Pretreatment method and mass spectrometry method

Abstract

Provided is a mass-spectrometry pretreatment method for a sample containing cells, the method including: a step for bringing the cells into contact with a first acidic solution containing an organic acid; and a step for extracting cytoplasmic components of the cells by heating the cells and the first acidic solution in the state in which said materials are in contact with each other.

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 technology]

[0002] A method of pretreating a sample containing cells with formic acid before mass spectrometry is known. Non-Patent Document 1 discloses that results suggest that the identification accuracy of a specific bacterial strain was improved by treating the strain with formic acid on a target plate or in a microtube and then performing mass spectrometry. Non-Patent Document 2 discloses a pretreatment method in which, for a specific bacterial strain cultured on a plate, cells are treated with formic acid on a sample plate or in a tube. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Comparison of the identification accuracy of nutritionally variant streptococci between MALDI-TOF MS2 and Vitec 2, and an examination of biochemical properties important for identification, Michiko Furukawa et al., Japanese Journal of Clinical Microbiology, 2016, vol. 26 No. 3, pp. 29-39 [Non-patent document 2] Rapid identification of microorganisms using MALDI-TOF MS for food microbiology, Hiroko Kawasaki, Journal of the Japanese Society for Food Microbiology, 2020, vol.37 No.4, pp.165-177 Summary of the Invention [Problem to be solved by the invention]

[0004] As disclosed in Non-Patent Documents 1 and 2, methods for treating cells with formic acid are roughly divided into methods in which cells are treated on a sample plate and methods in which cells are treated in a container such as a tube.

[0005] However, mass spectra obtained using the on-plate treatment method suffer from the problem of low peak intensities corresponding to cytoplasmic components. This is thought to reflect insufficient cell disruption and low extraction efficiency of cytoplasmic components. On the other hand, in-vessel treatment methods are thought to improve the extraction efficiency of cytoplasmic components by ensuring more reliable contact between individual cells and formic acid. However, it is known that in the case of microorganisms with strong cell walls, the in-vessel treatment method may result in insufficient peak intensities corresponding to the cytoplasmic components.

[0006] Ribosomal proteins, which are the main biomarkers for identifying and differentiating microorganisms, are contained in the cytoplasm, so there is a need for a method to improve the intensity of the peaks of cytoplasmic components.

[0007] The present disclosure has been made to solve such problems, and its purpose is to improve the intensity of peaks of cytoplasmic components by pre-treating a sample containing cells for mass spectrometry. [Means for solving the problem]

[0008] A pretreatment method according to a first aspect of the present disclosure is a method for pretreatment of a sample containing cells for mass spectrometry, comprising: Acid and heating the cells in contact with the first acidic solution to extract cytoplasmic components of the cells. [Effects of the Invention]

[0009] Pretreatment of cell-containing samples for mass spectrometry can enhance the intensity of peaks of cytoplasmic components. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a schematic diagram illustrating a configuration of an analysis device according to an embodiment. [Figure 2]10 is a flowchart showing a process relating to an on-plate heating method and a mass analysis method. [Figure 3] 10 is a flowchart showing processes relating to an intra-tube heating method and a mass analysis method. [Figure 4] FIG. 2 shows mass spectra obtained when the first acidic solution has different acid concentrations. [Figure 5] FIG. 10 shows a mass spectrum obtained after non-heated formic acid treatment. [Figure 6] FIG. 10 shows a mass spectrum obtained after heating with formic acid. [Figure 7] FIG. 1 shows changes in the intensity of ribosome peaks due to heating and formic acid treatment. [Figure 8] FIG. 8 shows changes in mass spectra caused by heating and formic acid treatment in a type of microorganism different from that in FIG. 7. [Figure 9] FIG. 1 shows the number of peaks when Escherichia coli is treated with formic acid under a variety of temperature conditions. [Figure 10] FIG. 1 shows the number of peaks when Janibacter limosus was treated with formic acid under a number of temperature conditions. [Figure 11] FIG. 1 shows changes in the mass spectrum of Aspergillus kawachii due to heating and formic acid treatment. DETAILED DESCRIPTION OF THE INVENTION

[0011] An embodiment of the present disclosure (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form "A to Z" means the upper and lower limits of a range (i.e., A or more and Z or less), and when no unit is specified for A and a unit is specified only for Z, the unit of A and the unit of Z are the same.

[0012] In addition, in this specification, "%" in a solution means "volume %" unless otherwise specified. The present embodiment will be described in detail below with reference to the drawings. In the following, the same or corresponding parts in the drawings are denoted by the same reference numerals, and their description will not be repeated in principle.

[0013] [1. Configuration of the analytical device] First, an example of an analytical apparatus 1 for carrying out a mass spectrometry method according to this embodiment is shown. The mass spectrometry method according to this embodiment includes a biological sample pretreatment method according to this embodiment. In this specification, unless otherwise specified, "pretreatment" refers to the preparation of a biological sample before mass spectrometry.

[0014] FIG. 1 is a schematic diagram showing the configuration of an analytical device 1. The analytical device 1 is a mass spectrometer for performing mass analysis of substances contained in a sample, and is, for example, a MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometer). Spectrometer The analysis device 1 uses the mass spectrum obtained by mass analysis to identify the type of organism.

[0015] The sample includes cells of a living organism. The cells contain a target substance, which is a molecule to be analyzed. In this embodiment, analysis by the analyzer 1 includes detecting peaks in a mass spectrum and measuring the mass-to-charge ratio (m / z) of a specific or non-specific substance contained in the sample. In one example, the substance is a protein. Analysis by the analyzer 1 includes identifying the type of microorganism from which the sample originates, based on the m / z corresponding to the peak in the mass spectrum. The m / z corresponding to the peak in the mass spectrum is generally also referred to as the "position" or "m / z position" of the peak.

[0016] Unless otherwise specified, the term "type of microorganism" as used herein includes at least one systematic taxonomic rank of the genotype, strain, subspecies, species, genus, and family of the microorganism. Furthermore, the determination of the type of microorganism includes classification, identification, and differentiation of the type of the microorganism. Hereinafter, the determination of the type of microorganism will also be referred to simply as the determination of the microorganism.

[0017] The analysis in the analysis device 1 may include determining whether or not a particular substance is contained in a sample.

[0018] Referring to FIG. 1, the analyzer 1 includes a control unit 10 and a measurement unit 20. The measurement unit 20 ionizes substances (e.g., proteins) in the sample using a high voltage, separates the ions S according to their time of flight, which correlates with m / z, and then detects them. The measurement unit 20 includes an ionization unit 21, an ion acceleration unit 22, a mass separation unit 23, and a detection unit 24. In Figure 1, the movement of the ions S in the measurement unit 20 is schematically indicated by arrow A1.

[0019] In one embodiment, the ionization unit 21 ionizes substances in the sample by matrix-assisted laser desorption ionization (MALDI). As will be described later, the MALDI method is a useful method for identifying microorganisms by mass spectrometry. As an ionization method, any soft ionization method such as electrospray ionization (ESI) can be used in addition to the MALDI method. When ionization is performed by the ESI method, it is preferable that the analyzer 1 further includes a liquid chromatograph and that the ionization unit 21 ionizes substances in the sample separated by the liquid chromatograph, as this can provide high separation ability.

[0020] The ionization unit 21 includes an ion source including a sample plate holder (not shown) that supports the sample plate and a laser device (not shown) that irradiates the sample plate with laser light. The analyst mixes a matrix solution with a sample that has been subjected to a pretreatment method according to the present embodiment (described later) and places the mixture on the sample plate. The matrix solution contains a matrix substance that easily absorbs and is easily ionized by laser light. Examples of matrix substances include, but are not limited to, α-cyano-4-hydroxycinnamic acid (4-CHCA), α-cyano-3-hydroxycinnamic acid (3-CHCA), sinapic acid, ferulic acid, 3-hydroxy-4-nitrobenzoic acid (3H4NBA), 2,5-dihydroxybenzoic acid, and 1,5-diaminonaphthalene.

[0021] The analyst obtains a dried sample by drying the sample-mixed matrix solution, which is a mixture of the sample and the matrix solution, on a sample plate. The sample plate is then placed in a sample plate holder in the vacuum chamber of the ionization unit 21.

[0022] The dried product obtained by mixing a sample with a matrix solution and drying the mixture is generally referred to as a "crystal," and more specifically, is variously called a "crystal," "mixed crystal," "sample crystal," "matrix crystal," "sample / matrix crystal," etc. In this specification, it will hereinafter be referred to as a "dried matrix product."

[0023] In the following, unless otherwise specified, the description of the sample-mixed matrix solution at the stage of being placed on the sample plate and forming a dried matrix product will be referred to as the "matrix solution," including the case where the matrix solution is mixed with the sample.

[0024] After depressurizing the vacuum chamber containing the sample plate, the ionization unit 21 irradiates the dried matrix on the sample plate with laser light to ionize the target substance in the dried matrix. The type of laser device that irradiates the laser light is not particularly limited as long as it can emit light that is absorbed by the matrix solution used. For example, when the matrix solution contains CHCA, an N2 laser (wavelength 337 nm) or the like can be suitably used. The ions S of the target substance ionized by the ionization unit 21 are extracted by an electric field generated by an extraction electrode or the like (not shown) and introduced into the ion acceleration unit 22.

[0025] 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.

[0026] The mass separation unit 23 includes a flight tube 231, and separates the ions S based on the difference in flight time when each ion S flies inside the flight tube 231. While a linear type flight tube 231 is shown in FIG. 1, a reflectron type, a multi-turn type, or the like may also be used. There are no particular limitations on the method of mass analysis as long as it is possible to separate and detect the ions S contained in the sample.

[0027] The detection unit 24 includes an ion detector such as a multi-channel plate, detects the ions S separated by the mass separation unit 23, and outputs a detection signal with an 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. In Fig. 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 arrow A2.

[0028] The control unit 10 includes a processing unit 11, a storage unit 12, and an input / output unit 13. The control unit 10 is configured, for example, by one or more computers.

[0029] The processing unit 11 includes a processor such as a CPU, and functions as a main unit for controlling the analysis device 1. The processing unit 11 performs various processes by executing programs stored in the storage unit 12 or the like.

[0030] The processing unit 11 includes a device control unit 111 and a mass spectrum analysis unit 113. The mass spectrum analysis unit 113 includes a discrimination unit 114.

[0031] The device control unit 111 controls the operation of the measurement unit 20 based on data relating to analysis conditions input from the input unit 131, which will be described later. In Fig. 1, the control of the measurement unit 20 by the device control unit 111 is schematically shown by arrow A3.

[0032] The mass spectrum analysis unit 113 converts the flight time into m / z from measurement data including the amount of ions detected by the detection unit 24 and the flight time of the ions, and creates a mass spectrum showing the amount of detection corresponding to each m / z.

[0033] The number of detection signals of the target substance detected by the detection unit 24 and the intensity of the detection signals correlate with the number of peaks corresponding to the target substance in the mass spectrum and the intensity of the peaks. That is, the greater the amount of the target substance contained in the dried matrix, the greater the peak intensity. Therefore, the greater the extraction efficiency of the target substance in the sample pretreatment, the greater the peak intensity.

[0034] The mass spectrum analysis unit 113 further determines the m / z corresponding to the peak in the mass spectrum. The mass spectrum analysis unit 113 may determine the substance to which the m / z indicated by the peak in the mass spectrum corresponds, based on a protein database or the like. That is, the mass spectrum analysis unit 113 can calculate the m / z of a specific or non-specific substance contained in the sample. The mass spectrum analysis unit 113 may further determine whether or not a specific substance is contained in the sample based on the m / z (identification of components in the sample).

[0035] The mass spectrum analysis unit 113 includes a discrimination unit 114. In one embodiment, the discrimination unit 114 creates a database including mass spectra and stores it in the memory unit 12. The database includes one or more, and preferably many, mass spectra of microorganisms whose types are known. The discrimination unit 114 uses the database of mass spectra to discriminate between microorganisms.

[0036] In one embodiment, the discrimination unit 114 discriminates microorganisms by fingerprinting. Specifically, the discrimination unit 114 discriminates microorganisms by comparing the mass spectrum pattern of an unknown microorganism with the mass spectrum patterns of known microorganisms stored in the database. The discrimination of microorganisms is performed by referring to the peaks of biomarkers, which are substances that exhibit characteristic expression patterns for each microorganism. In microorganisms, ribosomal proteins are mainly used as biomarkers.

[0037] The storage unit 12 includes a non-volatile storage medium. The storage unit 12 stores the mass spectrum generated by the mass spectrum analysis unit 113, the measurement data output from the measurement unit 20, and a program for the processing unit 11 to execute processing. The storage unit 12 corresponds to an embodiment of "memory" in the present disclosure. The storage unit 12 may include a storage medium that is removable from the analysis device 1. The storage medium may be any medium capable of storing various types of data, such as a CD (Compact Disc), a DVD (Digital Versatile Disc), or a USB (Universal Serial Bus) memory. In one embodiment, the storage unit 12 stores a database including the acquired mass spectra.

[0038] The input / output unit 13 is an interface for the analytical device 1 to input and output information from and to the outside. The input / output unit 13 includes an input unit 131, an output unit 132, and a communication unit 133.

[0039] The input unit 131 is configured to include input devices such as a mouse, a keyboard, various buttons and / or a touch panel, etc. The input unit 131 receives information necessary for controlling the operation of the measurement unit 20, information necessary for the processing performed by the processing unit 11, etc. from the analyst.

[0040] The output unit 132 includes a display device such as a liquid crystal monitor, a printer, etc. The output unit 132 displays information about the measurement by the measurement unit 20, the results of the processing by the processing unit 11, etc. on a display device or prints them on a paper medium.

[0041] The communication unit 133 includes a communication device capable of communicating via a wired or wireless connection such as the Internet. The communication unit 133 receives data necessary for processing by the processing unit 11, transmits data processed by the processing unit 11 such as a determination result, and transmits and receives necessary data as appropriate.

[0042] Some or all of the functions of the control unit 10 described above may be located in a computer, server, or the like that is physically separated from the measurement unit 20 .

[0043] The analyzer 1 used in this embodiment is preferably, but not limited to, one combined with a MALDI (matrix-assisted laser desorption ionization) ion source. Examples of the analyzer combined with a MALDI ion source include a MALDI-IT (matrix-assisted laser desorption ionization-ion trap) mass spectrometer, a MALDI-IT-TOF (matrix-assisted laser desorption ionization-ion trap-time-of-flight) mass spectrometer, or a MALDI-FTICR (matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance) mass spectrometer. The analytical conditions for the analyzer 1 are set within the range typically set by a person skilled in the art.

[0044] [2. Conventional sample pretreatment methods] Conventionally, there is a method for identifying microorganisms using mass spectrometry. Mass spectrometry allows for the easy and rapid acquisition of mass spectra as analytical results using a minute amount of microbial sample. Furthermore, the use of automated analysis allows for rapid and simple analysis of multiple samples.

[0045] Among these mass spectrometry methods, the analysis of microorganisms by MALDI-MS, a soft ionization method that ionizes biopolymers such as proteins with almost no degradation, is particularly widely used. Specifically, MALDI-MS is used to identify specific types of microorganisms in clinical microbiology analysis, food hygiene inspections, and other settings. Thus, while MALDI-MS is currently an excellent technology for identifying specific types of microorganisms, it has some difficulties in identifying specific types of microorganisms.

[0046] For example, the simplest and most commonly used pretreatment method involves simply mixing cells with a matrix solution. However, when this simplest pretreatment method is applied to microorganisms with rigid cell walls (Gram-positive bacteria, fungi, etc.), the ribosomal protein peaks may not be detected with sufficient intensity in the mass spectrum. This result is thought to reflect the fact that simply mixing with a matrix solution does not sufficiently destroy the microbial cell wall, preventing the release of components inside the cell wall and / or cell membrane, including ribosomal proteins. Hereinafter, in this specification, these "components inside the cell wall and / or cell membrane" are referred to as "intracellular components." Intracellular components include cytoplasmic components and nuclear components, and ribosomal proteins are contained in the cytoplasmic components.

[0047] Ribosomal proteins are used as biomarkers to identify species because slight differences in amino acid sequence and resulting mass can be decisive indicators for evaluating differences between species. Furthermore, ribosomal proteins are highly abundant structures within cells, so they are easily detected by mass spectrometry.

[0048] In addition, most ribosomal proteins are basic proteins with high proton affinity, so they are [M+H] in the MALDI process. + Furthermore, the molecular weight of ribosomal proteins is approximately 5,000 to 20,000, and MALDI-MS can determine the mass of ribosomal proteins with an error range of a few Da. Therefore, MALDI-MS mass spectrometry has the advantage of easily detecting ribosomal protein peaks from samples.

[0049] As described above, ribosomal proteins are excellent biomarkers, and therefore, in the identification of microorganisms by mass spectrometry, ribosomal proteins are generally used primarily as biomarkers.

[0050] As mentioned above, ribosomal proteins are primarily used to distinguish microorganisms, but other proteins, such as DNA-binding proteins, RNA-binding proteins, and molecular chaperones, are also known to be useful. These proteins, like ribosomal proteins, are also included in intracellular components.

[0051] Therefore, when performing mass spectrometry using the above-mentioned simplest pretreatment method, depending on the type of microorganism, the number and / or intensity of peaks of biomarkers, such as ribosomal proteins, may be insufficient, making it difficult to distinguish them.

[0052] In order to extract cytoplasmic components from cells with such a solid cell wall and obtain peaks with a sufficient number and / or intensity, a method of pretreating the cells with formic acid is known.

[0053] 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 involving heating according to the present embodiment described below, the conventional formic acid treatment on a sample plate will also be referred to as "on-plate non-heated treatment," and the conventional formic acid treatment in a tube will also be referred to as "in-tube non-heated treatment." Furthermore, a pretreatment method using "on-plate non-heated treatment" will also be referred to as "on-plate non-heated method," and a pretreatment method using "in-tube non-heated treatment" will also be referred to as "in-tube non-heated method."

[0054] In the on-plate non-heating method, for example, formic acid is dropped onto microbial cells applied to a sample plate, followed by on-plate non-heating treatment, followed by drying, and then a matrix solution is dropped onto the cells and dried again to obtain a dried matrix product.

[0055] In the in-tube non-heating method, cells are mixed with formic acid in a tube, and then acetonitrile is often added to the tube and mixed. The tube is then centrifuged, and the supernatant is dripped onto a sample plate and dried. A matrix solution is then dripped onto the plate and dried again to obtain a dried matrix.

[0056] In addition, Non-heating method inside the tube In the above, before adding formic acid, the cells may be dispersed in ethanol, centrifuged, and the supernatant removed.

[0057] However, the on-plate non-heating method has the problem that the peak intensity of cytoplasmic components is lower than that of the in-tube non-heating method, and may not be sufficient for distinguishing microorganisms.

[0058] The in-tube non-heating method is thought to improve extraction efficiency compared to the on-plate non-heating method because the sample and formic acid are mixed in the tube. However, peak intensity may be insufficient for gram-positive bacteria (e.g., Mycobacterium tuberculosis) and fungi (e.g., mold and yeast) with rigid cell walls. In particular, peak intensity is low for gram-positive bacteria (e.g., actinomycetes of the Corynebacterineae family, such as Mycobacterium and Nocardia) that have long-chain fatty acids called mycolic acids in their cell walls. In other words, even with the in-tube non-heating method, cytoplasm extraction efficiency appears to be insufficient. This is thought to be due to the thick fatty acid layer in gram-positive bacteria that have mycolic acids in their cell walls.

[0059] The group of bacteria containing mycolic acids includes the important pathogenic bacteria Mycobacterium tuberculosis and nontuberculous mycobacteria. Similarly, the Nocardia genus, which produces mycolic acids, is an important pathogenic bacterium responsible for skin and central nervous system infections (nocardiosis). Therefore, it is important in medical and clinical research settings to properly distinguish between these Gram-positive bacteria that have mycolic acids in their cell walls.

[0060] On the other hand, bead-based disruption is also known as a pretreatment method for disrupting (lysing) cells by a method other than chemical reaction with formic acid. In this method, cells, a solvent, and beads (e.g., 0.5 mm zirconia beads) are placed in a tube and shaken to disrupt the cells through physical friction. This is thought to improve the efficiency of extraction of cytoplasmic components.

[0061] However, during the bead crushing process, the beads may clog or be sucked into the pipette tip, making handling difficult. Furthermore, if beads become contaminated with the dried matrix, they may detach from the sample plate inside the instrument and adhere to or become contaminated within the instrument. For example, the impact of laser irradiation may cause beads to fly out of the dried matrix, potentially leading to instrument malfunction or breakdown.

[0062] As described above, in pretreatment methods using formic acid, depending on the type of microorganism, sufficient peak intensity of cytoplasmic components may not be obtained. Furthermore, bead-crushing treatments have problems such as leading to equipment failure. Therefore, there is a need for a method for improving the peak intensity of cytoplasmic components in the pretreatment of cell-containing samples for mass spectrometry without using beads.

[0063] 3. Sample Pretreatment Method According to the Embodiment In view of the above circumstances, the inventors conducted trial and error in sample pretreatment and discovered a method for improving the peak intensity of cytoplasmic components without using beads.

[0064] In the pretreatment method according to this embodiment, microorganisms are mixed with an acidic solution and then heated, which increases the peak intensity of the cytoplasmic components.

[0065] More specifically, the pretreatment method according to this embodiment is a pretreatment method for a sample containing cells for mass spectrometry, and includes a "contact process" in which the cells are brought into contact with a first acidic solution containing an acid, and a "heating process" in which the cells are heated while in contact with the first acidic solution.

[0066] (3-1. Types of microorganisms) The microbial cells contained in the sample are not limited to cells of microorganisms having a gram-negative cell wall structure, but may also be cells of microorganisms having a gram-positive cell wall structure. More specifically, the microbial cells contained in the sample may be cells of gram-negative bacteria (e.g., Escherichia coli), gram-positive bacteria (e.g., Mycobacterium tuberculosis), or fungi (e.g., molds and yeasts).

[0067] More specifically, the sample may contain cells derived from prokaryotes or eukaryotes. The sample is typically derived from microorganisms and may contain unknown microorganisms. Prokaryotes include bacteria and archaea. Bacteria include Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis), Lactobacillus (e.g., Lactic acid bacteria), Synechocystis (e.g., Cyanobacteria), and Mycobacteria (e.g., Actinomycetes). Archaea include Methanophilus, Methanococcus, Thermococcus, and Phyllococcus. Eukaryotes include animals, plants, fungi, and protists. Fungi include filamentous fungi, yeasts, mushrooms, molds, and the like, and include phyla such as Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, Glomeromycota, and Microsporidia. The Ascomycota includes the genus Aspergillus (such as Aspergillus), the genus Penicillium (such as blue mold), the genus Saccharomyces (such as budding yeast), etc. In one embodiment, the sample is a cell (bacterial body) of a fungus in the broad sense, including the bacteria, archaea, and fungi described above.

[0068] The cells of these microorganisms are disrupted and cytoplasmic components are released by the following two processes:

[0069] (3-2. Contact Process) The sample to be contacted with the first acidic solution may be in liquid or solid form. More specifically, the sample may be, for example, a liquid containing a bacterial culture solution, or a bacterial colony scooped with a toothpick from a solid medium. The bacterial culture solution contains the bacteria and a medium for culturing the bacteria.

[0070] The acid may be, for example, an organic acid such as formic acid, trifluoroacetic acid, or acetic acid, which is commonly used in biological experiments. These organic acids are easily available and familiar to researchers and clinical users who perform mass spectrometry of microorganisms. Furthermore, even if skilled artisans use them for pretreatment of mass spectrometry, they do not adversely affect the measurement results. Other examples of acids include acids with an acid dissociation constant (pKa) of 0.2 to 5, more preferably 0.2 to 4. Acids with an acid dissociation constant of 0.2 to 5 partially overlap with the examples of acids listed above, but include at least one of trifluoroacetic acid, oxalic acid, glycine, salicylic acid, formic acid, lactic acid, benzoic acid, acetic acid, and butyric acid. Of course, the examples of acids listed above are not limited to these. In one embodiment, the organic acid is formic acid. As described below, inorganic acids can also be used. The acid contained in the first acidic solution may be a single acid (pure substance) or a mixture of multiple acids.

[0071] In this specification, "acid" refers to a pure substance (e.g., 100% formic acid). A solution obtained by diluting an acid with a solvent to a predetermined concentration is referred to as a "first acidic solution." The solvent typically contains water. The water is preferably pure water, ultrapure water, or ion-exchanged water. The solvent may contain an organic solvent in addition to or instead of water, as long as the effects of the pretreatment method according to this embodiment are achieved. The organic solvent is, for example, a polar organic solvent. The organic solvent is, for example, acetonitrile, methanol, or ethanol.

[0072] A typical specific means for the "contact process" is mixing a sample with a first acidic solution. In this specification, the mixed solution of the sample and the first acidic solution is referred to as a "second acidic solution." By mixing the sample with the first acidic solution, cells can be dispersed in the first acidic solution. This ensures reliable contact between the cells and the first acidic solution, thereby improving the efficiency of treatment of the cells with the first acidic solution.

[0073] The mixing ratio of the first acidic solution to the sample is not limited as long as the effects of the pretreatment method according to this embodiment are achieved. For example, the sample is mixed in a volume ratio of 1 / 2 or less to 1 / 1000 or more of the first acidic solution. However, generally, when mixing with the first acidic solution, the amount of non-cellular components (e.g., liquid medium used for cell culture) contained in the sample is minimized. Specifically, for example, the sample before mixing with the first acidic solution is centrifuged, the supernatant is removed, the remaining precipitate is recovered, and the recovered precipitate is mixed with the first acidic solution. It is also preferable to further centrifuge and wash the precipitate with ultrapure water or the like. For these reasons, the acid content (concentration) and pH of the second acidic solution are almost unchanged from those of the first acidic solution. Hereinafter, the first acidic solution and the second acidic solution may be collectively referred to as "acidic solutions."

[0074] 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.

[0075] 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 at the above concentrations containing cells.

[0076] In one embodiment, the first acidic solution is 50-90% trifluoroacetic acid, preferably 60-80% trifluoroacetic acid, and more preferably 65-75% trifluoroacetic acid. In this embodiment, the second acidic solution is trifluoroacetic acid at the above-mentioned concentration containing cells.

[0077] 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 at the above concentration containing cells.

[0078] Whether the acidic solution has been mixed properly can be confirmed by measuring the hydrogen ion exponent (pH) of the acidic solution using, for example, a pH meter.

[0079] Another example of a specific means of the "contact process" is a means of contacting (e.g., placing) cells with an object (e.g., a cloth) impregnated with the first acidic solution. Thus, the specific means of the "contact process" is not limited as long as the effects of the pretreatment method according to this embodiment are achieved.

[0080] Next, two pretreatment methods included in the pretreatment method according to this embodiment will be described in order. The two pretreatment methods differ in the heating method, which will be described later.

[0081] (3-3. Heating process) The heating process is performed to enhance the peak intensity of the cytoplasmic components. More specifically, it is believed that heating the second acidic solution in the heating process enhances the efficiency of cell disruption and the efficiency of extraction of the cytoplasmic components.

[0082] The heating time is, for example, from 2 minutes to less than 20 minutes, preferably from 3 minutes to 15 minutes, and more preferably from 5 minutes to 10 minutes.

[0083] The heating temperature is, for example, 30 to 75° C., preferably 40 to 60° C., more preferably 45 to 55° C., and even more preferably about 50° C. In one aspect of the present embodiment, the heating temperature may be 35 to 55° C., or about 40 to about 50° C.

[0084] (3-4. On-plate heating method) The pretreatment method according to this embodiment includes a pretreatment method including a process of heating a second acidic solution placed on a sample plate. Hereinafter, the process of heating a second acidic solution placed on a sample plate will be referred to as an "on-plate heating process," and a pretreatment method including the on-plate heating process will be referred to as an "on-plate heating method." Next, the on-plate heating method will be described with reference to FIG. 2.

[0085] Figure 2 is a flowchart showing the processes related to the on-plate heating method and mass spectrometry method. The on-plate heating method shown in Figure 2 is one form of the pretreatment method according to this embodiment. The steps shown in Figure 2 are performed manually by an analyst using laboratory equipment and devices commonly used for pretreatment and mass spectrometry of microorganisms. In the figure, "S" is used as an abbreviation for "STEP."

[0086] In S11, the analyst applies a sample to a sample plate. For example, the analyst scoops up a bacterial colony on a solid medium with a toothpick and applies it to the sample plate.

[0087] In S12, the analyst drops the first acidic solution onto the applied sample to prepare a second acidic solution, which is a mixed solution of the sample and the first acidic solution. The mixing is performed, for example, by pipetting multiple times with a micropipette. S12 corresponds to one form of the above-mentioned "contact process."

[0088] 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 in S3 corresponds to "on-plate heating treatment" and also corresponds to one form of the above-mentioned "heating process."

[0089] In S14, the analyst dries the second acidic solution on the sample plate. In S15, the analyst adds the matrix solution to the dried product obtained by drying the second acidic solution (hereinafter also referred to as the "dried acid product"). For example, within a few minutes after the second acidic solution has dried, the analyst adds the matrix solution dropwise and mixes the dried acid product with the matrix solution by pipetting multiple times with a micropipette.

[0090] In S16, the analyst dries the matrix solution containing the sample on the sample plate to obtain a dried matrix.

[0091] 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.

[0092] (3-5. Heating method inside the tube) The pretreatment method according to this embodiment includes a pretreatment method including a process of heating the second acidic solution contained in a container such as a tube before it is placed on a sample plate. Hereinafter, the process of heating the second acidic solution contained in a container will be referred to as the "in-tube heating process," and the pretreatment method including the in-tube heating process will be referred to as the "in-tube heating method." Next, the in-tube heating method will be described with reference to FIG. 3.

[0093] Fig. 3 is a flowchart showing the process for the in-tube heating method and mass spectrometry method. The in-tube heating method shown in Fig. 3 is one form of the pretreatment method according to this embodiment. The steps shown in Fig. 3 are performed manually by an analyst using laboratory equipment and devices commonly used for pretreatment and mass spectrometry of microorganisms.

[0094] In S21, the analyst suspends a sample containing cells in water in a tube. For example, the analyst scoops up a colony cultured on solid medium using a quantitative loop or the like, adds it to a 1.5 mL tube containing 200 μL of water, and mixes it for 30 seconds using a vortex mixer. As a more specific example, the analyst suspends approximately 10 mg of bacterial cells in water by picking up a colony approximately 3 mm in diameter five or more times with a platinum loop.

[0095] After suspending the sample in water, the sample may be washed by adding ethanol, if necessary. Washing the sample with ethanol has the effect of dispersing cells that have aggregated due to secretions of microorganisms into the solution. However, in the pretreatment method according to this embodiment, peaks of sufficient intensity were obtained even without washing the sample with ethanol, so washing the sample with ethanol is not essential.

[0096] An example of sample washing with ethanol is shown below. When washing with ethanol, it is preferable that the ethanol concentration be 80% or higher, based on the mixture of sample and ethanol. The analyst first adds 800 μL of ethanol to a tube containing the sample (for example, a tube containing a suspension of approximately 10 mg of bacterial cells in 200 μL of water) and mixes it with a vortex mixer for 30 seconds. Next, the analyst centrifuges the tube at 10,000 g for 2 minutes and then removes as much of the supernatant as possible. If necessary, the analyst may centrifuge the tube again to completely remove the supernatant. After removing the supernatant, it is preferable to allow the tube to air dry for a few minutes to evaporate the ethanol.

[0097] In S22, the analyst mixes the sample with the first acidic solution in the tube to prepare a mixed solution, a second acidic solution. For example, the analyst adds 50 μL of the first acidic solution to the sample-containing sediment in the tube, and then mixes it for 10 seconds using a vortex mixer. S22 corresponds to one form of the "contact process" described above.

[0098] In S23, the analyst heats the second acidic solution. 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 in S24 corresponds to "in-tube heating treatment" and also corresponds to one form of the above-mentioned "heating process."

[0099] After step S23, the analyst may add an organic solvent such as acetonitrile to the second acidic solution. The organic solvent dissolves the matrix material in the matrix solution and reduces the surface tension of the matrix solution. adjustment However, in the pretreatment method according to the present embodiment, a peak of sufficient intensity was obtained even without the addition of an organic solvent, and therefore the addition of an organic solvent is not essential.

[0100] The process of adding an organic solvent is carried out, for example, as follows: The analyst adds the same amount of acetonitrile as the formic acid to the tube and mixes it for 10 seconds using a vortex mixer.

[0101] In S24, the analyst drops the second acidic solution onto the sample plate. For example, the analyst centrifuges the heated tube at 10,000 to 15,000 g for 2 minutes, and drops 0.5 to 1 μL of the resulting supernatant onto the sample plate.

[0102] The processing of S25 to S28 is as follows: Figure 2 Since this corresponds to the processes in S14 to S17, the description will not be repeated.

[0103] In the mass spectrum of a sample pretreated by the pretreatment method according to the embodiment described above, the peak intensity of intracellular components is increased. This is thought to improve the efficiency of cell destruction and the efficiency of intracellular component extraction depending on the pretreatment method according to the embodiment. It is also expected that the efficiency of identifying microorganisms using the peaks of biomarkers contained in intracellular components will be improved.

[0104] [4. Experimental Example] Next, the pretreatment method according to this embodiment and its effects will be described in more detail using experimental examples, but the pretreatment method according to this embodiment is not limited to these experimental examples.

[0105] (4-1. Experiment 1) Experiment 1 is an experiment showing the effect of the concentration of acid in the first acidic solution.

[0106] The samples in Experiment 1 were pretreated as follows. (1) A sample containing the mycolic acid-producing bacterium Rhodococcus erythropolis NBRC 15567T was dispersed in 500 μL of ultrapure water in a microtube. (2) The bacterial cells dispersed in ultrapure water were dispensed in 100 μL portions. (3) 400 μL of ethanol was added to each microtube containing the bacterial cells, and the mixture was mixed and centrifuged to obtain a bacterial cell precipitate. (4) After removing the supernatant, add 50 μL of 50% formic acid, 50 μL of 70% formic acid, and 50 μL of 90% formic acid to the resulting precipitate. Vortex mixer The samples contained in each tube were then subjected to the following treatments. (5) Heating was carried out at 50°C for 5 minutes. (6) Add another 50 μL of acetonitrile Vortex mixer The mixture was mixed with the above mixture and centrifuged to obtain the supernatant. (7) 1 μL of the supernatant was dropped onto a sample plate and dried, and then 1 μL of matrix solution (CHCA solution) was dropped onto the plate and dried to prepare a dried matrix product, which was then measured by MALDI-MS.

[0107] Figure 4 shows mass spectra obtained from Experiment 1 when the first acidic solution had different acid concentrations. In Figure 4, the horizontal axis represents m / z. The vertical axis represents the percentage intensity, which is the relative intensity when the highest intensity peak among the three mass spectra included in Figure 4 is set to 100%.

[0108] In addition, X% FA in Fig. 4 means a first acidic solution containing X% formic acid. In addition, in the explanation of Fig. 4, the mass spectrum obtained as a result of heating using a first acidic solution containing X% formic acid is also referred to as an "X% formic acid mass spectrum."

[0109] 4, the peak intensities in the 70% and 90% formic acid mass spectra were several to several tens of times higher than those in the 50% formic acid mass spectrum. For example, the peak intensity near m / z 6600 was 3 to 5 times higher. Furthermore, while the peak near m / z 5500 was almost absent in the 50% formic acid mass spectrum, a clear peak was detected in the 70% and 90% formic acid mass spectra, with an intensity approximately 8 to 20 times higher.

[0110] That is, according to the results of Experiment 1, the peak intensity was improved by heating using 70 to 90% formic acid compared to 50% formic acid. Therefore, it is considered more preferable to use 70 to 90% formic acid for heating.

[0111] In addition, considering that formic acid has a distinctive odor, it is considered more preferable to use 70% formic acid, which has a weaker odor than 90% formic acid.

[0112] (4-2. Experiment 2) Experiment 2 is an experiment to demonstrate the effect of heating time.

[0113] The sample pretreatment in Experiment 2 was a modification of the sample pretreatment in Experiment 1 (4) and (5).

[0114] The sample pretreatments (4) and (5) in Experiment 2 were carried out as follows. (4) Remove the supernatant and add 50 μL of 70% formic acid. Vortex mixer The second acidic solution was then dispensed into eight microtubes. (5) Four of the dispensed microtubes were treated with formic acid for 2, 5, 10, and 20 minutes without heating (room temperature) (Figure 5). The remaining four microtubes were heated to 50°C and treated with formic acid for 2, 5, 10, and 20 minutes (Figure 5). 6)。

[0115] Hereinafter, formic acid treatment without heating will also be referred to as "non-heated formic acid treatment," and formic acid treatment in a heated state will also be referred to as "heated formic acid treatment."

[0116] FIG. 5 shows a mass spectrum obtained after non-heated formic acid treatment. FIG. 6 shows a mass spectrum obtained after heated formic acid treatment. In FIGS. 5 and 6, the horizontal axis represents m / z. The vertical axis in FIG. 5 represents the relative intensity when the single peak with the highest intensity among the four mass spectra included in FIG. 5 is taken as 100%. The vertical axis in FIG. 6 represents the relative intensity when the single peak with the highest intensity among the four mass spectra included in FIG. 6 is taken as 100%. In FIGS. 5 and 6, the data above m / z 7000 are displayed with their intensities multiplied by 10.

[0117] Referring to Figure 5, almost no peaks were detected above m / z 7000, even when the intensity was multiplied by 10. On the other hand, referring to Figure 6, peaks were detected above m / z 7000.

[0118] Hereinafter, in the description of FIG. 6, the mass spectrum obtained as a result of heating and treating with formic acid for X minutes will also be referred to as the "X minute heating mass spectrum."

[0119] 6, the peak intensities near m / z 7000 to 7200 in the 5-minute heating mass spectrum and the 10-minute heating mass spectrum were several times higher than those in the 2-minute heating mass spectrum. For example, while almost no peak was detected near m / z 7100 in the 2-minute heating mass spectrum, a clear peak was detected in both the 5-minute heating mass spectrum and the 10-minute heating mass spectrum.

[0120] On the other hand, the 20-minute heating mass spectrum showed lower peak intensities than the 5-minute and 10-minute heating mass spectra, suggesting that excessive formic acid reaction may have resulted in protein degradation.

[0121] That is, according to the results of Experiment 2, adding formic acid to a sample and subjecting it to heated formic acid treatment for 2 to 20 minutes improved the intensities of peaks above m / z 7000 in each mass spectrum compared to non-heated formic acid treatment. Furthermore, even in the mass spectrum after heated formic acid treatment, the number and intensity of peaks in the m / z range from 7000 to 7200 were further improved, especially when the sample was heated formic acid for 5 to 10 minutes.

[0122] (4-3. Experiment 3) Experiment 3 is an experiment showing the change in intensity of the peaks corresponding to cytoplasmic components due to heating during formic acid treatment.

[0123] The samples in Experiment 3 were pretreated as follows. (1) 1 g of natto was dispersed in 9 mL of sterilized physiological saline in a microtube, mixed with a vortex mixer, and then centrifuged to obtain the supernatant. (2) The supernatant was smeared on a standard agar medium and cultured at 30°C for 24 hours to obtain natto-isolated bacteria. (3) Colonies of the natto-isolated bacteria were dispersed in ultrapure water in a microtube to an OD of approximately 1, and 100 μL of each was dispensed into two microtubes. This was centrifuged to remove the supernatant, and 100 μL of 25% formic acid and 100 μL of 70% formic acid were added to each. The sample with 25% formic acid was left to stand at room temperature for 5 minutes, then 1 μL was dropped onto a sample plate and dried. next, Drop 1 μL of matrix solution (CHCA solution) and let it dry. The dried matrix was prepared and measured by MALDI-MS. (Figure 7, top) The sample to which 70% formic acid was added was heated at 50°C for 5 minutes, and then 1 μL was dropped onto the sample plate and dried. next, Drop 1 μL of matrix solution (CHCA solution) and let it dry. A dried matrix was prepared byMeasurement was performed by MALDI-MS (lower panel of Figure 7).

[0124] FIG. 7 shows the change in intensity of ribosomal protein peaks due to heating, obtained as a result of Experiment 3. In FIG. 7, the horizontal axis represents m / z. The vertical axis represents the relative intensity, with the highest intensity peak in each mass spectrum included in FIG. 7 being set at 100%. The highest intensity peak in the upper panel of FIG. 7 is peak b, with an intensity of 0.6 mV. The highest intensity peak in the lower panel of FIG. 7 is peak b, with an intensity of 15.3 mV, more than 25 times that of 0.6 mV. In other words, even when compared to the peak showing the highest intensity after non-heated formic acid treatment, heated formic acid treatment yielded an intensity more than 25 times higher.

[0125] Furthermore, it can be seen that the ratio of peak a to peak b is approximately doubled when treated with heated formic acid.

[0126] Peak a is the peak of one of the ribosomal proteins most commonly used to distinguish microorganisms. More specifically, it corresponds to the L36 protein, a ribosomal protein. On the other hand, peak b was not identified as a protein contained in the cytoplasm. These results suggest that the heat-formic acid treatment promoted lysis of the microorganisms, allowing for efficient extraction of cytoplasmic components, including ribosomal proteins, from the bacterial cells.

[0127] The results of Experiment 3 showed that the formic acid treatment increased the peak intensity of ribosomal proteins, which are useful biomarkers for distinguishing microorganisms. In other words, it is believed that the formic acid treatment improves the efficiency of distinguishing microorganisms.

[0128] (4-4. Experiment 4) Experiment 4 shows the change in peak intensity due to heating during formic acid treatment for Bacillus subtilis subsp. subtilis NBRC 13719T.

[0129] Figure 8 shows the results of Experiment 4, showing the mass spectrum of a sample treated with 25% formic acid without heating, a mass spectrum of a sample treated with 70% formic acid without heating, and a mass spectrum of a sample treated with 70% formic acid for 5 minutes while heated. In Figure 8, the horizontal axis represents m / z. The vertical axis represents the relative intensity, with the highest intensity peak among all the mass spectra in Figure 8 taken as 100%.

[0130] Referring to FIG. 8, there is not much difference between the mass spectrum of the sample treated with 25% formic acid without heating and the mass spectrum of the sample treated with 70% formic acid without heating. heating It can be seen that the number and intensity of peaks increase significantly only in the mass spectrum when treated with 70% formic acid in the normal state.

[0131] In particular, the number and intensity of peaks corresponding to ribosomal proteins (peaks marked with an asterisk) were significantly increased. For example, the peak at m / z 6500 was heating When treated with 70% formic acid in the unheated state, the intensity was approximately 12 times higher than when treated with formic acid without heating. Similarly, the peak around m / z 7700 was approximately 18 times higher.

[0132] According to the results of Experiment 4, the heated formic acid treatment enabled the detection of more protein peaks with higher intensity. In particular, the intensity of ribosomal protein peaks was significantly improved. Since biomarkers, which are specific types of proteins, are used to identify microorganisms, the heated formic acid treatment is expected to improve the efficiency of identifying microorganisms.

[0133] (4-5. Experiment 5) Experiment 5 demonstrates the effect of heating during formic acid treatment on many types of microorganisms. Specifically, the experiment was conducted on microorganisms from the phylum Actinobacteria, which generally have Gram-positive cell walls and are known to be difficult to lyse.

[0134] The sample in Experiment 5 was pretreated as follows. (1) First, the Actinobacteria phylum included 14 strains of Agromyces rhizosphaerae (NBRC 16236), 168 strains of Arthrobacter globiformis (NBRC 12137), 1217 strains of Bifidobacterium longum E194bk (JCM 1217), 534 strains of Brachybacterium conglomeratum (NBRC 15472), 534 strains of Corynebacterium glutamicum (NBRC 12168), 534 strains of Glycomyces algeriensis (NBRC 103888), 5388 strains of Glycomyces arizonensis (NBRC 103886), 5388 strains of Glycomyces harbinensis (NBRC 14487), and 534 strains of Glycomyces algeriensis (NBRC 103888). Microbial strains: Microbial strain limosus HKI 83 (NBRC 16128), Microlunatus phosphovorus NM-1 (NBRC 101784), Nocardioides simplex AJ 1420 (NBRC 12069), Paenarthrobacter aurescens strain 579 (NBRC 12136), Paenarthrobacter histidinolovorans (NBRC 15510), Phycicoccus duodecadis (NBRC 12959), and Streptomyces griseus C1 (NBRC 12875) were purchased from the National Institute of Technology and Evaluation (NITE) Biotechnology Center or the RIKEN Microbial Materials Research Laboratory and cultured in liquid medium. (2) The cultured microorganisms were dispensed, and as a control experiment, mass spectrometry was performed on the sample that had been subjected to the non-heating method in the tube. (3) The cultured microorganisms were dispensed and subjected to experiments using the pretreatment method according to this embodiment. Specifically, mass spectrometry was performed on samples subjected to in-tube heating methods, including in-tube heating (50°C, 5 minutes).

[0135] Next, the results of Experiment 5 are shown. First, we will explain the results of control experiments using conventional pretreatment methods. For strains such as Agromyces rhizospherae NBRC16236, Arthrobacter globiformis NBRC12137, Glycomyces algeriensis NBRC103888, Glycomyces arizonensis NBRC103886, Glycomyces harbinensis NBRC14487, Janibacter limosus NBRC16128, Nocardioides simplex NBRC12069, Paenarthrobacter histidinolovorans NBRC15510, and Streptomyces griseus NBRC12875, in some samples, there were fewer than five peaks matching the ribosomal protein values ​​estimated from the theoretical genome information within a 200 ppm range. This suggests that in-tube formic acid treatment may not be sufficient for the extraction of ribosomal proteins. On the other hand, in the other strains, peaks corresponding to approximately 6 to 20 types of ribosomal proteins were obtained in the 200 ppm range, indicating that good extraction of ribosomal proteins was achieved even with in-tube formic acid treatment.

[0136] On the other hand, when the pretreatment method according to this embodiment was used, peaks matching approximately 6 to 20 types of ribosomal proteins were obtained in the range of 200 ppm for all of the strains tested.

[0137] These results indicate that formic acid treatment accompanied by heat treatment is effective not only for microorganisms with Gram-negative cell walls, such as Escherichia coli, whose intracellular components are relatively easy to extract, but also for microorganisms with strong cell walls, such as the Actinobacteria phylum. This suggests that the pretreatment method according to this embodiment is a highly versatile method that promotes bacteriolysis of microorganisms of various taxonomic groups under the same conditions.

[0138] (4-6. Experiment 6) In Experiment 6, the gram-negative bacterium Escherichia coli and the gram-positive bacterium Janibacter limosus were treated with formic acid under multiple temperature conditions.

[0139] The sample in Experiment 6 was pretreated as follows. (1) The bacterial cells used in Experiment 6 were Escherichia coli (ATCC 700926) cultured overnight at 37°C with shaking in GAM liquid medium prepared with GAM broth (Shimadzu Diagnostics 05422), and Janibacter limosus (NBRC 16128) cultured for two days at 30°C in 802 agar medium. For Janibacter limosus, 50 μL of the culture medium containing the bacterial cells was sampled and added to a 96-well U-bottom cell culture plate (FALCON 353077). The added culture medium was then measured using a SpectraMax iD3, and the corresponding liquid medium was added to each well to obtain a bacterial suspension with an OD (optical density) value of approximately 0.4 to 0.6. For Escherichia coli, GAM liquid medium The cells collected from the tube were suspended in distilled water and adjusted so that the OD value was similar to that of Janibacter limosus (approximately 0.4 to 0.6). (2) 400 μL of the obtained bacterial solution was placed in a 1.5 mL tube and centrifuged (15,000 rpm, room temperature, 5 minutes), and the supernatant was removed from the tube. 250 μL of pure water was added to the tube from which the supernatant was removed, and the bacterial pellet was resuspended. 750 μL of ethanol (99.5%) was then added to the tube to adjust the final ethanol concentration to 75% (v / v). The bacterial cells were washed in the tube, and then centrifuged (15,000 rpm, room temperature, 5 minutes) to remove the supernatant from the tube. The tube was then air-dried to remove the ethanol. 60 μL of ultrapure water (FUJIFILM 214-01301) was added to the tube, and the air-dried bacterial pellet was suspended. 140 μL of formic acid (FUJIFILM 067-04531) was added to the tube and mixed to a final formic acid concentration of 70% (v / v) (contact process). 10 μL of the mixed suspension was dispensed into 0.2 mL tubes. Each dispensed suspension was heated for 5 minutes at 20°C, 40°C, 50°C, or 60°C using the VeriFlex function of a MiniAmp Plus thermal cycler (Applied Biosystems) (heating process). Note that 20°C is typically below room temperature or near room temperature and does not qualify as "heating," but for convenience, it is referred to as "heating" in Experiment 6. After heating, the tubes containing the suspension were kept on ice. 10 μL of acetonitrile (FUJIFILM 012-19851) was added to each heated sample (suspension) and mixed. 1 μL of the sample was placed on a spot on the MALDI sample plate FlexiMass-DS (SHIMADZU BIOTECH TO-430) and air-dried. Meanwhile, α-cyano-4-hydroxycinnamic acid (CHCA) (TCI C1768) was added to an ultrapure aqueous solution of 50% acetonitrile (v / v) and 1% trifluoroacetic acid (Wako 206-1-731) (v / v) to a final concentration of 10 mg / mL to obtain a matrix solution ( When in use Preparation). 1 μL of the matrix solution was added to the spot where the air-dried sample was placed, and the matrix solution and sample were mixed by pipetting, followed by further air-drying. Through these steps, a MALDI sample plate with samples placed thereon was prepared.

[0140] The results of Experiment 6 are shown in Figures 9 and 10. Figure 9 shows the number of peaks when Escherichia coli was treated with formic acid under multiple temperature conditions. Figure 10 shows the number of peaks when Janibacter limosus was treated with formic acid under multiple temperature conditions. The horizontal axis in Figures 9 and 10 represents temperature (°C). The vertical axis in Figures 9A and 10A represents the total number of peaks detected in the mass spectrum. The vertical axis in Figures 9B and 10B represents the number of peaks identified as ribosomal proteins based on their m / z values ​​among all peaks detected in the mass spectrum. Two experiments were performed for each of Escherichia coli and Janibacter limosus under each temperature condition. The results are shown in Figures 9 and 10 using square and circle markers.

[0141] Referring to Figure 9, in Escherichia coli, which is easily disrupted, a similar number of ribosomes (10-13) were detected when treated with formic acid at 20°C, 40°C, and 50°C. On the other hand, when treated with formic acid at a higher temperature (e.g., 60°C), the number of detected ribosomal proteins was significantly reduced. The results in Figure 9 demonstrate that for Gram-negative bacteria such as Escherichia coli can be detected by treating with formic acid under various temperature conditions. In particular, formic acid treatment is possible with minimal protein degradation at temperatures above 20°C and up to approximately 50°C.

[0142] Referring to Figure 10, in the case of Janibacter limosus, which is relatively difficult to disrupt, the number of ribosomal proteins detected was greater when treated with formic acid at 40°C to 50°C than when treated with formic acid at 20°C. The results in Figure 10 show that even for Gram-positive bacteria such as Janibacter limosus, formic acid treatment under high temperature conditions (approximately 40°C to 50°C) for a short period of time (5 minutes) is effective for cell disruption.

[0143] As described above, formic acid treatment at approximately 40°C to 50°C did not significantly reduce the number of ribosomal proteins detected, even in Escherichia coli, whose cells are easily disrupted. Therefore, formic acid treatment at approximately 40°C to 50°C is a treatment condition that does not significantly damage MALDI measurements of easily disrupted microorganisms. As described above, heating at approximately 40°C to 50°C during formic acid treatment does not reduce the peaks of cytoplasmic components (e.g., proteins in the cytoplasm) even in easily disrupted microbial cells such as Escherichia coli, while promoting cell disruption in difficult-to-disrupt bacterial species such as Janibacter limosus. Therefore, formic acid treatment for 5 minutes at a temperature of approximately 40°C to 50°C was shown to be a pretreatment method that can be used for both easily disrupted and difficult-to-disrupt bacteria. In other words, formic acid treatment for 5 minutes at approximately 40°C to 50°C can be said to be a pretreatment method that can efficiently detect cytoplasmic protein peaks across a wide range of bacterial species.

[0144] The efficiency of formic acid detection of cytoplasmic protein peaks is related to temperature and time. For example, a long formic acid treatment at 20°C or a short formic acid treatment (e.g., 1 minute) at 60°C may be as effective as a 5-minute formic acid treatment at 50°C. As described above, adjusting the heating time and temperature may enable cell disruption while suppressing excessive protein degradation.

[0145] (4-7. Experiment 7) Experiment 7 was an experiment to demonstrate the effect of heating during formic acid treatment on a filamentous fungus (Aspergillus kawachii NBRC 4308) that is difficult to lyse.

[0146] The sample pretreatment in Experiment 7 was carried out as follows. (1) A filamentous fungus (Aspergillus kawachii NBRC 4308) was cultured on potato dextrose agar medium. (2) The mycelia were scraped off with a cotton swab and dispersed in 1000 μL of water to obtain a dispersion. 200 μL of the obtained dispersion was dispensed into three tubes. 800 μL of ethanol was further added to each of the three tubes. The supernatant was removed from each tube by centrifugation, and 50 μL of 70% formic acid was added to the precipitate (pellet of bacterial cells) to redisperse the cells (contact process). (3) After (2), one tube (the sample corresponding to the data (50°C, 0 min) in Figure 11) was heated to 50°C, and immediately 50 μL of acetonitrile was added, mixed, and centrifuged to obtain a supernatant. 0.5 μL of the supernatant was dropped onto a sample plate and dried. After drying, 1 μL of CHCA solution was dropped onto the plate and dried again. The CHCA solution was prepared by dissolving CHCA at 10 mg / mL in an aqueous solution containing 35% acetonitrile and 15% ethanol containing 1% trifluoroacetic acid. (4) After (2), the other tube (the sample corresponding to the data (room temperature, 5 minutes) in Figure 11) was left at room temperature (23°C) for 5 minutes. The other tube (the sample corresponding to the data (50°C, 5 minutes) in Figure 11) was heated at 50°C for 5 minutes (heating process). Then, 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 a sample plate and dried. After drying, 1 μL of the above CHCA solution was dropped and dried again. (5) Each sample dried after adding CHCA solution in (3) or (4) was measured by MALDI-8020.

[0147] Figure 11 shows the change in mass spectrum of Aspergillus kawachii due to heating and formic acid treatment. Referring to Figure 11, heating and formic acid treatment at 50°C for 5 minutes enabled the detection of multiple peaks from filamentous fungi. The results of Experiment 7 demonstrated that heating and formic acid treatment is a highly versatile method that is also effective for Aspergillus kawachii.

[0148] [5. Summary] As described above, according to the pretreatment method of this embodiment, even for microorganisms with strong cell walls, a mass spectrum with high peak intensity can be obtained by heating the sample in contact with an acidic solution. In particular, the number or intensity of peaks of ribosomal proteins, which are useful biomarkers for identifying microorganisms, is also increased. Since the increase in the number and intensity of peaks occurred when only the presence or absence of heating was changed while all other conditions were the same, it is believed that the increase in the number and intensity of peaks is due to the lysis by the acidic solution. heating Furthermore, as a result of the increase in the number and intensity of peaks, the efficiency of distinguishing microorganisms is improved.

[0149] In particular, the pretreatment method according to this embodiment was effective in increasing the number and intensity of peaks even for mycolic acid-producing bacteria, which are important bacteria in medical care and clinical research.

[0150] Typically, excessive heating of cells is thought to result in protein degradation, potentially breaking down into numerous fragments. In such cases, peaks corresponding to the numerous fragments are detected in the mass spectrum, potentially making it difficult to detect peaks representing the protein prior to degradation. The pretreatment method of this embodiment combines a predetermined concentration of acidic solution with moderate heating to promote cell wall destruction without excessively degrading proteins contained in the cytoplasm, etc. This allows for accurate detection of peaks of ribosomal proteins, which are biomarkers.

[0151] In the pretreatment method according to this embodiment, the three-dimensional structure of the protein molecule may change (denaturate) slightly, but the mass of the protein molecule itself does not change even if it is denatured, so this does not cause any problems in mass analysis.

[0152] The pretreatment method according to this embodiment is characterized by its ease of implementation. According to the on-plate heating method, the entire sample plate can be heated after the bacterial cells are dispersed in the first acidic solution. Furthermore, according to the in-tube heating method, heating can be performed using a PCR device, which is installed in most laboratories that handle microorganisms. Furthermore, since beads are not used, handling is easy and there is no need to release beads into a device such as a mass spectrometer, providing peace of mind. Since this method is compatible with a wide range of microorganisms, including those with strong cell walls, there is no need to consider a different pretreatment method depending on the type of microorganism. More specifically, the effect of improving peak intensity can be achieved for a wide range of microorganisms under the above-mentioned relatively uniform conditions (e.g., heating for 2 to 20 minutes). Considering these features, the pretreatment method according to this embodiment is also useful in that it can be easily implemented by a wide range of users.

[0153] In particular, because the pretreatment method according to this embodiment requires only simple liquid manipulation and heating, it is easy to automate some or all of the steps using machinery. More specifically, it is easy to incorporate the method into a pretreatment device. For the same reason, by preparing multiple containers containing a specific type of microorganism and simultaneously testing multiple conditions selected from the relatively uniform conditions described above, it is easy to determine the optimal conditions with the highest peak intensity in a single experiment and to obtain a mass spectrum under those optimal conditions.

[0154] Although the above examples have mainly shown that the pretreatment method according to this embodiment can improve the intensity of peaks corresponding to intracellular components of microorganisms, it is clear that the pretreatment method according to this embodiment can also be applied to biological samples containing cells of organisms other than microorganisms. For example, it is also useful for breaking down the cell walls and / or cell membranes of cells other than microorganisms to extract intracellular components.

[0155] In the above example, in the pretreatment method according to this embodiment, Cells and organic acid ContainsAlthough it has been described that cytoplasmic components are extracted by contacting with the first acidic solution and heating, a first acidic solution containing an inorganic acid may be used as long as it has the same effect as the pretreatment method according to this embodiment. Examples of the first acidic solution containing an organic acid are not limited to the above examples as long as it has the same effect as the pretreatment method according to this embodiment. Embodiment The first acidic solution that exhibits this effect is, for example, an acidic solution that improves the intensity of the peak of the cytoplasmic component by heat and acid treatment. Embodiment The first acidic solution that does not have the effect is, for example, a solution that cannot sufficiently destroy the cell wall. acidic solution , which degrades proteins to the extent that it interferes with the detection of peaks of cytoplasmic components. acidic solution , and / or acidic solutions that cause problems in processes other than cytoplasm extraction. For example, acidic solutions containing hydrochloric acid are generally considered unsuitable for use in the on-plate heat treatment of this embodiment because they may dissolve sample plates made of stainless steel. On the other hand, acidic solutions containing hydrochloric acid can be used for in-tube heat treatment in acid-resistant containers (e.g., glass or acid-resistant resin containers). In such cases, it is preferable to wash the sample after the in-tube heat treatment. Thus, the acid used in the pretreatment of this embodiment can be selected arbitrarily within the scope of those skilled in the art.

[0156] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0157] (Item 1) A pretreatment method according to one embodiment is a method for pretreatment of a sample containing cells for mass spectrometry, comprising: Acid and heating the cells in contact with the first acidic solution to extract cytoplasmic components of the cells.

[0158] According to the pretreatment method described in paragraph 1, the extraction efficiency of cytoplasmic components is improved. Therefore, the intensity of the peaks corresponding to the cytoplasmic components in the mass spectrum is improved. In other words, the pretreatment of a sample containing cells for mass spectrometry can improve the intensity of the peaks of the cytoplasmic components.

[0159] (Item 2) In the pretreatment method described in Item 1, the contacting step includes preparing a second acidic solution, which is a mixed solution of the sample and the first acidic solution, and the extracting step includes heating the second acidic solution.

[0160] According to the pretreatment method described in paragraph 2, cells can be dispersed in the first acidic solution by mixing the sample with the first acidic solution, thereby ensuring contact between the cells and the first acidic solution, thereby improving the efficiency of treatment of the cells with the first acidic solution.

[0161] (Item 3) In the pretreatment method according to item 1 or 2, the organic acid includes at least one of formic acid, trifluoroacetic acid, and acetic acid.

[0162] According to the pretreatment method described in Section 3, the pretreatment method of this embodiment can be carried out using these acids, which are easily available and familiar to users in research and clinical settings who perform mass spectrometry of microorganisms. These acids are also useful in that they have been empirically proven to not adversely affect the measurement results when used by those skilled in the art for pretreatment of mass spectrometry.

[0163] (Item 4) In the pretreatment method according to 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.

[0164] According to the pretreatment method described in item 4, the pretreatment method can be carried out using an acidic solution having the above-mentioned concentration.

[0165] (Item 5) In the pretreatment method according to item 4, the concentration of the organic acid is 65% by volume or more and 75% by volume or less, based on the first acidic solution or the second acidic solution.

[0166] According to the pretreatment method described in item 5, the pretreatment method according to this embodiment can be carried out using an acidic solution having the above concentration.

[0167] (Item 6) In the pretreatment method according to any one of items 1 to 5, the first acidic solution contains water or an organic solvent.

[0168] According to the pretreatment method described in item 6, the pretreatment method according to this embodiment can be carried out using the first acidic solution prepared using these solvents.

[0169] (Item 7) In the pretreatment method according to any one of items 1 to 6, the heating time is 2 minutes or more and less than 20 minutes.

[0170] According to the pretreatment method described in item 7, the pretreatment method according to this embodiment can be carried out by heating for the above-mentioned period of time.

[0171] (Item 8) In the pretreatment method according to item 7, the heating time is 5 minutes or more and 10 minutes or less.

[0172] According to the pretreatment method described in item 8, the pretreatment method according to this embodiment can be carried out by heating for the above-mentioned period of time.

[0173] (Item 9) In the pretreatment method according to any one of items 1 to 8, the heating temperature is 30°C or higher and 75°C or lower.

[0174] According to the pretreatment method described in item 9, the pretreatment method according to this embodiment can be carried out by heating at the above temperature.

[0175] (Item 10-1) In the pretreatment method according to item 9, the heating temperature is 40°C or higher and 60°C or lower.

[0176] (Item 10-2) In the pretreatment method according to item 9, the heating temperature is 35°C or higher and 55°C or lower.

[0177] According to the pretreatment methods described in items 10-1 and 10-2, the pretreatment method according to this embodiment can be carried out by heating at the above-mentioned temperatures.

[0178] (Item 11) In the pretreatment method described in Item 2, the step of heating the second acidic solution includes a step of heating the second acidic solution placed on a sample plate for mass spectrometry, or a step of heating the second acidic solution contained in a container before being placed on the sample plate.

[0179] According to the pretreatment method described in item 11, the second acidic solution can be heated by the above two simple methods, and users can select a suitable method from the above two simple methods.

[0180] (Item 12) In the pretreatment method according to any one of items 1 to 11, the cells are microbial cells.

[0181] According to the pretreatment method described in item 12, the pretreatment method according to this embodiment can also be carried out when microorganisms are subjected to mass spectrometry in clinical microbiological analysis, food hygiene inspection, and other similar situations.

[0182] (Item 13) In the pretreatment method according to any one of items 1 to 12, the cells are cells having a cell wall.

[0183] According to the pretreatment method described in item 13, the cell walls can be destroyed using the pretreatment method according to this embodiment, and the intensity of the peaks of cytoplasmic components can be improved.

[0184] (Item 14) In the pretreatment method according to any one of items 1 to 13, the mass spectrometry is matrix-assisted laser desorption / ionization mass spectrometry.

[0185] According to the pretreatment method described in item 14, mass spectrometry can be carried out using the MALDI method, which is suitable for identifying microorganisms by mass spectrometry.

[0186] (Item 15) A mass spectrometry method comprising a step of obtaining a mass spectrum by performing matrix-assisted laser desorption ionization mass spectrometry on a sample that has been pretreated by the pretreatment method described in any one of items 1 to 14.

[0187] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0188] 1 analytical device, 10 control unit, 11 processing unit, 12 memory unit, 13 input / output unit, 20 measurement unit, 21 ionization unit, 22 ion acceleration unit, 23 mass separation unit, 24 detection unit, 111 device control unit, 113 mass spectrum analysis unit, 114 discrimination unit, 131 input unit, 132 output unit, 133 communication unit, 221 acceleration electrode, 231 flight tube.

Claims

1. A method for pre-treating a sample containing cells for mass spectrometry, comprising: mixing the sample with a first acidic solution containing an organic acid to prepare a suspension; and a step of heating the suspension contained in the container to extract cytoplasmic components of the cells before the suspension is placed on a sample plate for mass spectrometry.

2. The pretreatment method of claim 1 , wherein the organic acid comprises at least one of formic acid, trifluoroacetic acid, and acetic acid.

3. The pretreatment method according to claim 1 or 2, wherein a 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 suspension.

4. The pretreatment method according to claim 3 , wherein a concentration of the organic acid is 65% by volume or more and 75% by volume or less based on the first acidic solution or the suspension.

5. The pretreatment method according to claim 1 or 2, wherein the first acidic solution contains water or an organic solvent.

6. The pretreatment method according to claim 1 or 2, wherein the heating time is from 2 minutes to less than 20 minutes.

7. The pretreatment method according to claim 6 , wherein the heating time is from 5 minutes to 10 minutes.

8. The pretreatment method according to claim 1 or 2, wherein the heating temperature is 30°C or higher and 75°C or lower.

9. The pretreatment method according to claim 8 , wherein the heating temperature is 35° C. or higher and 55° C. or lower.

10. The pretreatment method according to claim 1 or 2, wherein the cells are microbial cells.

11. The pretreatment method according to claim 1 or 2, wherein the cells have a cell wall.

12. The pretreatment method according to claim 1 or 2, wherein the mass spectrometry is matrix-assisted laser desorption / ionization mass spectrometry.

13. A mass spectrometry method comprising the step of obtaining a mass spectrum by performing matrix-assisted laser desorption / ionization mass spectrometry on a sample that has been pretreated by the pretreatment method according to claim 1 or 2.

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