Pretreatment method and mass analysis method

Abstract

The present invention pertains to a method for pre-treating a cell-containing sample for mass analysis, the method comprising: a step in which cells are brought into contact with a first acidic solution containing an organic acid; and a step in which the cells are heated in a state in which the cells are brought into contact with the first acidic solution to extract cytoplasm components of the cells.

Description

Technical Field

[0001] The present invention relates to a pretreatment method and a mass spectrometry method, and more particularly to a pretreatment method and a mass spectrometry method for a sample containing cells. Background Art

[0002] A method of pre-treating a sample containing cells with formic acid before mass analysis is known. Non-patent document 1 discloses that a predetermined strain is subjected to formic acid treatment on a target plate or in a microtube and then mass analysis is performed, thereby obtaining a result indicating improved identification accuracy. Non-patent document 2 discloses a pre-treatment method of treating cells with formic acid on a sample plate or in a tube for a predetermined strain cultured on a plate.

[0003] Prior art literature

[0004] Non-patent literature

[0005] Non-patent document 1: Comparative study on the identification accuracy of nutritionally variant streptococci between MALDI-TOF MS2 and VITEK2 and study on important biochemical characteristics in identification, Michiko Furugauchi et al., Japanese Journal of Clinical Microbiology, 2016, vol. 26 No. 3, pp. 29-39

[0006] Non-patent document 2: Rapid identification of microorganisms using MALDI-TOF MS: Advances in the field of food microorganisms, Hiroko Kawasaki, Journal of the Japanese Society of Food Microbiology, 2020, vol.37No.4, pp.165-177 Summary of the invention

[0007] Technical problem to be solved by the invention

[0008] As disclosed in Non-Patent Documents 1 and 2, methods for treating cells with formic acid are roughly divided into a method of treating cells on a sample plate and a method of treating cells in a container such as a tube.

[0009] However, there is a problem that the intensity of the peak corresponding to the cytoplasm component in the mass spectrum obtained by the method of processing on the sample plate is low. It is believed that this reflects that the cells are not fully destroyed and the extraction efficiency of the cytoplasm component is low. On the other hand, it is believed that if the method of processing in a container is used, each cell can be more reliably contacted with formic acid, so the extraction efficiency of the cytoplasm component is improved. However, it is known that in fact, in microorganisms with strong cell walls, even if the method of processing in a container is used, the intensity of the peak corresponding to the cytoplasm component is sometimes insufficient.

[0010] Ribosomal proteins, which are major biomarkers for identifying and recognizing microorganisms, are contained in the cytoplasm, and therefore a means for increasing the intensity of the peak of the cytoplasmic component is required.

[0011] The present disclosure has been made to solve the above technical problems, and an object of the present disclosure is to increase the peak intensity of cytoplasmic components by pre-treating a sample containing cells for mass analysis.

[0012] Solutions for solving the above technical problems

[0013] The first pretreatment method of the present disclosure is a pretreatment method for a sample containing cells for mass spectrometry, comprising: a step of contacting the cells with a first acidic solution containing an organic acid; and a step of heating the cells while they are in contact with the first acidic solution to extract cytoplasmic components of the cells.

[0014] Effects of the Invention

[0015] By pre-treating a sample containing cells for mass analysis, the intensity of the peak of the cytoplasmic component can be increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram showing the configuration of an analysis device according to an embodiment.

[0017] Figure 2 This is a flowchart showing processes related to an on-plate heating method and a mass analysis method.

[0018] Figure 3 This is a flowchart showing the processing related to the in-tube heating method and the mass spectrometry method.

[0019] Figure 4 It is a figure which shows the mass spectrum in the case where the concentration of the acid in the first acidic solution is different.

[0020] Figure 5 This is a diagram showing a mass spectrum when a non-heated formic acid treatment is performed.

[0021] Figure 6 This is a diagram showing a mass spectrum when a heated formic acid treatment is performed.

[0022] Figure 7 It is a graph showing the change in the intensity of the ribosome peak caused by the heat formic acid treatment.

[0023] Figure 8 is shown with Figure 7 Plot of changes in mass spectra caused by heating formic acid treatment in different types of microorganisms.

[0024] Fig. 9This is a graph showing the number of peaks when Escherichia coli (Escherichia coli) is treated with formic acid under various temperature conditions.

[0025] Fig.10 This is a graph showing the number of peaks when Janibacter limosus is treated with formic acid under various temperature conditions.

[0026] Fig.11 The graph shows changes in mass spectrum caused by heating and formic acid treatment in Aspergillus kawachii (Aspergillus kawachii). DETAILED DESCRIPTION

[0027] Hereinafter, an embodiment of the present disclosure (hereinafter referred to as "this embodiment") is described. However, this embodiment is not limited thereto. In this specification, the expression of the form "A to Z" refers to the upper and lower limits of the range (i.e., above A and below Z). When there is no unit recorded in A and only a unit is recorded in Z, the unit of A is the same as the unit of Z.

[0028] In addition, in this specification, "%" of a solution means "volume %" unless otherwise specified.

[0029] Hereinafter, the present embodiment will be described in detail with reference to the accompanying drawings. In addition, the same reference numerals are attached to the same or corresponding parts in the drawings, and the description will not be repeated in principle.

[0030] [1. Configuration of the analysis device]

[0031] First, an example of an analysis device 1 for implementing the mass spectrometry method of the present embodiment is shown. The mass spectrometry method of the present embodiment includes a pretreatment method of a biological sample of the present embodiment. In addition, in this specification, unless otherwise specified, "pretreatment" means the preparation of a biological sample before mass analysis.

[0032] Figure 1 1 is a schematic diagram showing the structure of the analysis device 1. The analysis device 1 is a mass analysis device for performing mass analysis of substances contained in a sample, for example, MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry). The analysis device 1 uses the mass spectrum obtained by mass analysis to distinguish the type of organisms.

[0033] The sample contains biological cells. The cells contain target substances that are molecules to be analyzed. In addition, in the present embodiment, the analysis in the analysis device 1 includes detecting peaks in the mass spectrum and measuring the mass-to-charge ratio (m / z) of specific or non-specific substances contained in the sample. In one embodiment, the substance is a protein. The analysis in the analysis device 1 includes distinguishing the type of microorganism that is the source of the sample based on the m / z corresponding to the peak on the mass spectrum. In addition, the m / z corresponding to the peak on the mass spectrum is also generally referred to as the "position" or "m / z position" of the peak.

[0034] In this specification, unless otherwise specified, the type of microorganism refers to at least one systematic taxonomic level including the genotype, strain, subspecies, species, genus and family of the microorganism. In addition, the discrimination of the type of microorganism includes the classification, identification and recognition related to the type of the microorganism. Hereinafter, the discrimination of the type of microorganism is also briefly recorded as the discrimination of the microorganism.

[0035] Furthermore, the analysis performed by the analyzing device 1 may include determining whether a specific substance is contained in the sample.

[0036] Reference Figure 1 The analyzing device 1 includes a control unit 10 and a measuring unit 20 .

[0037] The measuring unit 20 ionizes the substance (e.g., protein) in the sample by high voltage, separates the ions S according to the flight time associated with m / z, and then detects them. The measuring unit 20 includes an ionizing unit 21, an ion accelerating unit 22, a mass separating unit 23, and a detecting unit 24. Figure 1 , the movement of ions S in the measurement unit 20 is schematically shown by arrows A1.

[0038] In one embodiment, the ionization unit 21 ionizes the substance in the sample by the matrix-assisted laser desorption ionization (MALDI) method. As described later, the MALDI method is a method useful in the discrimination of microorganisms based on the mass analysis of microorganisms. As a method of ionization, in addition to the MALDI method, any soft ionization method such as the electrospray ionization (ESI: Electrosprayionization) method can be used. In the case of ionization by the ESI method, if it is set that the analysis device 1 is further equipped with a liquid chromatograph, and the ionization unit 21 ionizes the substance in the sample separated by the liquid chromatograph, a high separation ability can be obtained, so it is preferred.

[0039] The ionization unit 21 includes a sample plate holder (not shown) for supporting the sample plate, and an ion source including a laser device (not shown) for irradiating the sample plate with laser light. The analyst mixes a matrix solution with a sample subjected to the pretreatment method of the present embodiment described later, and arranges the sample plate. The matrix solution contains a matrix substance that easily absorbs laser light and is easily ionized by the laser light. The matrix substance is, for example, α-cyano-4-hydroxycinnamic acid (4-CHCA), α-cyano-3-hydroxycinnamic acid (3-CHCA), sinapinic acid, ferulic acid, 3-hydroxy-4-nitrobenzoic acid (3H4NBA), 2,5-dihydroxybenzoic acid or 1,5-diaminonaphthalene, but is not limited thereto.

[0040] The analyst dries the sample-matrix solution obtained by mixing the sample and the matrix solution as described above on the sample plate to obtain a dried product. Thereafter, the sample plate is placed on a sample plate holder in the vacuum container of the ionization unit 21 .

[0041] In addition, the dried product obtained by mixing a sample with a matrix solution and drying them is also generally referred to as "crystals", and more specifically, it is referred to as "crystals", "mixed crystals", "sample crystals", "matrix crystals", "sample / matrix crystals", etc. In this specification, it is hereinafter referred to as "matrix dried product".

[0042] In the following description, the sample-mixed matrix solution at the stage of forming a matrix dried product disposed on a sample plate is referred to as "matrix solution" including the matrix solution mixed with the sample unless otherwise specified.

[0043] The ionization unit 21 irradiates the matrix dried material on the sample plate with laser light after decompressing the vacuum container provided with the sample plate, thereby ionizing the target substance in the matrix dried material. The type of laser device for irradiating the laser light is not particularly limited as long as it can oscillate the light absorbed by the matrix solution used. For example, when the matrix solution contains CHCA, N2 laser (wavelength 337nm) can be appropriately used. The ions S of the target substance ionized by the ionization unit 21 are extracted by an electric field formed by an extraction electrode, etc., which is not shown in the figure, and are introduced into the ion acceleration unit 22.

[0044] The ion acceleration unit 22 includes an acceleration electrode 221 , and accelerates the introduced ions S. The flow of the accelerated ions S is appropriately converged by an ion lens (not shown) and introduced into the mass separation unit 23 .

[0045] The mass separation unit 23 includes a flight tube 231 , and separates the ions S according to the difference in flight time when the ions S fly inside the flight tube 231 . Figure 1A linear flight tube 231 is shown in the figure, but it may be a reflectron type or a multi-type type, etc. The mass analysis method is not particularly limited as long as the ions S contained in the sample can be separated and detected.

[0046] The detection unit 24 includes a multi-channel plate plasma detector, detects the ions S separated by the mass separation unit 23, and outputs a detection signal of intensity corresponding to the number of ions incident on the detection unit 24. The detection signal output from the detection unit 24 is input to the processing unit 11 of the control unit 10. Figure 1 , the flow of the detection signal of the ions S from the detection unit 24 of the measurement unit 20 is schematically shown by the arrow A2.

[0047] The control unit 10 includes a processing unit 11, a storage unit 12, and an input / output unit 13. The control unit 10 is constituted by, for example, one or a plurality of computers.

[0048] The processing unit 11 is configured to include a processor such as a CPU, and functions as a main body for controlling the operation of the analysis device 1. The processing unit 11 performs various processes by executing a program stored in the storage unit 12 or the like.

[0049] The processing unit 11 includes an apparatus control unit 111 and a mass spectrometer 113. The mass spectrometer 113 includes a determination unit 114.

[0050] The device control unit 111 controls the operation of the measurement unit 20 based on data related to analysis conditions input from the input unit 131 described later. Figure 1 In FIG. 1 , the control of the measuring unit 20 by the device control unit 111 is schematically shown by arrow A3 .

[0051] The mass spectrometer 113 converts the flight time into m / z based on the measurement data including the detected amount of ions detected by the detector 24 and the flight time of the ions, and creates a mass spectrum showing the detected amount corresponding to each m / z.

[0052] The number of detection signals of the target substance detected by the detection unit 24 and the intensity of the detection signals are related to the number of peaks corresponding to the target substance in the mass spectrum and the intensity of the peaks. That is, there is a relationship that the greater the amount of the target substance contained in the matrix dry product, the greater the peak intensity. Therefore, there is a relationship that the higher the extraction efficiency of the target substance in the pretreatment of the sample, the greater the peak intensity.

[0053] The mass spectrometer 113 further obtains the m / z corresponding to the peak of the mass spectrum. The mass spectrometer 113 can also identify the substance corresponding to the m / z shown in the peak on the mass spectrum based on a protein database, etc. That is, the mass spectrometer 113 can calculate the m / z of a specific or non-specific substance contained in the sample. The mass spectrometer 113 can also further determine whether the sample contains a specific substance (component identification in the sample) based on the m / z.

[0054] The mass spectrometer 113 includes a discriminating unit 114. In one embodiment, the discriminating unit 114 creates a database including mass spectra and stores it in the storage unit 12. The database includes mass spectra of one or more known microorganisms, preferably a plurality of mass spectra. The discriminating unit 114 discriminates microorganisms using the mass spectra database.

[0055] In one embodiment, the identification unit 114 identifies the microorganisms according to the fingerprint method. Specifically, the identification unit 114 compares the mass spectrum pattern of the unknown microorganism with the mass spectrum pattern of the known microorganisms stored in the database, thereby identifying the microorganisms. The identification of the microorganisms is performed by referring to the peak of the substance showing a characteristic expression pattern in each microorganism, that is, the biomarker. Ribosomal proteins are mainly used as biomarkers in microorganisms.

[0056] The storage unit 12 has a non-volatile storage medium. The storage unit 12 stores the mass spectrum produced by the mass spectrometry unit 113, the measurement data output from the measurement unit 20, and the program for the processing unit 11 to perform the processing. The storage unit 12 corresponds to an embodiment of the "memory" in the present disclosure. The storage unit 12 may also include a storage medium that can be removed from the analysis device 1. The storage medium may be a CD (Compact Disc), a DVD (Digital Versatile Disc), a USB (Universal Serial Bus) memory, etc., as long as it can store various data. In one embodiment, the storage unit 12 stores a database containing the acquired mass spectra.

[0057] The input / output unit 13 is an interface for inputting and outputting information between the analysis device 1 and the outside. The input / output unit 13 includes an input unit 131 , an output unit 132 , and a communication unit 133 .

[0058] The input unit 131 includes input devices such as a mouse, a keyboard, various buttons, and / or a touch panel. The input unit 131 receives information necessary for controlling the operation of the measurement unit 20 and information necessary for processing by the processing unit 11 from the analyst.

[0059] The output unit 132 is configured to include a display device such as a liquid crystal monitor, a printer, etc. The output unit 132 displays information related to the measurement of the measuring unit 20, the result of the processing of the processing unit 11, etc. on the display device or prints on a paper medium.

[0060] The communication unit 133 includes a communication device capable of communicating via wireless or wired connection such as the Internet, etc. The communication unit 133 receives data required for processing by the processing unit 11, transmits data such as determination results processed by the processing unit 11, and appropriately transmits and receives required data.

[0061] A part or all of the functions of the control unit 10 may be arranged on an electronic computer, a server, or the like that is physically separated from the measurement unit 20 .

[0062] In addition, as the analytical device 1 used in the present embodiment, it is preferably a device combined with a MALDI (matrix-assisted laser desorption ionization) ion source, but is not limited thereto. As a device combined with a MALDI ion source, for example, a MALDI-IT (matrix-assisted laser desorption ionization-ion trap) type mass spectrometer, a MALDI-IT-TOF (matrix-assisted laser desorption ionization-ion trap-time of flight) type mass spectrometer or a MALDI-FTICR (matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance) type mass spectrometer. In addition, the analysis conditions in the analytical device 1 are usually set within the range set by those skilled in the art.

[0063] [2. Conventional sample pretreatment method]

[0064] In the past, there was a method of distinguishing microorganisms using mass spectrometry. In mass spectrometry, a micro-amount of microorganism samples is used to obtain a mass spectrum as an analysis result in a simple and short time. In addition, by using automatic analysis, rapid and simple multi-specimen analysis can be performed.

[0065] Among such mass spectrometry methods, analysis of microorganisms based on MALDI-MS, which is one of the soft ionization methods for ionizing biological macromolecules such as proteins without almost decomposing them, is particularly widely used. Specifically, at the scene of clinical microbiological analysis, food hygiene inspection, etc., the identification of microorganisms of specified types using MALDI-MS is performed. Thus, as a current situation, MALDI-MS is a very excellent technology in the identification of microorganisms of specified types, but on the other hand, it is also difficult to identify specific types of microorganisms.

[0066] For example, as the simplest and commonly used pre-treatment method, there is a method of only mixing cells with a matrix solution. However, when this simplest pre-treatment method is applied to a microorganism (Gram-positive bacteria, fungi, etc.) with a solid cell wall, in a mass spectrum, it is sometimes impossible to detect the peak of ribosomal proteins with sufficient intensity. It is believed that the result reflects that the cell wall of the microorganism is not fully destroyed by simply mixing with a matrix solution, and the components that are more inside than the cell wall and / or the cell membrane, including ribosomal proteins, do not flow out. Hereinafter, in this manual, the "components that are more inside than the cell wall and / or the cell membrane" are referred to as "intracellular components". Intracellular components include cytoplasmic components and nuclear components, and ribosomal proteins are included in the cytoplasmic components.

[0067] Ribosomal proteins are used as biomarkers for identifying species because of minute differences in amino acid sequences and the resulting differences in mass. Ribosomal proteins are structures that are present in large quantities in cells, so they are easily detected during mass analysis.

[0068] In addition, most ribosomal proteins are basic proteins with high proton affinity, so [M+H] is easily generated in the MALDI process. + ions. In addition, the molecular weight of ribosomal proteins is about 5000 to 20000, and the mass of ribosomal proteins can be determined within an error range of several Da by MALDI-MS. Therefore, when the mass analysis is performed by MALDI-MS, there is an advantage that the peak of ribosomal proteins can be easily detected from the sample.

[0069] As described above, ribosomal proteins are very excellent biomarkers, and therefore, in the identification of microorganisms based on mass analysis, ribosomal proteins are generally mainly used as biomarkers.

[0070] In addition, as described above, it is known that ribosomal proteins are mainly used in the discrimination of microorganisms, but in addition, DNA binding proteins, RNA binding proteins and molecular chaperones can also be used. These proteins are also contained in the intracellular components like ribosomal proteins.

[0071] Therefore, when mass analysis is performed using the simplest pretreatment method described above, it may be difficult to distinguish the biomarker represented by ribosomal proteins due to insufficient number and / or intensity of peaks, depending on the type of microorganism.

[0072] In order to extract cytoplasmic components from cells having strong cell walls and obtain peaks with sufficient quantity and / or intensity, a method of pre-treating with formic acid is known.

[0073] Non-patent documents 1 and 2 disclose a method of treating cells with formic acid on a sample plate and a method of treating cells with formic acid in a tube. Hereinafter, for comparison with the formic acid treatment accompanied by heating of the present embodiment described later, the conventional formic acid treatment on the sample plate is also described as "non-heating treatment on the sample plate", and the conventional formic acid treatment in a tube is described as "non-heating treatment in a tube". In addition, the pretreatment method using "non-heating treatment on the sample plate" is also described as "non-heating method on the sample plate", and the pretreatment method using "non-heating treatment in a tube" is described as "non-heating method in a tube".

[0074] In the non-heating method on the sample plate, for example, formic acid is dripped onto microbial cells coated on the sample plate to perform non-heating treatment on the sample plate, followed by drying, and then a matrix solution is dripped and dried again to obtain a matrix dried product.

[0075] In the in-tube non-heating method, after the in-tube non-heating treatment is performed by mixing formic acid and cells in the tube, acetonitrile is further added to the tube and mixed in many cases. Thereafter, the tube is centrifuged, and the supernatant is dripped onto a sample plate and dried. Thereafter, a matrix solution is dripped and dried again, thereby obtaining a matrix dried product.

[0076] Alternatively, in the in-tube non-addition method, the cells may be dispersed in ethanol and centrifuged to remove the supernatant before adding formic acid.

[0077] However, the on-sample plate non-heating method has a problem in that the peak intensity of the cytoplasmic component is sometimes low and insufficient for discrimination of microorganisms, compared with the in-tube non-heating method.

[0078] It is believed that the non-heating method in the tube is compared with the non-heating method on the sample plate, and the sample is mixed with formic acid in the tube, and the extraction efficiency is also improved accordingly. However, sometimes, the peak intensity cannot be fully obtained in the bacterium (such as tubercle bacillus) and fungi (such as mold and yeast) of Gram-positive bacteria with a solid cell wall. In particular, there is a problem of low peak intensity in Gram-positive bacteria (such as actinomycetes of Corynebacterineae (Corynebacterium) suborders such as Mycobacterium (mycobacterium) or Nocardia (Nocardia)) having a long-chain fatty acid called mycolic acid in the cell wall. That is, it is believed that in the non-heating treatment in the tube, the extraction efficiency of the cytoplasm is also insufficient. It is believed that the reason is that the layer of fatty acid in the Gram-positive bacteria with mycolic acid in the above-mentioned cell wall is thicker.

[0079] In addition, the bacterial flora containing mycolic acid includes tuberculosis bacillus and nontuberculous mycobacteria, which are important pathogens. Similarly, as mycolic acid-producing bacteria, Nocardia bacteria are important pathogens that are proinflammatory bacteria of infectious diseases of the skin and central nervous system (Nocardia disease). Therefore, it is important to properly distinguish these Gram-positive bacteria that have mycolic acid in their cell walls in the field of medical treatment and clinical research.

[0080] On the other hand, as a pretreatment for disrupting (lysing) cells by a method other than chemical reaction using formic acid, bead disruption is also known. In the bead disruption, cells, a solvent, and beads (e.g., 0.5 mm zirconium oxide beads) are added to a tube, and the cells are disrupted by physical friction by oscillation. It is believed that this improves the extraction efficiency of cytoplasmic components.

[0081] However, in the bead crushing process, there is a situation where the beads are blocked in the pipette tip or are sucked, so the process may become complicated. In addition, when the beads are mixed into the matrix dry material, the beads may be peeled off from the sample plate in the device and attached to or mixed into the device. As an example, it is believed that the beads may fly out from the matrix dry material due to the impact generated by laser irradiation. In this case, there is a possibility of causing an undesirable condition or failure of the device.

[0082] As described above, in the pretreatment method using formic acid, there is a case where the peak intensity of the cytoplasmic component cannot be sufficiently obtained depending on the type of microorganism. In addition, the bead crushing process also has problems such as causing device failure. Therefore, a method for improving the peak intensity of the cytoplasmic component without using beads in the pretreatment of a sample containing cells for mass analysis is required.

[0083] [3. Sample Pretreatment Method According to Embodiment]

[0084] In view of the above circumstances, the inventors conducted trial and error on sample pretreatment and found a method for increasing the peak intensity of cytoplasmic components without using beads.

[0085] The pretreatment method of this embodiment is to heat the mixture after mixing the microorganisms and the acidic solution. The peak intensity of the cytoplasmic component is increased by the heating.

[0086] More specifically, the pretreatment method of this embodiment is a pretreatment method for a sample containing cells for mass analysis, including: a "contact process" of contacting the cells with a first acidic solution containing an acid; and a "heating process" of heating the cells while they are in contact with the first acidic solution.

[0087] (3-1. Types of microorganisms)

[0088] The microbial cells contained in the sample are not limited to those having a Gram-negative cell wall structure, but may also be those having a Gram-positive cell wall structure. More specifically, the microbial cells contained in the sample may also be Gram-negative bacteria (e.g., Escherichia coli), Gram-positive bacteria (e.g., Mycobacterium tuberculosis), or fungi (e.g., mold, yeast).

[0089] In more detail, the sample may contain cells derived from prokaryotes or cells derived from eukaryotes. Typically, the sample is derived from a microorganism or may contain an unknown microorganism. Prokaryotes include bacteria and archaea. As bacteria, they include Escherichia bacteria (Escherichia coli, etc.), Bacillus bacteria (Bacillus subtilis, etc.), Lactobacillus bacteria (lactic acid bacteria, etc.), Synechocystis bacteria (cyanobacteria, etc.), Mycobacterium bacteria (actinomycetes, etc.), etc. As archaea, they include Methanophilic thermophiles (Methanopyrus), Methanogenic cocci, Thermococcus, Staphylococcus, etc. Eukaryotes include animals, plants, fungi and protozoa. Fungi include filamentous fungi, yeast, mushrooms, mold, etc., including Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, Glomeromycota, Microsporidia, etc. Ascomycota include Aspergillus (Aspergillus oryzae, etc.), Penicillium (Penicillium, etc.), Saccharomyces (Budding yeast, etc.), etc. In one embodiment, the sample is a cell (bacterial body) of bacteria in a broad sense including the bacteria, archaea, and fungi described above.

[0090] The cells of these microorganisms are disrupted by the two processes shown below, and the cytoplasmic components are eluted.

[0091] (3-2. Contact process)

[0092] The sample contacting the first acidic solution may be in liquid or solid form. More specifically, the sample may be, for example, a liquid containing a culture solution of bacteria, or a sample obtained by picking a colony of bacteria cultured on a solid culture medium with a toothpick, etc. The culture solution of the bacteria contains the bacteria and a culture medium for culturing the bacteria.

[0093] Acids are, for example, organic acids such as formic acid, trifluoroacetic acid, and acetic acid, which are commonly used in biological experiments. These organic acids are also easily available and accustomed to handling acids for users who conduct research on the quality analysis of microorganisms and clinical sites. In addition, even if a person skilled in the art uses it in the pre-treatment of the quality analysis, it will not have an adverse effect on the measurement result. Other examples of acids are acids with an acid dissociation constant (pKa) of 0.2 or more and 5 or less, and more preferably acids with an acid dissociation constant of 0.2 or more and 4 or less. Acids with an acid dissociation constant of 0.2 or more and 5 or less are repeated with examples of some of the above acids, such as trifluoroacetic acid, oxalic acid, glycine, salicylic acid, formic acid, lactic acid, benzoic acid, acetic acid, and at least one of butyric acid. In addition, the examples of the above acids are certainly not limited to this. In one embodiment, the organic acid is formic acid. In addition, as described later, inorganic acids can also be used in the same manner. In addition, the acid contained in the first acidic solution can be one acid (pure substance) or a mixture of multiple acids.

[0094] In this specification, "acid" means a pure substance (e.g., 100% formic acid). In addition, a solution obtained by diluting an acid to a specified concentration using a solvent is referred to as the "first acidic solution". Typically, the solvent contains water. Water is preferably pure water, ultrapure water, or ion-exchanged water. The solvent may also contain an organic solvent other than or in place of water within the scope of the effect of the pretreatment method of the present embodiment. The organic solvent is, for example, a polar organic solvent. The organic solvent is, for example, acetonitrile, methanol, or ethanol.

[0095] Typically, as a specific means of the "contact process", a means of mixing the sample with the first acidic solution is used. In this specification, the mixed solution of the sample and the first acidic solution is referred to as the "second acidic solution". By mixing the sample with the first acidic solution, the cells can be dispersed in the first acidic solution. As a result, the cells can be reliably brought into contact with the first acidic solution, and thus the efficiency of treating the cells with the first acidic solution is improved.

[0096] The mixing ratio of the first acidic solution and the sample is not limited within the scope of the effect of the pretreatment method of the present embodiment. For example, a sample whose volume ratio is less than 1 / 2 and more than 1 / 1000 of the first acidic solution is mixed. However, usually when mixed with the first acidic solution, the sample is prepared in a manner in which the amount of components other than cells contained in the sample (for example, a liquid culture medium for culturing cells) is as small as possible. Specifically, for example, the sample before mixing with the first acidic solution is centrifuged and the supernatant is removed, the residual precipitate is recovered, and the recovered precipitate is mixed with the first acidic solution. In addition, it is further preferred to further centrifuge and wash the precipitate with ultrapure water in advance. Based on the above reasons, the acid content ratio (concentration) and pH in the second acidic solution are almost unchanged from the first acidic solution. Hereinafter, the first acidic solution and the second acidic solution are sometimes collectively referred to as "acidic solution".

[0097] The concentration of the acid in the acidic solution is, for example, 50 to 90% by volume, preferably 60 to 80% by volume, and more preferably 65 to 75% by volume.

[0098] In one embodiment, the first acidic solution is 50-90% formic acid, preferably 60-80% formic acid, and more preferably 65-75% formic acid. In this embodiment, the second acidic solution is formic acid of the above concentration containing cells.

[0099] In one embodiment, the first acidic solution is 50-90% trifluoroacetic acid, preferably 60-80% trifluoroacetic acid, more preferably 65-75% trifluoroacetic acid. In this embodiment, the second acidic solution is trifluoroacetic acid of the above concentration containing cells.

[0100] In one embodiment, the first acidic solution is 50-90% acetic acid, preferably 60-80% acetic acid, more preferably 65-75% acetic acid. In this embodiment, the second acidic solution is acetic acid of the above concentration containing cells.

[0101] Whether the acidic solution is properly mixed can be confirmed by measuring the hydrogen ion index (pH) of the acidic solution using a pH meter, for example.

[0102] Another example of a specific means of the "contact process" is to contact (eg place) the cells on an object (eg, cloth) impregnated with the first acidic solution. Thus, the specific means of the "contact process" is not limited within the scope of exerting the effect of the pretreatment method of this embodiment.

[0103] Next, two pretreatment methods included in the pretreatment method of this embodiment are described in order. The two pretreatment methods have different heating methods. The heating method is described below.

[0104] (3-3. Heating process)

[0105] The heating process is performed to increase the peak intensity of the cytoplasm component. More specifically, it is considered that by heating the second acidic solution in the heating process, the cell destruction efficiency is improved, and the extraction efficiency of the cytoplasm component is improved.

[0106] The heating time is, for example, 2 minutes or more and less than 20 minutes, preferably 3 minutes or more and 15 minutes or less, and more preferably 5 minutes or more and 10 minutes or less.

[0107] The heating temperature is, for example, 30 to 75° C., preferably 40 to 60° C., more preferably 45 to 55° C., and further preferably about 50° C. In one aspect of this embodiment, the heating temperature may be 35 to 55° C., or about 40 to 50° C.

[0108] (3-4. Heating method on sample plate)

[0109] The pretreatment method of this embodiment includes the following pretreatment method: a treatment including heating the second acidic solution disposed on the sample plate. Hereinafter, the treatment including heating the second acidic solution disposed on the sample plate is referred to as "heating treatment on the sample plate", and the pretreatment method including heating treatment on the sample plate is referred to as "heating method on the sample plate". Next, using Figure 2 The method of heating the sample plate is described below.

[0110] Figure 2 This is a flowchart showing processes related to the sample plate heating method and the mass analysis method. Figure 2 The method of heating the sample plate shown is one form of the pretreatment method of the present embodiment. Figure 2 The steps shown are performed by manual operation of the analyst using laboratory instruments and experimental devices used in the general pretreatment and quality analysis of microorganisms. In addition, in the figure, "S" is used as an abbreviation for "step (STEP)".

[0111] In S11, the analyst applies the sample to the sample plate. For example, the analyst picks up a bacterial colony on the solid culture medium with a toothpick and applies the colony to the sample plate.

[0112] In S12, the analyst drips the first acidic solution onto the applied sample to prepare a second acidic solution as a mixed solution of the sample and the first acidic solution. The mixing is performed by, for example, pipetting several times using a micropipette. S12 corresponds to one aspect of the above-mentioned "contact process".

[0113] In S13, the analyst heats the second acidic solution. The heating is performed, for example, by placing the sample plate in an incubator set to a predetermined temperature for a predetermined time. The process of S3 corresponds to "heating treatment on the sample plate". In addition, it corresponds to one mode of the above-mentioned "heating process".

[0114] In S14, the analyst dries the second acidic solution on the sample plate.

[0115] In S15, the analyst adds the matrix solution to the dried product obtained by drying the second acidic solution (hereinafter also referred to as "acid dried product"). For example, the analyst drips the matrix solution within several minutes after the second acidic solution is dried, and performs multiple pipetting with a micropipette to mix the acid dried product and the matrix solution.

[0116] In S16, the analyst dries the matrix solution containing the sample on the sample plate to obtain a matrix dry product.

[0117] In S17, the analyst places the sample plate in a mass spectrometer and performs mass analysis to obtain a mass spectrum. Preferably, the mass analysis is MALDI mass analysis.

[0118] (3-5. Heating method inside the tube)

[0119] The pretreatment method of this embodiment includes the following pretreatment method: a treatment including heating the second acidic solution contained in a container such as a tube before being placed on the sample plate. Hereinafter, the treatment of heating the second acidic solution contained in the container is referred to as "in-tube heating treatment", and the pretreatment method including the in-tube heating treatment is referred to as "in-tube heating method". Next, using Figure 3 The method of heating the inside of the tube is described.

[0120] Figure 3 This is a flowchart showing the processing related to the in-tube heating method and the mass spectrometry method. Figure 3 The in-tube heating method shown is one aspect of the pretreatment method of the present embodiment. Figure 3 The steps shown are performed by manual operations of an analyst using laboratory instruments and experimental devices commonly used in pretreatment and quality analysis of microorganisms.

[0121] In S21, the analyst suspends a sample containing cells in water in a tube. The analyst, for example, scoops a colony cultured in a solid medium using a quantitative loop, adds it to a 1.5 mL tube containing 200 μL of water, and mixes it with a vortex mixer for 30 seconds. As a more specific example, the analyst suspends about 10 mg of bacterial cells in water by picking a colony of about 3 mm in diameter with a platinum loop for more than 5 times.

[0122] After suspending the sample in water, ethanol may be added to wash the sample as needed. Washing the sample with ethanol has the effect of dispersing cells that have aggregated due to secretions of microorganisms, etc. in the solution. However, in the pretreatment method of this embodiment, even if the sample is not washed with ethanol, a peak of sufficient intensity can be obtained, so this sample washing is not necessary.

[0123] Below, an example of a case where sample cleaning using ethanol is performed is shown. In the cleaning using ethanol, the concentration of ethanol is preferably 80% or more, based on the mixture of the sample and ethanol. The analyst first adds 800 μL of ethanol to a tube containing the sample (for example, a tube containing a suspension obtained by suspending about 10 mg of bacteria in 200 μL of water) and mixes it with a vortex mixer for 30 seconds. Next, the analyst performs centrifugation at 10,000 g for 2 minutes and removes the supernatant as much as possible. The analyst may also perform centrifugation again as needed to completely remove the supernatant. After removing the supernatant, it is expected that the ethanol will evaporate by natural drying for about a few minutes.

[0124] In S22, the analyst mixes the sample with the first acidic solution in the tube to prepare a second acidic solution as a mixed solution. For example, the analyst adds 50 μL of the first acidic solution to the precipitate containing the sample in the tube and mixes it with a vortex mixer for 10 seconds. S22 corresponds to one aspect of the above-mentioned "contact process".

[0125] In S23, the analyst heats the second acidic solution. The heating is performed, for example, by placing the tube containing the second acidic solution in a PCR (polymerase chain reaction) device set to a predetermined temperature for a predetermined time. The process of S24 corresponds to "heating treatment in the tube". In addition, it corresponds to one mode of the above-mentioned "heating process".

[0126] After S23, the analyst may also add an organic solvent such as acetonitrile to the second acidic solution. The organic solvent may be added for purposes such as dissolving the matrix material in the matrix solution, preparing the surface tension of the matrix solution, destroying cell membranes, etc. However, in the pre-treatment method of the present embodiment, even without adding the organic solvent, a peak of sufficient intensity is obtained, so adding the organic solvent is not necessary.

[0127] The treatment of adding an organic solvent is performed, for example, as follows: the analyst adds an equal amount of acetonitrile to formic acid to a tube and mixes it with a vortex mixer for 10 seconds.

[0128] In S24, the analyst drops the second acidic solution onto the sample plate. For example, the analyst drops 0.5 to 1 μL of a supernatant obtained by centrifuging a heated tube at 10,000 to 15,000 g for 2 minutes onto the sample plate.

[0129] Processing of S25 to S28 Figure 3 The processing of S14 to S17 corresponds to that of S14 to S17, so the description will not be repeated.

[0130] In the mass spectrum of the sample pre-treated by the pre-treatment method of the embodiment described above, the intensity of the peak of the intracellular component is improved. It is believed that the pre-treatment method of the embodiment improves the efficiency of cell destruction and the efficiency of extraction of intracellular components. In addition, it is expected that the efficiency of distinguishing microorganisms using the peak of the biomarker contained in the intracellular component will be improved.

[0131] [4. Experimental example]

[0132] Next, the pretreatment method of the present embodiment and its effects will be described in more detail with reference to experimental examples. However, the pretreatment method of the present embodiment is not limited to the experimental examples.

[0133] (4-1. Experiment 1)

[0134] Experiment 1 is an experiment to show the influence of the concentration of the acid in the first acidic solution.

[0135] The pretreatment of the samples in Experiment 1 was performed as follows.

[0136] (1) A sample containing mycolic acid-producing bacteria Rhodococcus erythropolis NBRC15567T was dispersed in 500 μL of ultrapure water in a microtube.

[0137] (2) The bacterial cells dispersed in ultrapure water were dispensed in 100 μL portions.

[0138] (3) 400 μL of ethanol was added to each microtube into which the bacterial cells were dispensed, and the mixture was mixed and centrifuged to obtain a bacterial cell precipitate.

[0139] (4) 50 μL of 50% formic acid, 50 μL of 70% formic acid, and 50 μL of 90% formic acid were added to the precipitate obtained by removing the supernatant, and mixed by vortexing. Then, the samples contained in each tube were subjected to the following treatments.

[0140] (5) Heating was performed at 50°C for 5 minutes.

[0141] (6) 50 μL of acetonitrile was further added, mixed by vortexing, and centrifuged to obtain a supernatant.

[0142] (7) After 1 μL of the supernatant was dropped onto the sample plate and dried, 1 μL of the matrix solution (CHCA solution) was dropped and dried to prepare a matrix dry product, which was then measured by MALDI-MS.

[0143] Figure 4 1 is a diagram showing mass spectra obtained when the concentration of the acid in the first acidic solution is different as a result of performing Experiment 1. Figure 4 In the figure, the horizontal axis is m / z. The vertical axis is Figure 4 The % intensity is the relative intensity when the peak with the highest intensity among the three mass spectra included in is taken as 100%.

[0144] in addition, Figure 4 The X%FA in the formula refers to the first acidic solution containing X% formic acid. Figure 4 In the description of , a mass spectrum obtained as a result of heating the first acidic solution containing X% formic acid is also described as “X% formic acid mass spectrum”.

[0145] Reference Figure 4 , the peak intensity in the 70% formic acid mass spectrum and the 90% formic acid mass spectrum increased by several to dozens of times relative to the 50% formic acid mass spectrum. For example, the peak intensity of the peak near m / z6600 increased by 3 to 5 times. In addition, the peak near m / z5500 was almost not detected in the 50% formic acid mass spectrum, while clear peaks were detected in the 70% formic acid mass spectrum and the 90% formic acid mass spectrum, and their intensities were about 8 to 20 times or more.

[0146] That is, according to the results of Experiment 1, the peak intensity is improved by heating with 70 to 90% formic acid compared with 50% formic acid. Therefore, it is considered more preferable to use 70 to 90% formic acid during heating.

[0147] In addition, considering that formic acid has a unique odor, it is considered more preferable to use 70% formic acid, which has a lighter odor than 90% formic acid.

[0148] (4-2. Experiment 2)

[0149] Experiment 2 is an experiment to show the influence of heating time.

[0150] The pretreatment of the samples in Experiment 2 was performed by changing the pretreatments (4) and (5) of the samples in Experiment 1.

[0151] The pretreatments (4) and (5) of the samples in Experiment 2 were performed as follows.

[0152] (4) The supernatant was removed, and 50 μL of 70% formic acid was added and mixed by vortexing to prepare a second acidic solution. The second acidic solution was then dispensed into 8 microtubes.

[0153] (5) Four of the dispensed microtubes were treated with formic acid for 2 minutes, 5 minutes, 10 minutes, and 20 minutes, respectively, without heating (room temperature). Figure 5 ). In addition, the remaining 4 microtubes were treated with formic acid for 2 minutes, 5 minutes, 10 minutes, and 20 minutes respectively while being heated to 50°C ( Figure 6 ). Afterwards, the samples contained in each tube were subjected to the following treatments.

[0154] In addition, hereinafter, the formic acid treatment without heating is also referred to as "non-heated formic acid treatment", and the formic acid treatment in a heated state is also referred to as "heated formic acid treatment".

[0155] Figure 5 This is a diagram showing a mass spectrum when a non-heated formic acid treatment is performed. Figure 6 is a diagram showing a mass spectrum when a heated formic acid treatment is performed. Figure 5 and Figure 6 , the horizontal axis is m / z. Figure 5 The vertical axis is Figure 5 The relative intensity is when the peak with the highest intensity among the four mass spectra included in is set as 100%. Figure 6 The vertical axis is Figure 6 The relative intensity when the peak with the highest intensity among the four mass spectra included in is set as 100%. Figure 5 and Figure 6 The data above m / z 7000 show 10 times the intensity.

[0156] Reference Figure 5 Even when the intensity is increased by 10 times, almost no peaks above m / z 7000 are detected. Figure 6 , peaks were also detected above m / z 7000.

[0157] Below, in Figure 6 In the description of , the mass spectrum obtained as a result of performing the formic acid heating treatment for X minutes is also described as "X-minute heating mass spectrum".

[0158] Reference Figure 6 Compared with the 2-minute heating mass spectrum, the peak intensity near m / z7000-7200 in the 5-minute heating mass spectrum and the 10-minute heating mass spectrum increased by several times. For example, with respect to the peak near m / z7100, almost no peak was detected in the 2-minute heating mass spectrum, while clear peaks were detected in the 5-minute heating mass spectrum and the 10-minute heating mass spectrum.

[0159] On the other hand, the peak intensity in the 20-minute heating mass spectrum was lower than that in the 5-minute heating mass spectrum and the 10-minute heating mass spectrum. From this result, it is considered that the protein may be decomposed as a result of excessive reaction of formic acid.

[0160] That is, according to the results of Experiment 2, by adding formic acid to the sample and performing a heated formic acid treatment for 2 minutes or more and less than 20 minutes, the intensity of the peaks above m / z 7000 in each mass spectrum is improved compared to the case of non-heated formic acid treatment. In addition, in the mass spectrum subjected to the heated formic acid treatment, especially when the heated formic acid treatment is performed for 5 minutes or more and less than 10 minutes, the number of peaks and the peak intensity near m / z 7000 to 7200 are further improved.

[0161] (4-3. Experiment 3)

[0162] Experiment 3 is an experiment showing the change in intensity of the peak corresponding to the cytoplasmic component caused by heating during formic acid treatment.

[0163] The pretreatment of the samples in Experiment 3 was performed as follows.

[0164] (1) 1 g of natto was dispersed in 9 mL of sterilized physiological saline in a microtube, mixed with a vortex mixer, and centrifuged to obtain a supernatant.

[0165] (2) The supernatant was spread on a standard agar medium and cultured at 30°C for 24 hours to obtain natto isolates.

[0166] (3) Disperse the colony of natto isolate in ultrapure water in a microtube at an OD of about 1, and dispense 100 μL into two microtubes. Centrifuge to remove the supernatant, and add 100 μL of 25% formic acid and 100 μL of 70% formic acid, respectively. After the sample with 25% formic acid was allowed to stand at room temperature for 5 minutes, 1 μL was added to the sample plate and dried, and 1 μL of the matrix solution (CHCA solution) was added and dried ( Figure 7 The sample with 70% formic acid added was heated at 50°C for 5 minutes, 1 μL was dropped onto the sample plate and dried, 1 μL of the matrix solution (CHCA solution) was dropped and dried, and then the measurement based on MALDI-MS was performed ( Figure 7 lower part).

[0167] (4) After the formic acid was dried, a matrix solution (CHCA solution) was added dropwise and dried to prepare a matrix dry product, and measurement by MALDI-MS was performed.

[0168] Figure 7 3 is a graph showing the intensity change of the ribosomal protein peak caused by heating obtained as a result of Experiment 3. Figure 7 In the figure, the horizontal axis is m / z. The vertical axis is Figure 7 The relative intensity when the peak with the highest intensity in each mass spectrum included in is set as 100%. Figure 7 The highest peak in the upper part is peak b, which has an intensity of 0.6 mV. Figure 7 The peak with the highest intensity in the lower part is peak b, which has an intensity of 15.3 mV, which is more than 25 times that of 0.6 mV. That is, even if the peak with the highest intensity when the non-heated formic acid treatment is used as a reference, the intensity is more than 25 times when the heated formic acid treatment is performed.

[0169] Furthermore, it can be seen that the ratio of peak a to peak b increases to about 2 times when the heating formic acid treatment is performed.

[0170] Peak a is a peak of ribosomal proteins most commonly used for identification of microorganisms. More specifically, it corresponds to L36 protein, which is one of the ribosomal proteins. On the other hand, peak b is not included as a protein contained in the cytoplasm. From this result, it is believed that the lysis of microorganisms is promoted by heating formic acid treatment, and the cytoplasmic components containing ribosomal proteins can be efficiently extracted from the bacteria.

[0171] According to the results of Experiment 3, the intensity of the peak of ribosomal protein, which is a biomarker useful for distinguishing microorganisms, is increased by the heating and formic acid treatment. That is, it is considered that the efficiency of distinguishing microorganisms is improved by the heating and formic acid treatment.

[0172] (4-4. Experiment 4)

[0173] Experiment 4 shows the change in peak intensity caused by heating during formic acid treatment of Bacillus subtilis subsp. subtilis NBRC13719T.

[0174] Figure 8 The figure shows the results of Experiment 4, showing the mass spectra of the case where 25% formic acid treatment was performed without heating, the mass spectra of the case where 70% formic acid treatment was performed without heating, and the mass spectra of the case where 70% formic acid treatment was performed for 5 minutes under heating. Figure 8 In the figure, the horizontal axis is m / z. The vertical axis is Figure 8 The relative intensity is when the peak with the highest intensity among all the mass spectra included in is set as 100%.

[0175] Reference Figure 8It can be seen that there is no significant difference between the mass spectrum of the case where the 25% formic acid treatment was performed without heating and the mass spectrum of the case where the 70% formic acid treatment was performed without heating, but the number and intensity of peaks increased significantly only in the mass spectrum of the case where the 70% formic acid treatment was performed under superheating.

[0176] In particular, the number and intensity of peaks corresponding to ribosomal proteins (peaks with asterisks) increased significantly. For example, the peak near m / z 6500 showed an intensity of about 12 times greater when 70% formic acid was treated in a superheated state than when formic acid was treated without heating. Similarly, the peak near m / z 7700 showed an intensity of about 18 times greater.

[0177] According to the results of Experiment 4, more protein peaks can be detected with higher intensity by heating formic acid treatment. In particular, the intensity of the peak of ribosomal protein is significantly improved. In the identification of microorganisms, it is used as a biomarker of a specified type of protein, and it is expected that the efficiency of microorganism identification can be improved by heating formic acid treatment.

[0178] (4-5. Experiment 5)

[0179] Experiment 5 is an experiment to show the effect of heating in formic acid treatment on various microorganisms. Specifically, the experiment was conducted on microorganisms of the Actinobacteria phylum including actinomycetes, which are generally known to have a Gram-positive cell wall and are difficult to lyse.

[0180] The pretreatment of the samples in Experiment 5 was performed as follows.

[0181] (1) First, as microorganisms of the phylum Actinobacteria, the following microorganisms were purchased from the Biotechnology Center of the National Institute for Product Evaluation and Research or the Microbial Material Development Laboratory of the RIKEN Institute for Physical and Chemical Research and cultured in liquid culture using designated culture media: Agromyces rhizosphaerae (rhizosphere soil fungi) 14 strains (NBRC16236), Arthrobacter globiformis (spherical arthrobacterium) 168 strains (NBRC12137), Bifidobacterium longum (Bifidobacterium longum) E194bk strain (JCM1217), Brachybacterium conglomeratum (Brachybacterium conglomeratum) 5-2 strain (NBRC15472), Corynebacterium glutamicum (Corynebacterium glutamicum) 534 strain (NBRC12168), Glycomyces algeriensis (Algerian Saccharomyces) LLR-39Z-86 strain (NBRC103888), Glycomyces arizonensis (Arizona Saccharomyces) DPL-G-76 strain (NBRC 103886), Glycomyces harbinensis (Harbin Saccharomyces) LL-DO5139 strain (NBRC 14487), Janibacter limosus (Janibacter limosus) HKI 83 strain (NBRC16128), Microlunatus phosphovorus (Microlunatus phosphovorus) NM-1 strain (NBRC101784), Nocardioides simplex (Nocardioides simplex) AJ 1420 strain (NBRC12069), Paenarthrobacter aurescens (Paenarthrobacter aureus) 579 strain (NBRC 12136), Paenarthrobacter histidinolovorans (NBRC 15510), Phycicoccus duodecadis (NBRC 12959), Streptomyces griseus (Streptomyces griseus) C1 strain (NBRC 12875).

[0182] (2) The cultured microorganisms were dispensed and the quality of the sample subjected to the in-tube non-heating method was analyzed as a control experiment.

[0183] (3) The cultured microorganisms were dispensed and an experiment using the pretreatment method of the present embodiment was conducted. Specifically, the mass analysis was conducted on samples subjected to the in-tube heating method including the in-tube heating treatment (50° C., 5 minutes).

[0184] Next, the results of Experiment 5 are shown.

[0185] First, the results of the control experiment using the conventional pretreatment method are described. Agromyces rhizospherae (rhizosphere soil fungi) NBRC16236, Arthrobacter globiformis (spherical Arthrobacter) NBRC12137, Glycomyces algeriensis (Algerian Saccharomyces) NBRC103888, Glycomyces arizonensis (Arizona Saccharomyces) NBRC103886, Glycomyces harbinensis (Harbin Saccharomyces) NBRC14487, Janibacter limosus (Janibacter limosus) NBRC16128, Nocardioides simplex (Simple Nocardia) NBRC12069, Paenarthrobacter Histidinolovorans NBRC15510, Streptomyces griseus NBRC12875, etc., sometimes had less than 5 peaks that matched the ribosomal proteins inferred from the theoretical values ​​in the genome information in the range of 200 ppm, depending on the sample, indicating that the ribosomal proteins may not have been fully extracted in the in-tube formic acid treatment. On the other hand, in other strains, peaks that matched about 6 to 20 ribosomal proteins in the range of 200 ppm were obtained, indicating that the ribosomal proteins were well extracted in the in-tube formic acid treatment.

[0186] On the other hand, when the pretreatment method of the present embodiment was used, peaks corresponding to about 6 to 20 kinds of ribosomal proteins were obtained in the range of 200 ppm in all the strains tested.

[0187] These results show that formic acid treatment accompanied by heat treatment is effective not only for microorganisms with Gram-negative cell wall structures such as Escherichia coli, which are relatively easy to extract intracellular components, but also for microorganisms with strong cell walls such as Actinobacteria. Therefore, it is shown that the pretreatment method of the present embodiment is a highly versatile method for promoting the lysis of microorganisms of various taxa under the same conditions.

[0188] (4-6. Experiment 6)

[0189] Experiment 6 is an experiment in which Escherichia coli (Escherichia coli) which is a Gram-negative bacterium and Janibacter limosus (Janibacterium limosus) which is a Gram-positive bacterium were treated with formic acid under various temperature conditions.

[0190] The pretreatment of the samples in Experiment 6 was performed as follows.

[0191] (1) As the bacterial cells used in Experiment 6, Escherichia coli (ATCC700926) cultured overnight at 37°C in a GAM liquid medium prepared with GAM broth (Shimadzu Diagnostics Co., Ltd. 05422) and Janibacter limosus (NBRC 16128) cultured for two days at 30°C in an 802 agar medium were used. For Janibacter limosus, 50 μL of the culture solution containing the bacterial cells was collected and added to a 96-well cell culture plate with a U bottom (FALCON 353077). Then, the added culture solution was measured using a SpectraMax iD3 (multi-function microplate reader), and the liquid culture medium corresponding to each well was added so that the OD (Optical Density) value was about 0.4 to 0.6 to obtain a bacterial solution. In addition, Escherichia coli was prepared by suspending bacterial cells collected from 802 agar medium in distilled water so that the OD value was comparable to that of Janibacter limosus (about 0.4 to 0.6).

[0192] (2) Add 400 μL of the obtained bacterial solution to a 1.5 mL tube, centrifuge it (15,000 rpm, room temperature, 5 minutes), and remove the supernatant from the tube. Add 250 μL of pure water to the tube from which the supernatant has been removed, and resuspend the bacterial pellets. Thereafter, add 750 μL of ethanol (99.5%) to the tube, and prepare it in such a way that the final concentration of ethanol becomes 75% (v / v). After washing the bacteria in the tube, centrifuge it (15,000 rpm, room temperature, 5 minutes), and remove the supernatant from the tube. Then, air-dry it, and remove the ethanol from the tube. Add 60 μL of ultrapure water (Fujifilm 214-01301) to the tube, and suspend the air-dried bacterial pellets. 140 μL of formic acid (Fujifilm 067-04531) was added to the tube and mixed in such a way that the final concentration of formic acid became 70% (v / v) (contact process). The mixed suspension was dispensed into 0.2 mL tubes at 10 μL each. The dispensed suspension was heated at 20°C, 40°C, 50°C or 60°C for 5 minutes (heating process) using the VeriFlex (temperature module control) function of the MiniAmp Plus thermal cycler (Applied Biosystems, Inc., USA). In addition, 20°C is usually a temperature below or around room temperature and does not belong to "heating", but it is expressed as "heating" for convenience in Experiment 6. After heating, the tube containing the suspension was kept cold on ice. 10 μL of acetonitrile (Fujifilm 012-19851) was added to each heated sample (suspension) and mixed. 1 μL of sample is placed on the spot of MALDI sample plate FlexiMass-DS (Shimadzu Biotech TO-430) and air-dried. On the other hand, in an ultrapure aqueous solution of 50% acetonitrile (v / v), 1% trifluoroacetic acid (Fujifilm Wako Pure Chemical Industries, Ltd. 206-1-731) (v / v), α-cyano-4-hydroxycinnamic acid (CHCA) (Tokyo Chemical Industry Co., Ltd. C1768) is added in a way that the final concentration becomes 10 mg / mL to obtain a matrix solution (prepared if necessary). 1 μL of the above-mentioned matrix solution is added to the spot where the sample after air-drying is set, the matrix solution is mixed with the sample by pipetting, and further air-dried. The MALDI sample plate provided with the sample is made by the above process.

[0193] (3) The prepared MALDI sample plate was inserted into MALDI-8020 manufactured by Shimadzu Corporation, and measurement was performed under the following conditions: The Acquire tab was set to Mass range: 2000-20000, Accumulate 5 shot(s)@200Hz blast shot, Profiles: 100 profiles (mass range: 2000-20000, accumulation 5 times@200Hz pulse laser, number of acquisitions: 100 times) (20220601_Microbio). The Process tab was set to Subtractbaseline using filter width 500, Smoothing method: Gaussian, Peak width: 80, Peakdelimiter method: Threshold Apex, Threshold offset and response: 0.015mV1.2000mV (baseline subtraction using a filter with a width value of 500, smoothing method: Gaussian, peak width: 80, peak definition method: peak threshold, threshold offset and response calibration: 0.015mV 1.2000mV) settings (20221223microbio). For the calibration of MALDI-8020, Escherichia coli was used as a standard sample. Specifically, the m / z axis was calibrated using multiple peaks known to be observed in Escherichia coli. More specifically, a suspension of Escherichia coli prepared with 50% acetonitrile (v / v) was used for calibration, and the peaks corresponding to 4365.3 Da, 6316.2 Da, 6411.6 Da, 7158.8 Da, 7274.5 Da, 8369.8 Da, 8994.3 Da, 9060.4 Da, 10138.6 Da, 10300.1 Da, 10694.4 Da, 11450.3 Da, 12227.3 Da, and 12770.6 Da (Ec221122) were used for calibration.

[0194] (4) The peaks obtained from the samples measured by the above-mentioned MALDI-8020 were analyzed to calculate the total number of peaks and the number of peaks of ribosomal proteins. At this time, MALDI data analysis was performed using GPMsDB-tkv1.0.1 (https: / / github.com / ysekig / GPMsDB-tk).

[0195] The results of Experiment 6 are shown in Fig. 9 and Fig.10 . Fig. 9 This is a graph showing the number of peaks when Escherichiacoli (Escherichia coli) is treated with formic acid under various temperature conditions. Fig.10 This is a graph showing the number of peaks when Janibacter limosus is treated with formic acid under various temperature conditions. Fig. 9 and Fig.10 The horizontal axis represents temperature (°C). Fig. 9 A and Fig.10 The vertical axis in A represents the total number of peaks detected in the mass spectrum, that is, the total number of peaks found. Fig. 9 B and Fig.10 The vertical axis in B represents the number of peaks identified as ribosomal proteins based on their m / z among all peaks detected in the mass spectrum (ribosomal proteins). For Escherichia coli and Janibacter limosus, the results of two experiments conducted at each temperature condition are shown in the figure with squares and circles. Fig. 9 , Fig.10 middle.

[0196] Reference Fig. 9 In Escherichia coli (Escherichia coli) cells that are easily disrupted, the same number of ribosomes (10 to 13) were detected when formic acid treatment was performed at 20°C, 40°C, and 50°C. On the other hand, when formic acid treatment was performed at high temperature conditions (e.g., 60°C), the number of ribosomal proteins detected was significantly reduced. Fig. 9 The results show that for Gram-negative bacteria such as Escherichia coli, formic acid treatment under various temperature conditions can detect bacterial-derived proteins. In particular, formic acid treatment can be performed at a temperature of 20°C or more to about 50°C to minimize protein degradation.

[0197] Reference Fig.10 In Janibacter limosus, which is difficult to disrupt cells, the number of ribosomal proteins detected was greater when formic acid treatment was performed at 40°C to 50°C than when formic acid treatment was performed at 20°C. Fig.10 The results show that formic acid treatment under high temperature conditions (about 40°C to 50°C) for a short time (5 minutes) is effective in cell disruption even for Gram-positive bacteria such as Janibacter limosus.

[0198] As described above, formic acid treatment at about 40°C to 50°C did not significantly reduce the number of detected ribosomal proteins even in Escherichia coli (Escherichia coli) whose cells are easily disrupted. Therefore, formic acid treatment at about 40°C to 50°C is a treatment condition that does not cause major damage even in MALDI measurement of microorganisms whose cells are easily disrupted. As described above, in formic acid treatment, heating at about 40°C to 50°C does not reduce the peak of cytoplasmic components (proteins in the cytoplasm, etc.) for microbial cells that are easily disrupted, such as Escherichia coli (Escherichia coli), but promotes cell disruption in strains that are difficult to disrupt, such as Janibacter limosus (Janibacter limosus). Therefore, it is shown that formic acid treatment for 5 minutes in a state heated to about 40°C to 50°C is a pretreatment method that can be used universally regardless of whether it is a bacteria that is easy to disrupt or a bacteria that is difficult to disrupt. In other words, it can be said that the heating formic acid treatment at about 40°C to 50°C for 5 minutes is a pretreatment method that can cope with a wide range of bacterial species and efficiently detect the peak of protein in the cytoplasm.

[0199] In addition, the detection efficiency of the peak of protein in the cytoplasm by formic acid is related to temperature and time. For example, it is considered that the same effect as the case of formic acid treatment at 50°C for 5 minutes can be shown by performing formic acid treatment for a long time at 20°C or for a short time (for example, 1 minute) at 60°C. As described above, it is shown that the possibility of cell disruption can be achieved while suppressing excessive protein decomposition by adjusting the heating time and temperature.

[0200] (4-7. Experiment 7)

[0201] Experiment 7 is an experiment to show the effect of heating during formic acid treatment on lysable filamentous fungi ( Aspergillus kawachii (white mold) NBRC 4308).

[0202] The pretreatment of the samples in Experiment 7 was performed as follows.

[0203] (1) The filamentous fungus (Aspergillus kawachii (white mold) NBRC 4308) was cultured in a potato dextrose agar medium.

[0204] (2) Scrape the mycelium with a cotton swab and disperse it in 1000 μL of water to obtain a dispersion. Pour 200 μL of the obtained dispersion into three tubes. Further add 800 μL of ethanol to each of the three tubes. For each tube, remove the supernatant by centrifugation, and add 50 μL of 70% formic acid to the precipitate (bacterial particles) to redisperse it (contact process).

[0205] (3) After (2), immediately after setting to 50°C, Fig.11 50 μL of acetonitrile was added to the sample with data of (50°C, 0 minutes) and mixed, and centrifuged to obtain a supernatant. 0.5 μL of the supernatant was dripped onto the sample plate and dried. After drying, 1 μL of CHCA solution was dripped and dried again. The CHCA solution was prepared by dissolving CHCA in an aqueous solution containing 35% acetonitrile and 15% ethanol containing 1% trifluoroacetic acid to a concentration of 10 mg / mL.

[0206] (4) After (2), place another tube (equivalent to Fig.11 The sample with the data of (room temperature, 5 minutes) was placed at room temperature (23 degrees) for 5 minutes. Fig.11 The sample with the data of (50°C, 5 minutes) was heated at 50°C for 5 minutes (heating process). After that, 50 μL of acetonitrile was added to each of the two tubes, mixed, and centrifuged to obtain a supernatant. 0.5 μL of the supernatant was dropped onto the sample plate and dried. After drying, 1 μL of the above CHCA solution was added and dried again.

[0207] (5) Each sample after adding CHCA solution in (3) or (4) and drying was measured using MALDI-8020.

[0208] Fig.11 This is a graph showing changes in the mass spectrum caused by heating and formic acid treatment in Aspergillus kawachii (white mold). Fig.11 , multiple peaks can be detected from filamentous fungi by performing a heating formic acid treatment at 50° C. for 5 minutes. The results of Experiment 7 show that the heating formic acid treatment is a highly versatile method that is also effective for Aspergillus kawachii (Aspergillus kawachii).

[0209] [5. Conclusion]

[0210] As described above, according to the pretreatment method of the present embodiment, even for microorganisms with strong cell walls, a mass spectrum with high peak intensity can be obtained by heating the sample while it is in contact with an acidic solution. In particular, the number or intensity of the peaks of ribosomal proteins, which are useful biomarkers for microbial discrimination, is also increased. When all conditions other than heating are kept the same and only the presence or absence of heating is changed, the increase in the number and intensity of the peaks occurs, and it is believed that the increase in the number and intensity of the peaks is the result of the lysis caused by the acidic solution being appropriately promoted due to overheating. In addition, the result of the increase in the number and intensity of the peaks is that the discrimination efficiency of microorganisms is improved.

[0211] In particular, the pretreatment method of the present embodiment is effective for improving the number and intensity of peaks of mycolic acid-producing bacteria, which are important bacteria in medical and clinical research.

[0212] It is generally believed that if cells are overheated, proteins will decompose and may become multiple fragments. In this case, peaks corresponding to the multiple fragments are detected in the mass spectrum, and it may be difficult to detect the peaks representing the proteins before decomposition. The pretreatment method of the present embodiment promotes cell wall destruction by combining an acidic solution of a specified concentration with appropriate heating, and can prevent the proteins contained in the cytoplasm from being overly decomposed. Therefore, the peaks of ribosomal proteins as biomarkers can be detected with high accuracy.

[0213] In the pretreatment method of the present embodiment, the three-dimensional structure of the protein molecule may slightly change (denature), but even if denatured, the mass of the protein molecule itself does not change, so there is no hindrance in mass analysis.

[0214] The pretreatment method of the present embodiment has the feature that it is easy to implement for the user. According to the heating method on the sample plate, after the bacteria are dispersed in the first acidic solution, they can be heated together with the sample plate. In addition, according to the heating method in the tube, heating can be performed by using a PCR device that is installed in most laboratories that handle microorganisms. Furthermore, since beads are not used, it is easy to handle, and the beads will not be released into devices such as mass spectrometers, so it is safe. Since it corresponds to a wide range of microorganisms including microorganisms with strong cell walls, there is no need to study the pretreatment method according to the type of microorganism. More specifically, in a wide range of microorganisms, under the above-mentioned relatively uniform conditions (e.g., heating for 2 to 20 minutes), the effect of improving the peak intensity can be obtained. In view of these characteristics, the pretreatment method of the present embodiment is also useful in that a wide range of users can easily implement this point.

[0215] In particular, the pretreatment method of the present embodiment can be completed by simple liquid operation and heating, so it is also easy to automate some or all of the steps by machinery. More specifically, it is also easy to assemble into a pretreatment device. In addition, for the same reason, multiple containers containing prescribed types of microorganisms are prepared, and multiple conditions selected from the above-mentioned relatively uniform conditions are tried at the same time, so that it is also easy to find the best condition with the highest peak intensity and obtain the mass spectrum under the best condition in one experiment.

[0216] In addition, in the above examples, it is mainly shown that the intensity of the peak corresponding to the intracellular component of the microorganism can be increased by using the pretreatment method of the present embodiment, but it is obvious that the pretreatment method of the present embodiment can also be applied to biological samples containing cells of organisms other than microorganisms. For example, it is also useful in destroying the cell wall and / or cell membrane of cells other than microorganisms to extract intracellular components.

[0217] In addition, in the above examples, in the pretreatment method of the present embodiment, the extraction of cytoplasmic components by contacting an organic acid with the first acidic solution and heating is described, but as long as the same effect as the pretreatment method of the present embodiment is exerted, the first acidic solution containing an inorganic acid can also be used. As for the example of the first acidic solution containing an organic acid, as long as the same effect as the pretreatment method of the present embodiment is exerted, it is not limited to the above example. In addition, the first acidic solution exerting the effect of the present application refers to an acidic solution that increases the intensity of the peak of the cytoplasmic component, for example, by heating acid treatment. In addition, the first acidic solution that does not exert the effect of the present application refers to an acid that cannot fully destroy the cell wall, an acid that decomposes protein to the extent that the detection of the peak of the cytoplasmic component is hindered, and / or an acidic solution that has problems in the process other than cytoplasmic extraction. For example, an acidic solution containing hydrochloric acid is usually likely to dissolve a sample plate containing stainless steel, and is therefore considered unsuitable for use in the heating treatment on the sample plate of the present embodiment. On the other hand, an acidic solution containing hydrochloric acid can be used for heating treatment in a tube in an acid-resistant container (for example, a glass or acid-resistant resin container). In this case, it is preferred to perform sample washing after the heating treatment in the tube. As described above, the acid in the pretreatment of the present embodiment can be arbitrarily selected within the scope of consideration of those skilled in the art.

[0218] [plan]

[0219] Those skilled in the art will appreciate that the above-mentioned multiple exemplary embodiments are specific examples of the following schemes.

[0220] (Item 1) A pretreatment method of one scheme is a pretreatment method of a sample containing cells for mass analysis, comprising: a step of contacting the cells with a first acidic solution containing an organic acid; and a step of heating the cells while they are in contact with the first acidic solution to extract cytoplasmic components of the cells.

[0221] According to the pretreatment method described in Item 1, the extraction efficiency of the cytoplasmic component is improved. Therefore, the intensity of the peak corresponding to the cytoplasmic component in the mass spectrum is improved. That is, by pretreating the sample containing cells for mass analysis, the intensity of the peak of the cytoplasmic component can be increased.

[0222] (Item 2) In the pretreatment method according to Item 1, the contacting step includes the step of preparing a second acidic solution as a mixed solution of the sample and the first acidic solution. The extracting step includes the step of heating the second acidic solution.

[0223] According to the pretreatment method described in Item 2, the cells can be dispersed in the first acidic solution by mixing the sample with the first acidic solution. This allows the cells to be reliably brought into contact with the first acidic solution, thereby improving the efficiency of treating the cells with the first acidic solution.

[0224] (Item 3) In the pretreatment method described in Item 1 or 2, the organic acid contains at least one of formic acid, trifluoroacetic acid and acetic acid.

[0225] According to the pretreatment method described in Item 3, the pretreatment method of this embodiment can be implemented using these acids that are easily available and accustomed to being handled by users who conduct research and clinical field analysis of microorganisms. In addition, even if a person skilled in the art uses these acids for pretreatment of mass analysis, it is known from experience that they will not adversely affect the measurement results, which is also useful in this regard.

[0226] (Item 4) In the pretreatment method described in any one of Items 1 to 3, the concentration of the organic acid is 50% by volume or more and 90% by volume or less based on the first acidic solution or the second acidic solution.

[0227] According to the pretreatment method described in Item 4, the pretreatment method can be carried out using the acidic solution having the above concentration.

[0228] (Item 5) In the pretreatment method described in Item 4, the concentration of the organic acid is 65% by volume or more and 75% by volume or less based on the first acid solution or the second acid solution.

[0229] According to the pretreatment method described in Item 5, the pretreatment method of the present embodiment can be implemented using the acidic solution having the above-mentioned concentration.

[0230] (Item 6) In the pretreatment method described in any one of Items 1 to 5, the first acidic solution contains water or an organic solvent.

[0231] According to the pretreatment method described in Item 6, the pretreatment method of the present embodiment can be implemented using the first acidic solution prepared using these solvents.

[0232] (Item 7) In the pretreatment method described in any one of Items 1 to 6, the heating time is 2 minutes or more and less than 20 minutes.

[0233] According to the pretreatment method described in Item 7, the pretreatment method of the present embodiment can be implemented by performing heating for the above-mentioned time.

[0234] (Item 8) In the pretreatment method described in Item 7, the heating time is 5 minutes to 10 minutes.

[0235] According to the pretreatment method described in Item 8, the pretreatment method of the present embodiment can be implemented by performing heating for the above-mentioned time.

[0236] (Item 9) In the pretreatment method described in any one of Items 1 to 8, the heating temperature is 30°C to 75°C.

[0237] According to the pretreatment method described in Item 9, the pretreatment method of the present embodiment can be implemented by heating at the above-mentioned temperature.

[0238] (Item 10-1) In the pretreatment method described in Item 9, the heating temperature is 40°C to 60°C.

[0239] (Item 10-2) In the pretreatment method described in Item 9, the heating temperature is 35°C to 55°C.

[0240] The pretreatment method of the present embodiment can be implemented by heating at the above-mentioned temperature according to the pretreatment method described in Items 10-1 and 10-2.

[0241] (Item 11) In the pretreatment method described in Item 2, the step of heating the second acidic solution includes: the step of heating the second acidic solution set on a sample plate for mass analysis; or the step of heating the second acidic solution contained in a container before being set on the sample plate.

[0242] According to the pretreatment method described in Item 11, the second acidic solution can be heated by the two simple methods described above. The user can select an appropriate method from the two simple methods described above.

[0243] (Item 12) In the pretreatment method described in any one of Items 1 to 11, the cells are microbial cells.

[0244] According to the pretreatment method described in Item 12, the pretreatment method of this embodiment can be implemented even when quality analysis of microorganisms is performed on-site in clinical microbiological analysis, food hygiene inspection, and the like.

[0245] (Item 13) In the pretreatment method described in any one of Items 1 to 12, the cells are cells having a cell wall.

[0246] According to the pretreatment method described in Item 13, the cell wall can be destroyed using the pretreatment method of this embodiment, thereby increasing the intensity of the peak of the cytoplasmic component.

[0247] (Item 14) In the pretreatment method described in any one of Items 1 to 13, the mass analysis is mass analysis by matrix-assisted laser desorption ionization.

[0248] According to the pretreatment method described in Item 14, mass analysis can be performed using the MALDI method suitable for identification of microorganisms by mass analysis of microorganisms.

[0249] (Item 15) A mass spectrometry method comprising the step of performing matrix-assisted laser desorption ionization mass spectrometry on a sample pretreated by the pretreatment method according to any one of Items 1 to 14 to obtain a mass spectrum.

[0250] The embodiments disclosed this time should be considered in all respects as illustrative and non-restrictive. The scope of the present invention is indicated not by the above description but by the claims, which means that all modifications within the meaning and scope equivalent to the claims are also included.

[0251] Description of Reference Numerals

[0252] 1Analytical device

[0253] 10. Control Unit

[0254] 11 Processing Department

[0255] 12 Storage unit

[0256] 13 Input and output unit

[0257] 20 Measurement Department

[0258] 21Ionization unit

[0259] 22Ion Acceleration Unit

[0260] 23Mass separation unit

[0261] 24 Detection Department

[0262] 111 Device Control Unit

[0263] 113 Mass Spectrometry Department

[0264] 114 Identification Department

[0265] 131 Input unit

[0266] 132 Output unit

[0267] 133 Ministry of Communications

[0268] 221 Accelerating Electrode

[0269] 231 flight tube.

Claims

1. A pretreatment method for a sample containing cells for mass analysis, characterized in that: have: a step of contacting the cells with a first acidic solution comprising an organic acid; and The step of extracting cytoplasmic components of the cells by heating the cells while they are in contact with the first acidic solution.

2. The pre-treatment method according to claim 1, characterized in that: The contacting step includes the step of preparing a second acidic solution as a mixed solution of the sample and the first acidic solution, The extracting step includes the step of heating the second acidic solution.

3. The pre-treatment method according to claim 1 or 2, characterized in that: The organic acid includes at least one of formic acid, trifluoroacetic acid and acetic acid.

4. The pre-treatment method according to claim 2, characterized in that: The concentration of the organic acid is 50 volume % or more and 90 volume % or less based on the first acidic solution or the second acidic solution.

5. The pre-treatment method according to claim 4, characterized in that: The concentration of the organic acid is 65 volume % or more and 75 volume % or less based on the first acidic solution or the second acidic solution.

6. The pre-treatment method according to claim 1 or 2, characterized in that: The first acidic solution includes water or an organic solvent.

7. The pre-treatment method according to claim 1 or 2, characterized in that: The heating time is greater than 2 minutes and less than 20 minutes.

8. The pre-treatment method according to claim 7, characterized in that: The heating time is 5 minutes to 10 minutes.

9. The pre-treatment method according to claim 1 or 2, characterized in that: The heating temperature is 30° C. or higher and 75° C. or lower.

10. The pre-treatment method according to claim 9, characterized in that: The heating temperature is 35° C. or higher and 55° C. or lower.

11. The pre-treatment method according to claim 2, characterized in that: The step of heating the second acidic solution comprises: a step of heating the second acidic solution disposed on a sample plate for mass analysis; or A step of heating the second acidic solution contained in a container before placing the second acidic solution on the sample plate.

12. The pretreatment method according to claim 1 or 2, characterized in that: The cells are cells of microorganisms.

13. The pretreatment method according to claim 1 or 2, characterized in that: The cells are cells having cell walls.

14. The pre-treatment method according to claim 1 or 2, characterized in that: The mass analysis is mass analysis by matrix-assisted laser desorption ionization.

15. A quality analysis method, characterized in that: The method comprises the step of performing mass analysis by matrix-assisted laser desorption ionization on the sample pretreated by the pretreatment method according to claim 1 or 2 to obtain a mass spectrum.

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