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

Pretreatment method and mass spectrometry method

[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.

[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.

[0003] Comparison of the Identification Accuracy of Nutritionally Variant Streptococci Between Two MALDI-TOF MS Models and Vitec 2, and Study of Biochemical Properties Important for Identification, Michiko Furukawa et al., Japanese Journal of Clinical Microbiology, 2016, Vol. 26 No. 3, pp. 29-39; Application of Rapid Identification of Microorganisms Using MALDI-TOF MS to the Field of Food Microbiology, Hiroko Kawasaki, Journal of the Japanese Society for Food Microbiology, 2020, Vol. 37 No. 4, pp. 165-177

[0004] As disclosed in Non-Patent Documents 1 and 2, methods for treating cells with formic acid are broadly 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.

[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, and includes the steps of contacting the cells with a first acidic solution containing an organic acid, and heating the cells while they are in contact with the first acidic solution to extract cytoplasmic components of the cells.

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

[0010] 7 is a schematic diagram showing the configuration of an analytical device according to an embodiment; FIG. 8 is a flowchart showing processes relating to an on-plate heating method and a mass spectrometry method; FIG. 9 is a flowchart showing processes relating to an in-tube heating method and a mass spectrometry method; FIG. 10 is a diagram showing mass spectra when the acid concentration in a first acidic solution is different; FIG. 11 is a diagram showing a mass spectrum when non-heated formic acid treatment is performed; FIG. 12 is a diagram showing a mass spectrum when heated formic acid treatment is performed; FIG. 13 is a diagram showing changes in the intensity of the ribosome peak due to heated formic acid treatment; FIG. 14 is a diagram showing changes in the mass spectrum due to heated formic acid treatment in a type of microorganism different from that in FIG. 7; FIG. 15 is a diagram showing the number of peaks when Escherichia coli is treated with formic acid under multiple temperature conditions; FIG. 16 is a diagram showing the number of peaks when Janibacter limosus is treated with formic acid under multiple temperature conditions; and FIG. 17 is a diagram showing changes in the mass spectrum when Aspergillus kawachii is treated with heated formic acid.

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

[0013] [1. Configuration of the Analytical Apparatus] First, an example of an analytical apparatus 1 that performs the 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] 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, such as a MALDI-TOF MS (Matrix-Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry) device. The analytical 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, in this specification, the type of microorganism 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 simply referred to as the determination of the microorganism.

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

[0018] Referring to Fig. 1, the analytical device 1 includes a control unit 10 and a measurement unit 20. The measurement unit 20 ionizes substances (e.g., proteins) in a sample using a high voltage, separates the ions S according to a time of flight correlated 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 Fig. 1, the movement of the ions S in the measurement unit 20 is schematically indicated by an arrow A1.

[0019] In one embodiment, the ionization unit 21 ionizes substances in a sample by matrix-assisted laser desorption / ionization (MALDI). As described below, MALDI 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 MALDI. When ionization is performed by ESI, 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, since this can provide high separation ability.

[0020] The ionization unit 21 includes an ion source including a sample plate holder (not shown) that supports a 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 dries the sample-mixed matrix solution, which is a mixture of the sample and the matrix solution, on a sample plate to obtain a dried product. The sample plate is then placed in a sample plate holder in a 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," a "mixed crystal," a "sample crystal," a "matrix crystal," a "sample / matrix crystal," etc. In the present 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] The ionization unit 21 irradiates the dried matrix on the sample plate with laser light after depressurizing the vacuum chamber containing the sample plate, thereby ionizing 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 accelerator 22 includes an acceleration electrode 221 and accelerates the introduced ions S. The flow of the accelerated ions S is appropriately focused by an ion lens (not shown) and introduced into the mass separator 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 by, for example, 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 an apparatus 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 detection amount 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 intensity of the peaks. Therefore, the greater the extraction efficiency of the target substance in the sample pretreatment, the greater the intensity of the peaks.

[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 storage 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 example 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 example, the storage unit 12 stores a database including the acquired mass spectra.

[0038] The input / output unit 13 is an interface for the analysis 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 includes 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 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 wirelessly or via a wired connection such as the Internet, etc. 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 analytical apparatus 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 apparatus 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 analytical apparatus 1 are set within the range typically set by a person skilled in the art.

[0044] [2. Conventional Sample Pretreatment Methods] Conventionally, there are methods for identifying microorganisms using mass spectrometry. Mass spectrometry allows for the easy and rapid acquisition of mass spectra, which are 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, which is a soft ionization method that ionizes biopolymers such as proteins with almost no decomposition, is particularly widely used. Specifically, MALDI-MS is used to distinguish specific types of microorganisms in clinical microorganism analysis, food hygiene inspections, and other similar situations. Thus, while MALDI-MS is currently an extremely excellent technology for distinguishing specific types of microorganisms, it also has some difficulties when it comes to distinguishing 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 (e.g., 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 easily degraded by [M+H] during 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 several Da. Therefore, when mass spectrometry is performed using MALDI-MS, there is an advantage in that peaks of ribosomal proteins can be easily detected from a sample.

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

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

[0051] Therefore, when mass spectrometry is performed 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 of 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-heating treatment," and the conventional formic acid treatment in a tube will also be referred to as "in-tube non-heating treatment." Furthermore, a pretreatment method using "on-plate non-heating treatment" will also be referred to as "on-plate non-heating method," and a pretreatment method using "in-tube non-heating treatment" will also be referred to as "in-tube non-heating 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 the in-tube non-addition method, the cells may be dispersed in ethanol, centrifuged, and the supernatant removed before adding formic acid.

[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, since 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 suborder, such as Mycobacterium and Nocardia) that contain long-chain fatty acids called mycolic acids in their cell walls. Thus, 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 contain 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 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 extraction efficiency 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 method for pretreatment of 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., mold, yeast).

[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, including chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, Glomeromycota, and Microsporidia. The Ascomycota includes the genus Aspergillus (such as Aspergillus mold), 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 eluted by the following two processes.

[0069] (3-2. Contact Process) The sample to be contacted with the first acidic solution may be in a 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 or the like from a bacterial culture on 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 of microorganism mass spectrometry. 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 overlap in part 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 one type of acid (pure substance) or a mixture of multiple types of 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 specific means for the "contact process" typically involves 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 may be 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, and in this embodiment, the second acidic solution is trifluoroacetic acid at the above-mentioned concentrations 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, and in this embodiment, the second acidic solution is acetic acid at the above concentrations containing cells.

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

[0079] Another example of a specific means for the "contact process" is a means for contacting (e.g., placing) cells on an object (e.g., a cloth) impregnated with the first acidic solution. Thus, the specific means for 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. The heating method will be described later.

[0081] (3-3. Heating Process) The heating process is carried out to improve the peak intensity of the cytoplasmic components. More specifically, it is believed that heating the second acidic solution in the heating process improves 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 this 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 the second acidic solution placed on the sample plate. Hereinafter, the process of heating the second acidic solution placed on the sample plate will be referred to as the "on-plate heating process," and the pretreatment method including the on-plate heating process will be referred to as the "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 an "on-plate heating process" 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 (hereinafter also referred to as the "dried acid product") obtained by drying the second acidic solution. For example, within a few minutes after the second acidic solution has dried, the analyst drops the matrix solution 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. Intra-tube heating method) 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 "intra-tube heating process," and the pretreatment method including the intra-tube heating process will be referred to as the "intra-tube heating method." Next, the intra-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 colonies cultured on solid medium using a quantitative loop or the like, adds them to a 1.5 mL tube containing 200 μL of water, and mixes them 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 colonies 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 (e.g., 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 evaporate the ethanol by air-drying the tube for a few minutes.

[0097] In S22, the analyst mixes the sample with the first acidic solution in the tube to prepare a mixed solution, that is, 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 above-mentioned "contact process."

[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 S23, the analyst may add an organic solvent such as acetonitrile to the second acidic solution. The organic solvent can be added for the purposes of dissolving the matrix substance in the matrix solution, adjusting the surface tension of the matrix solution, destroying cell membranes, etc. However, in the pretreatment method according to this embodiment, a peak of sufficient intensity was obtained even without adding an organic solvent, so adding the 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 that of 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 may centrifuge the heated tube at 10,000 to 15,000 g for 2 minutes, and drop 0.5 to 1 μL of the resulting supernatant onto the sample plate.

[0102] The processes of S25 to S28 correspond to the processes of S14 to S17 in FIG. 3, and therefore will not be described repeatedly.

[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 Examples 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 acid concentration in the first acidic solution.

[0106] The sample pretreatment in Experiment 1 was performed 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 aliquots. (3) 400 μL of ethanol was added to each microtube containing the bacterial cells, mixed, and centrifuged to obtain a bacterial cell precipitate. (4) The supernatant was removed, and 50 μL of 50% formic acid, 50 μL of 70% formic acid, or 50 μL of 90% formic acid was added to the resulting precipitate, and the mixture was mixed using a vortex mixer. The sample contained in each tube was then subjected to the following treatment: (5) Heating at 50°C for 5 minutes was performed. (6) An additional 50 μL of acetonitrile was added, mixed using a vortex mixer, 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 the 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] 4 shows mass spectra obtained as a result of Experiment 1, where the first acidic solution had different acid concentrations. In FIG. 4, the horizontal axis represents m / z. The vertical axis represents the percentage intensity, which is the relative intensity when the highest peak among the three mass spectra included in FIG. 4 is set to 100%.

[0108] 4 indicates a first acidic solution containing X% formic acid. In the description of FIG. 4, the mass spectrum obtained by heating the 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% formic acid mass spectrum and the 90% formic acid mass spectrum 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 almost no peak was detected near m / z 5500 in the 50% formic acid mass spectrum, a clear peak was detected in the 70% formic acid mass spectrum and the 90% formic acid mass spectrum, 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 pretreatments (4) and (5) in Experiment 1.

[0114] Sample pretreatments (4) and (5) in Experiment 2 were performed as follows. (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 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 treated with formic acid for 2, 5, 10, and 20 minutes while heated to 50°C (Figure 6). The samples contained in each tube were then subjected to the following treatments.

[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] Figure 5 shows mass spectra obtained with non-heated formic acid treatment. Figure 6 shows mass spectra obtained with heated formic acid treatment. In Figures 5 and 6, the horizontal axis represents m / z. The vertical axis in Figure 5 represents relative intensity when the single peak with the highest intensity among the four mass spectra included in Figure 5 is taken as 100%. The vertical axis in Figure 6 represents relative intensity when the single peak with the highest intensity among the four mass spectra included in Figure 6 is taken as 100%. In Figures 5 and 6, data with m / z equal to or greater than 7000 is displayed with its intensity 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 in the vicinity of 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 in the 2-minute heating mass spectrum near m / z 7100, a clear peak was detected in 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 decomposition.

[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 at m / z 7000 or more 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 of 7000 to 7200 were further improved, particularly when the sample was heated formic acid treatment for 5 to 10 minutes.

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

[0123] The sample pretreatment in Experiment 3 was performed as follows: (1) 1 g of natto was dispersed in 9 mL of sterilized 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 isolates. (3) Colonies of the natto isolates were dispersed in ultrapure water in a microtube to an OD of approximately 1, and 100 μL 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, respectively. The sample to which 25% formic acid had been added was left to stand at room temperature for 5 minutes, after which 1 μL was dropped onto a sample plate and dried, and 1 μL of matrix solution (CHCA solution) was dropped onto the plate and dried (top panel of Figure 7). The sample to which 70% formic acid had been added was heated at 50°C for 5 minutes, after which 1 μL was dropped onto a sample plate and dried, and 1 μL of matrix solution (CHCA solution) was dropped onto the sample plate and dried, followed by measurement by MALDI-MS (Figure 7, bottom panel). (4) After the formic acid had dried, the matrix solution (CHCA solution) was dropped onto the sample plate and dried to prepare a dried matrix product, which was then measured by MALDI-MS.

[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 when treated with non-heated formic acid, an intensity more than 25 times higher was obtained when treated with heated formic acid.

[0125] Furthermore, it can be seen that the ratio of peak a to peak b is approximately doubled when the sample is 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. From these results, it is believed that the heated formic acid treatment promoted lysis of the microorganisms, and that cytoplasmic components including ribosomal proteins were efficiently extracted from the bacterial cells.

[0127] The results of Experiment 3 showed that the formic acid treatment improved 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 under heating. 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 included in Figure 8 taken as 100%.

[0130] Referring to FIG. 8, it can be seen that there is not much difference between the mass spectrum obtained when treated with 25% formic acid without heating and the mass spectrum obtained when treated with 70% formic acid without heating, but the number and intensity of peaks are significantly increased only in the mass spectrum obtained when treated with 70% formic acid in a superheated state.

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

[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 of the phylum Actinobacteria, which generally have Gram-positive cell walls and are known to be difficult to lyse.

[0134] The samples in Experiment 5 were pretreated as follows. (1) First, the Actinobacteria phylum microorganisms were selected, including Agromyces rhizosphaerae strain 14 (NBRC 16236), Arthrobacter globiformis strain 168 (NBRC 12137), Bifidobacterium longum strain E194bk (JCM 1217), Brachybacterium conglomeratum strain 5-2 (NBRC 15472), Corynebacterium glutamicum strain 534 (NBRC 12168), Glycomyces algeriensis strain LLR-39Z-86 (NBRC 103888), Glycomyces arizonensis strain DPL-G-76 (NBRC 103886), Glycomyces harbinensis strain LL-DO5139 (NBRC 14487), and Janibacter limosus HKI. Strain 83 (NBRC 16128), Microlunatus phosphovorus strain NM-1 (NBRC 101784), Nocardioides simplex strain AJ 1420 (NBRC 12069), Paenarthrobacter aurescens strain 579 (NBRC 12136), Paenarthrobacter histidinolovorans (NBRC 15510), Phycicoccus duodecadis (NBRC 12959), and Streptomyces griseus strain 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 using the designated medium. (2) The cultured microorganisms were aliquoted, and mass spectrometry was performed on a control sample that had been subjected to the non-heated in-tube method. (3) The cultured microorganisms were aliquoted and experiments using the pretreatment method of this embodiment were performed. Specifically, mass spectrometry was performed on samples that had been subjected to an in-tube heating method including in-tube heating treatment (50° C., 5 minutes).

[0135] Next, we present the results of Experiment 5. First, we explain the results of a control experiment using a conventional pretreatment method. 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, depending on the sample, there were five or fewer peaks that matched the ribosomal protein values ​​estimated from theoretical genome information within a 200 ppm range. This suggests that in-tube formic acid treatment may not adequately extract 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 range of 200 ppm, 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, Escherichia coli, a gram-negative bacterium, and Janibacter limosus, a gram-positive bacterium, were treated with formic acid under multiple temperature conditions.

[0139] The samples in Experiment 6 were 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 on 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. The corresponding liquid medium was added to each well to obtain a bacterial suspension with an optical density (OD) of approximately 0.4 to 0.6. For Escherichia coli, cells collected from the 802 agar medium were suspended in distilled water and adjusted to an OD value similar to that of Janibacter limosus (approximately 0.4-0.6). (2) 400 μL of the resulting cell suspension 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 cell 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 cells were washed in the tube, then centrifuged (15,000 rpm, room temperature, 5 minutes), and the supernatant was removed 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 at 20°C, 40°C, 50°C, or 60°C for 5 minutes using the VeriFlex function of a MiniAmp Plus thermal cycler (Applied Biosystems) (heating process).Although 20°C is typically below room temperature or at room temperature and does not qualify as "heating," it is referred to as "heating" in Experiment 6 for convenience. After heating, the tubes containing the suspensions 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 a MALDI sample plate FlexiMass-DS (SHIMADZU BIOTECH TO-430) and air-dried. Separately, a matrix solution was prepared by adding α-cyano-4-hydroxycinnamic acid (CHCA) (TCI C1768) to an ultrapure solution of 50% acetonitrile (v / v) and 1% trifluoroacetic acid (Wako 206-1-731) (v / v) to a final concentration of 10 mg / mL (prepared fresh). 1 μL of the matrix solution was added to the spot where the air-dried sample was located, and the matrix solution and sample were mixed by pipetting, followed by further air-drying. A MALDI sample plate with samples was prepared using the above steps. (3) The prepared MALDI sample plate was inserted into a MALDI-8020 manufactured by Shimadzu Corporation and measured under the following conditions. The Acquire tab was set to a mass range of 2000-20000, accumulate 5 shots (s) @ 200 Hz blast shot, and 100 profiles (20220601_Microbio). The Process tab was set to a Subtract baseline using a filter width of 500, a Gaussian smoothing method, a Peak width of 80, a Peak delimiter method with a Threshold Apex, and a Threshold offset and response of 0.015 mV and 1.2000 mV (20221223 microbio). For calibration of the 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 in 50% acetonitrile (v / v) was used for calibration, and 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. (4) The peaks obtained from the samples measured with the MALDI-8020 described above were analyzed, and the total number of peaks and the number of peaks corresponding to ribosomal proteins were calculated. At this time, GPMsDB-tk v1.0.1 (https: / / github.com / ysekig / GPMsDB-tk) was used for MALDI data analysis.

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

[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, or 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 can be performed at temperatures from 20°C to approximately 50°C, minimizing protein degradation.

[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 the cells were treated with formic acid at 40°C to 50°C than when the cells were 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, which is 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 microorganisms whose cells are easily disrupted. As described above, heating at approximately 40°C to 50°C in formic acid treatment does not reduce the peaks of cytoplasmic components (such as proteins in the cytoplasm) even in microbial cells that are easily disrupted, such as Escherichia coli, while promoting cell disruption in bacterial species that are difficult to disrupt, 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 in both bacteria that are easily disrupted and bacteria that are difficult to disrupt. 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 for 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 is 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 performed as follows: (1) Filamentous fungus (Aspergillus kawachii NBRC 4308) was cultured on potato dextrose agar medium. (2) Mycelia were scraped with a cotton swab and dispersed in 1,000 μL of water to obtain a dispersion. 200 μL of the resulting dispersion was dispensed into three tubes. 800 μL of ethanol was 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) for redispersion (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. Another 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 onto the sample and dried again. (5) Each sample to which the CHCA solution was added and dried in (3) or (4) was measured using a 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, mass spectra with high peak intensities 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 microbial identification, also increases. Since the increase in the number and intensity of peaks occurred when heating was used or not, while keeping all other conditions the same, it is believed that the increase in the number and intensity of peaks is the result of bacteriolysis by the acidic solution being appropriately promoted by overheating. Furthermore, the increase in the number and intensity of peaks improves the efficiency of microbial identification.

[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. The on-plate heating method allows for heating the entire sample plate after dispersing the bacterial cells in the first acidic solution. Furthermore, the in-tube heating method allows for heating using a PCR device, which is installed in most laboratories that handle microorganisms. Furthermore, since beads are not used, handling is easy and the method is safe because beads are not released into instruments such as mass spectrometers. Since the method is compatible with a wide range of microorganisms, including those with strong cell walls, there is no need to consider different pretreatment methods 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., 2 to 20 minutes of heating). In light of 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] Furthermore, in the above example, the pretreatment method according to the present embodiment was described as extracting cytoplasmic components by contacting an organic acid with a first acidic solution and heating. However, a first acidic solution containing an inorganic acid may also be used as long as it provides the same effect as the pretreatment method according to the present embodiment. Examples of the first acidic solution containing an organic acid are not limited to the above examples, as long as it provides the same effect as the pretreatment method according to the present embodiment. A first acidic solution that provides the effects of the present application is, for example, an acidic solution that improves the intensity of the peaks of cytoplasmic components when subjected to a heat acid treatment. A first acidic solution that does not provide the effects of the present application is, for example, an acid that does not sufficiently destroy cell walls, an acid that degrades proteins to the extent that it interferes with the detection of peaks of cytoplasmic components, and / or an acidic solution that causes problems in processes other than cytoplasm extraction. For example, an acidic solution containing hydrochloric acid is generally considered unsuitable for use in the on-plate heating treatment according to the present embodiment because it may dissolve sample plates containing stainless steel. On the other hand, an acidic solution containing hydrochloric acid can be used for in-tube heating treatment in an acid-resistant container (e.g., a glass or acid-resistant resin container). In this case, it is preferable to perform sample washing after the in-tube heating treatment. Thus, the acid used in the pretreatment according to this embodiment can be selected arbitrarily within the scope of those skilled in the art.

[0156] Aspects It will be understood 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, and includes the steps of contacting the cells with a first acidic solution containing an organic acid, and heating the cells while they are 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 efficiency of extraction of cytoplasmic components is improved. Therefore, the intensity of the peaks corresponding to the cytoplasmic components in the mass spectrum is increased. In other words, the pretreatment of a sample containing cells for mass spectrometry can increase 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 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.

[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 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 acidic solution or the second acidic solution.

[0166] According to the pretreatment method described in Section 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 Section 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 described in 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 described in item 9, the heating temperature is 40°C or higher and 60°C or lower.

[0176] (Item 10-2) In the pretreatment method described in 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 Section 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.

[0188] REFERENCE SIGNS LIST 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 pretreatment of a sample containing cells for mass spectrometry, comprising the steps of: 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 , 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 are cells having cell walls.

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

13. 3. 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.