9610 could process 96 samples simultaneously. Automated processing using the equipment resulted in less error and more stable processing compared to manual methods. This is crucial for the stable and rapid processing of large sample volumes in neonatal screening and cohort studies.

[0110] Figure 5 shows the results of comparing the number of proteins identified using manual and automated processing in the FPBP method. 4782 proteins were detected using the manual method, while 5817 proteins were detected using the automated method, meaning the automated method detected 1.2 times more proteins than the manual method (Figure 5A). To investigate the reason for the increase in the number of identified proteins, the total ionic intensities of representative proteins (HBB, HBA2, IGHG1, APOA1, ALB) that are present in high concentrations in blood and interfere with protein identification depth were compared. The results showed that the total ionic intensities in the automated method were reduced to less than half of those in the manual method, suggesting that the washing operation effectively removed abundant hydrophilic proteins from the blood (Figure 5B). Furthermore, to examine the stability of protein detection and quantification in the manual and automated methods, the cv values ​​of the ionic intensities of individual proteins were examined (proteins detected in 3 or more out of 4 were targeted; 4376 / 4782 for the manual method and 5463 / 5817 for the automated method). As a result, while 57% (2495 / 4376) of all identified proteins had a cv value <20% using the manual method, this figure increased significantly to 81% (4438 / 5463) using the automated method, indicating that proteins are detected and quantified more stably using the automated method (Figure 5C).

[0111] The characteristics of proteins detected by the SCP method, FPBP method (manual), and FPBP method (automated) were compared. The results are shown in Figures 6A to 6C. First, the origin of the proteins detected by each method in the blood was examined (Figure 6A). The number of plasma-derived proteins identified was 273, 266, and 285, which was almost the same for all methods. However, the number of blood cell-derived proteins identified was 2697, 3826, and 4420, which was 1.4 times and 1.6 times higher for the FPBP method (manual) and FPBP method (automated) than for the SCP method, respectively. This suggests that the filter paper has the property of adsorbing more blood cell-derived proteins.

[0112] When the number of identified membrane proteins was examined, the SCP method identified 1080, the FPBP (manual) method identified 1374, and the FPBP (automated) method identified 1558. The FPBP (manual) and FPBP (automated) methods identified 1.3 times and 1.4 times more membrane proteins than the SCP method, respectively (Figure 6B). The proportion of membrane proteins to total detected proteins was 32% (1080 / 3352) for the SCP method, 28% (1374 / 4782) for the FPBP (manual) method, and 20% (1558 / 5817) for the FPBP (automated) method, with the SCP method having the highest proportion. This suggests that the SCP method has a greater effect on enriching membrane proteins than the FPBP method. The FPBP (automated) method identified the most membrane proteins. The main component of filter paper is cellulose. Cellulose becomes hydrophobic and insoluble when hydrophilic β-glucose units polymerize linearly via β-1,4 glucosidic bonds. It is believed that the filter paper was able to adsorb proteins of various properties because it possessed both hydrophilic and hydrophobic properties.

[0113] To determine whether disease-causing proteins were detected, we performed matching against Online Mendelian Inheritance in Man (OMIM). As a result, the number of OMIM hit proteins identified increased significantly: 1204 with the SCP method, 1649 with the FPBP (manual) method, and 1895 with the FPBP (automated) method (Figure 6C). Tables 2-1 to 2-7 summarize the OMIM hit proteins that were not detected by the SCP method but were detected by the FPBP method (either manual, automated, or both). Furthermore, Table 3 summarizes some of the proteins detected by the FPBP method (including those also detected by the SCP method) that are expected to be useful for neonatal screening. This suggests that the FPBP method may be applicable to screening a wider range of diseases.

[0114] Reproducibility is crucial for disease screening. Therefore, we confirmed the reproducibility of the automated FPBP method both daily and between instruments (Figure 7). Twelve samples were prepared independently three times using the automated FPBP method, with intervals of at least four weeks between preparations. These samples were then subjected to proteomic analysis using DIA-MS on two identical LC-MS instruments. Pearson's r was checked for all obtained data. Samples prepared on the same day showed a high correlation, with a mean Pearson's r of 0.98, a median of 0.98, a maximum of 0.99, and a minimum of 0.96. Considering the blood volume held by the DBS, subsequent protein extraction, peptidization, desalting, and variability in LC-MS conditions, the daily and instrument-to-instrument reproducibility was considered high. In neonatal screening, it is necessary to examine DBS samples received daily and report results within a limited time; therefore, the high reproducibility of this automated FPBP method is considered important.

[0115] [Test Example 1] Magnetic separation of filter paper and gel pieces In a test tube, 1 ml of water, iron powder (particle size 3-5 μm, 5 mg), and filter paper (diameter 3.2 mm, 1.5 mg) or gel piece (1 mm x 5 mm x 1 mm, 5 mg) were added and stirred. When a magnet (cylindrical neodymium magnet (outer diameter 5 mm, thickness 5 mm), surface magnetic flux density (T) = 0.4, adsorption force (N) = 9.29) was brought near the side of the tube, the filter paper that had softened and incorporated the iron powder inside, and the gel piece with iron powder adhering to its surface, were attracted to the magnet (Figure 8).

[0116] [Table 1-1]

[0117] [Table 1-2]

[0118] Table 1-3

[0119] Table 1-4

[0120] Table 1-5

[0121] Table 1-6

[0122] Table 1-7

[0123] Table 1-8

[0124] Table 1-9

[0125] Table 1-10

[0126] Table 1-11

[0127] Table 1-12

[0128] Table 1-13

[0129] Table 1-14

[0130] Table 1-15

[0131] Table 1-16

[0132] Table 1-17

[0133] Table 1-18

[0134] Table 1-19

[0135] Table 1-20

[0136] [Table 2-1]

[0137] [Table 2-2]

[0138] [Table 2-3]

[0139] [Table 2-4]

[0140] [Table 2-5]

[0141] [Table 2-6]

[0142] [Table 2-7]

[0143] [Table 3]

[0144] [Test Example 2] Pretreatment of filter paper-attached proteins for proteomic analysis The samples and solutions were added to a deep 96-well plate for automated processing using a Maelstrom 8 Autostage (MS8) (manufactured by Taiwan Advanced Nanotech) as follows: Row 1: TBST (500 μl), iron powder (particle size 3-5 μm, 5 mg), dried filter paper soaked in blood (diameter 3.2 mm, 1.5 mg) Second row: TBST (500 μl) 3rd column: 50 mM Tris-HCl pH8.0 (500 μl) 4th column: 50 mM Tris-HCl pH8.0 (500 μl) 5th column: 50 mM Tris-HCl pH8.0 (200 μl)

[0145] A 96-well plate was mounted on an auto-stage stand, and the pretreatment protocol shown in Figure 8 (right) was performed using the MS8 automated processing system. When iron powder and dried filter paper impregnated with blood were added to the TBST and stirred in the MS8, high-concentration components unnecessary for analysis, such as hemoglobin, albumin, and immunoglobulins, were removed from the TBST (Figure 9). The stirring caused the filter paper to soften and its fibers to unravel, and the iron powder was incorporated into the gaps between the fibers. When the electromagnet of the MS8 was turned on, the filter paper with the attached iron powder was attracted to the electromagnet, separated from the liquid phase, and moved to the next row as the electromagnet moved (Figure 10). In conventional methods, manual centrifugation and replacement of the washing solution were required when washing the filter paper multiple times (Figure 8 (left)), but by adding iron powder to the washing solution, it became possible to automatically pretreat the filter paper using the MS8, an automated device for magnetic beads. The surface magnetic flux density of the electromagnet equipped on the magnetic rod of the MS8 is 3000 gauss.

[0146] [Test Example 3] Investigation of adsorption and recovery efficiency of suspension filter paper Using iron powder suspended in a liquid phase (water), the efficiency of filter paper recovery was evaluated by varying the number of filter papers, the amount of iron powder added, and the particle size of the iron powder. A cylindrical neodymium magnet with a diameter of 5 mm and a height of 5 mm (surface magnetic flux density (T) = 0.4, adsorption force (N) = 9.29) was used to recover the filter paper. The test conditions and evaluation results are summarized in Table 1.

[0147] [Table 4]

[0148] In experiments (1), (2), and (3), the amount of iron powder (magnetic material) (particle size 3-5 μm) per sheet of filter paper (non-magnetic material) (diameter 3.2 mm, 1.5 mg) was increased to 1 mg, 3 mg, and 5 mg, and the adsorption and recovery rates of the filter paper containing the iron powder to a magnet were investigated. As a result, the filter paper could be recovered with a magnet regardless of the amount of iron powder used, but the adsorption and recovery efficiency of the filter paper to the magnet improved as the amount of iron powder increased, and all of the filter paper was recovered with 5 mg.

[0149] In experiments (3), (4), (5), and (6), the number of filter papers used per 5 mg of iron powder was increased from 1, 2, 4, and 8 to investigate how the adsorption and recovery efficiency of the filter paper containing the iron powder changed. As a result, the adsorption and recovery efficiency of the filter paper decreased with each increase in the number of filter papers. From these two experiments, it was found that the adsorption and recovery rate improved as the ratio of magnetic material to non-magnetic material increased.

[0150] In experiments (3), (7), and (8), the adsorption and recovery efficiency of the filter paper containing the iron powder was investigated by changing only the particle size (3-5 μm, 45 μm, 150 μm) while keeping the weight of the added iron powder constant. As a result, it was observed that as the particle size of the iron powder increased, the settling velocity of the iron powder increased, and the iron powder did not adsorb evenly onto the filter paper, instead accumulating at the bottom of the container. Therefore, it was considered that magnetic materials with smaller particle sizes and slower settling velocities are suitable for evenly adsorbing magnetic materials onto non-magnetic materials and improving the recovery rate.

[0151] In experiments (9) and (10), carboxyl-modified magnetic beads (particle size 1 μm, 0.3 or 0.6 mg) (Cytiva, Sera-Mag) were used instead of iron powder. TM SpeedBeads and Sera-Mag TMWe investigated whether filter paper could be adsorbed and recovered using carboxylate-modified magnetic particles. As a result, we found that filter paper could be adsorbed and recovered using carboxylate-modified magnetic beads.

[0152] These experimental results demonstrate that if there is at least 0.3 mg / ml of magnetic particles (iron powder) per 1.5 mg of filter paper (at least 0.2 mg / ml of magnetic particles (iron powder) per 1.0 mg of non-magnetic carrier), the filter paper can be adsorbed and recovered by magnetism.

[0153] Furthermore, when suspending iron powder in a solution, adding glycerol at a final concentration of 50% (v / v) reduces the settling rate of the iron powder, making precipitation less likely and allowing for the acquisition of a more uniform iron powder suspension, thus improving handling. The viscosity (at 20°C) of the glycerol aqueous solution at a final concentration of 50% (v / v) is 6.00 mPa·s. [Industrial applicability]

[0154] The present invention makes it possible to easily prepare a peptide-containing sample from a fibrous aggregate to which a biological sample has been adsorbed, in which the content of peptides derived from hydrophilic proteins such as albumin, which are present in large quantities in the biological sample, is suppressed, and peptides derived from trace proteins are abundant. By subjecting the peptide-containing sample obtained by the preparation method of the present invention to LC-MS analysis, it becomes possible to identify numerous proteins, including trace proteins. Furthermore, the present invention makes it possible to separate non-magnetic solid-phase supports from liquids in a magnetic-dependent manner, thus enabling the washing, recovery, and removal of non-magnetic solid-phase supports without the use of centrifugation or the like. Conventionally, when continuously washing, extracting, or otherwise processing non-magnetic solid-phase supports with liquid, the non-magnetic solid-phase supports are left in the container and precipitated by centrifugation before the washing and extraction liquid is added and recovered. As a result, when removing or recovering the liquid from the container, the precipitated non-magnetic solid-phase supports may be sucked in or float to the surface, leading to loss of the non-magnetic solid-phase supports. In the method of the present invention, the non-magnetic solid-phase supports are removed from the container while the washing and extraction liquid remains in the container, thus minimizing the loss of non-magnetic solid-phase supports while continuously washing and extracting them with liquid. Since automated devices for handling magnetic solid-phase supports such as magnetic beads are already widely available, combining this method with them enables large-scale automated processing of non-magnetic solid-phase supports, which was previously difficult. Furthermore, by applying this method of separating non-magnetic solid-phase supports using magnetic particles to the preparation of peptide-containing samples from fiber aggregates to which biological samples have been adsorbed, and by attaching magnetic particles to the fiber aggregates, operations such as washing, recovery, and buffer replacement of fiber aggregates to which biological samples have been adsorbed by magnetic adsorption can be easily performed. In addition, by automating the process using an automated nucleic acid extraction device, it becomes possible to process a large number of samples in a short time, and the reproducibility of the results is also improved, making it possible to apply this to mass screening of neonatal congenital diseases using dried blood spots.

[0155] This application is based on Japanese Patent Application No. 2022-199020 (filing date: December 13, 2022), the contents of which are fully incorporated herein.

Claims

1. A method for preparing a peptide-containing sample for identifying proteins contained in a biological sample by mass spectrometry, comprising the following steps: 1) Washing the fibrous aggregate to which a protein-containing biological sample has adsorbed with an aqueous solvent to remove the hydrophilic protein contained in the biological sample from the fibrous aggregate, and recovering the fibrous aggregate to which the residual protein is bound; 2) Digesting the residual proteins bound to the fiber aggregates with proteolytic enzymes by treating the recovered residual proteins bound to the fiber aggregates with proteolytic enzymes; and 3) Recover the peptide mixture as a digestion product by removing fibrous aggregates from the digestion reaction mixture.

2. The preparation method according to claim 1, wherein the fiber aggregate is a cellulose fiber aggregate.

3. The preparation method according to claim 2, wherein the cellulose fiber aggregate is filter paper.

4. The preparation method according to claim 1, wherein a fibrous aggregate on which a biological sample containing protein has been adsorbed is a dried blood spot.

5. The preparation method according to claim 1, further comprising subjecting the fibrous aggregate to which residual protein is bound to a reductive alkylation treatment.

6. A method for separating a non-magnetic solid support from a liquid containing a non-magnetic solid support, comprising the following steps, wherein the non-magnetic solid support is a fibrous aggregate or gel composed of any material selected from the group consisting of paper, cloth, resin, cotton, swab, glass, non-magnetic metal, and ceramics: 1) To provide a liquid containing magnetic particles and a non-magnetic solid support; 2) Stir the liquid containing magnetic particles and a non-magnetic solid support to cause the magnetic particles to adhere to the non-magnetic solid support; 3) Bringing a magnet close to a liquid containing magnetic particles and a non-magnetic solid support, and magnetically adsorbing the non-magnetic solid support to which the magnetic particles have adhered; and 4) Separating the magnetically adsorbed non-magnetic solid support from the liquid.

7. The method according to claim 6, wherein the non-magnetic solid support has a porous structure, and magnetic particles enter the voids within the porous structure by stirring.

8. The method according to claim 6, wherein the nonmagnetic solid support is filter paper.

9. The method according to claim 6, wherein the nonmagnetic solid support is in the form of a sheet.

10. The method according to claim 6, wherein the liquid contains a thickening agent.

11. The method according to claim 10, wherein the thickening agent is glycerol.

12. The method according to claim 6, wherein the magnetic particles are ferromagnetic metal particles or ferromagnetic metal oxide particles.

13. The method according to claim 12, wherein the magnetic particles are iron powder.

14. The method according to claim 6, wherein the average particle diameter of the magnetic particles is 150 μm or less.

15. The method according to claim 6, wherein 0.3 mg or more of magnetic particles are dispersed in 1 ml of liquid.

16. The method according to claim 6, wherein 0.2 mg or more of magnetic particles are dispersed in a liquid per 1 mg of a non-magnetic solid support.

17. The preparation method according to claim 1, further comprising stirring a fibrous aggregate on which a biological sample containing magnetic particles and protein has been adsorbed in an aqueous solvent to cause the magnetic particles to adhere to the fibrous aggregate.

18. The preparation method according to claim 17, wherein the aqueous solvent used in step 1 contains magnetic particles, and step 1 is performed by stirring the fibrous aggregate on which a biological sample containing protein has been adsorbed with the aqueous solvent containing magnetic particles to wash the fibrous aggregate with the aqueous solvent, thereby removing the hydrophilic protein contained in the biological sample from the fibrous aggregate, and attaching the magnetic particles to the fibrous aggregate, and then recovering the fibrous aggregate to which the magnetic particles have been attached and to which residual protein has been bound.

19. The preparation method according to claim 17, wherein the fiber aggregate to which residual protein from step 1 is bound is recovered by magnetic adsorption.

20. The preparation method according to claim 17, wherein the fibrous aggregates in step 3 are removed by magnetic adsorption.

21. The preparation method according to claim 17, wherein the magnetic particles are iron powder.

22. The preparation method according to claim 17, wherein the aqueous solvent contains a thickening agent.

23. The preparation method according to claim 22, wherein the thickening agent is glycerol.

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