Matrix solution, mass analysis method, storage medium, and determination method

Abstract

A matrix solution for mixing with a sample in matrix-assisted laser desorption ionization mass spectrometry. The matrix solution is an aqueous solution containing acetonitrile, ethanol, trifluoroacetic acid and water. The content ratio of acetonitrile is 30-40% by volume based on the matrix solution. Based on the matrix solution, the content ratio of the ethanol is 10-20% by volume. The content ratio of trifluoroacetic acid is 1-3% by volume based on the matrix solution. The matrix solution comprises 5 to 20 mg / m < l > of alpha-cyano-4-hydroxycinnamic acid, 2, 5-dihydroxybenzoic acid or sinapic acid.

Description

Technical Field

[0001] The present invention relates to a matrix solution, a mass analysis method, a storage medium and a discrimination method, and more particularly to a matrix solution, a mass analysis method, a storage medium and a discrimination method related to microbial discrimination using matrix-assisted laser desorption ionization mass spectrometry. Background Art

[0002] There are known methods for distinguishing microorganisms based on the pattern of mass spectra obtained by mass analysis of microorganisms. In particular, among mass analysis methods, methods for distinguishing microorganisms using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) disclosed in non-patent documents 1 to 3 are widely used in, for example, clinical microbiological analysis, food hygiene inspection, and other fields.

[0003] In MALDI-MS, a matrix solution is used to ionize the target substance of the analysis object contained in the sample. A general matrix solution also contains a component for dissolving the sample. A mixed solution of such a matrix solution and the sample is placed on a sample plate and dried to obtain a dried product of the mixed solution. By irradiating the dried product with a laser, the target substance in the dried product is ionized. The ionized target substance is detected as a peak of a mass spectrum. The analyst can distinguish microorganisms based on the difference in the pattern of the peak.

[0004] In such MALDI-MS for microbial identification, a CHCA solution obtained by dissolving CHCA (α-cyano-4-hydroxycinnamic acid) in an organic solvent is used as a matrix solution. Non-patent document 4 discloses a CHCA solution containing acetonitrile and ethanol as organic solvents at a predetermined ratio.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Victor Ryzhov and Catherine Fenselau, "Characterization of the Protein Subset Desorbed by MALDI from Whole Bacterial Cells", Analytical Chemistry, 2001, 73, 746-750.

[0008] Non-patent document 2: Fernando J. Pineda et al., "Microorganism Identification by Matrix-Assisted Laser / Desorption Ionization Mass Spectrometry and Model-Derived Ribosomal Protein Biomarkers", Analytical Chemistry, 2003, 75, 3817-3822.

[0009] Non-patent document 3: Teramoto K et al., "Phylogenetic Classification of Pseudomonas putida Strains by MALDI-MS Using Ribosomal Subunit Proteins as Biomarkers", Analytical Chemistry, 2007, 79, 22, 8712-8719.

[0010] Non-patent document 4: VITEK MS Matrix-CHCA Safety Data Sheet, BioMérieux Japan Ltd., June 3, 2021, [retrieved May 12, 2022], Internet <URL:https: / / www.biomerieux.co.jp / sites / subsidiary_jp / files / SPS-revamp / 411071_vitek_ms_matrix-chca_sds_04_bmxj.pdf> Summary of the invention

[0011] Technical problem to be solved by the invention

[0012] In the method of using MALDI-MS to distinguish microorganisms, it is expected to detect the peak of the target substance with high sensitivity. However, when MALDI-MS is performed on a dried product formed using a conventional matrix solution, there is a problem that sufficient sensitivity cannot be obtained, especially in the high-mass region. Therefore, in MALDI-MS, a means is required to detect the peak of the target substance with high sensitivity.

[0013] The present disclosure has been made to solve the above-mentioned technical problems, and its purpose is to provide a matrix solution that enables high-sensitivity mass spectrometry in matrix-assisted laser desorption ionization mass spectrometry and improve the accuracy of discrimination of samples derived from microorganisms.

[0014] Solutions for solving the above technical problems

[0015] The matrix solution involved in the first scheme of the present disclosure is a matrix solution for mixing with a sample in matrix-assisted laser desorption ionization mass spectrometry. The matrix solution is an aqueous solution containing acetonitrile, ethanol, trifluoroacetic acid and water. Based on the matrix solution, the acetonitrile content is 30 to 40 volume%. Based on the matrix solution, the ethanol content is 10 to 20 volume%. Based on the matrix solution, the trifluoroacetic acid content is 1 to 3 volume%. The matrix solution contains 5 to 20 mg / ml of α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid or sinapinic acid.

[0016] The mass spectrometry method involved in the first scheme of the present disclosure comprises: a step of mixing a sample with a matrix solution; a step of drying the matrix solution mixed with the sample on a sample plate to form a dried product; and a step of acquiring a mass spectrum by performing matrix-assisted laser desorption ionization mass analysis on the dried product. The matrix solution is an aqueous solution containing acetonitrile, ethanol, trifluoroacetic acid and water. Based on the matrix solution, the content of acetonitrile is 30 to 40% by volume. Based on the matrix solution, the content of ethanol is 10 to 20% by volume. Based on the matrix solution, the content of trifluoroacetic acid is 1 to 3% by volume. The matrix solution contains 5 to 20 mg / ml of α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid or sinapinic acid.

[0017] Effects of the Invention

[0018] According to the matrix solution of the present disclosure, it is possible to provide a matrix solution that enables high-sensitivity mass spectrometry in matrix-assisted laser desorption ionization mass spectrometry, and to improve the accuracy of discrimination of samples derived from microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a schematic diagram showing the configuration of an analysis device according to an embodiment.

[0020] Figure 2 is a diagram illustrating the composition of the base solvent.

[0021] Figure 3 It is a diagram for explaining a method of forming a dried product on a sample plate.

[0022] Figure 4 The figure shows the photographed image of the dried material on the sample plate.

[0023] Figure 5 It is a figure which shows the mass spectrum of Escherichia coli.

[0024] Figure 6 is a graph showing the amount of ribosomal proteins detected.

[0025] Figure 7 It is a flowchart showing the mass analysis process and the microorganism identification process.

[0026] Figure 8 It is a diagram showing an experimental example of the mass spectrometry method according to the embodiment. DETAILED DESCRIPTION

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

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

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

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

[0031] First, an example of an analysis device 1 for implementing the method for distinguishing microorganisms according to the present embodiment will be described. The method for distinguishing microorganisms according to the present embodiment includes the mass spectrometry method according to the present embodiment.

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

[0033] The sample may be derived from a prokaryotic organism or a eukaryotic organism. The sample is preferably derived from a microorganism and may contain an unknown microorganism. Prokaryotes include bacteria and archaea. As bacteria, they include Escherichia bacteria (Escherichia coli, etc.), Bacillus bacteria (Bacillus subtilis, etc.), Lactobacillus bacteria (lactic acid bacteria, etc.), Synechocystis bacteria (cyanobacteria, etc.), Mycobacterium bacteria (actinomycetes, etc.), etc. As archaea, they include Methanophilic thermophilic bacteria (Methanopyrus), Methanogenic cocci, Thermococcus, Staphylococcus, etc. Eukaryotic organisms include animals, plants, fungi and protozoa. Fungi include filamentous fungi, yeasts, mushrooms, molds, etc., including Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, Glomeromycota, Microsporidia, etc. Ascomycota include Aspergillus (Aspergillus oryzae, etc.), Penicillium (Penicillium, etc.), Saccharomyces (Budding yeast, etc.), etc.

[0034] The sample contains a target substance as a molecule to be analyzed. In addition, in the present embodiment, the analysis in the analysis device 1 includes: detecting a peak of 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 embodiment, the substance is a protein. The analysis in the analysis device 1 includes determining the type of microorganism that is the source of the sample based on the m / z corresponding to the peak on the mass spectrum. In addition, the m / z corresponding to the peak on the mass spectrum is also generally referred to as the "position" or "m / z position" of the peak.

[0035] In this specification, unless otherwise specified, the type of microorganism includes at least one systematic taxonomic level of the genotype, strain, subspecies, species, genus and family of the microorganism. In addition, the discrimination of the type of microorganism includes the classification, identification and recognition related to the type of the microorganism. Hereinafter, the discrimination of the type of microorganism is also referred to as the discrimination of the microorganism.

[0036] The analysis performed by the analyzing device 1 may include: determining whether a specific substance is contained in the sample; and calculating the concentration of the specific substance in the sample.

[0037] Reference Figure 1 The analyzing device 1 includes a control unit 10 and a measuring unit 20 .

[0038] The measuring unit 20 ionizes a substance (e.g., protein) in a sample by high voltage, separates the ions S according to the flight time associated with m / z, and then detects them. The measuring unit 20 includes an ionizing unit 21, an ion accelerating unit 22, a mass separating unit 23, and a detecting unit 24. Figure 1 In FIG. 2 , the movement of ions S in the measurement unit 20 is schematically shown by arrows A1 .

[0039] The ionization unit 21 ionizes the substance in the sample by the matrix-assisted laser desorption ionization (MALDI) method. As the ionization method, in addition to the MALDI method, any soft ionization method such as the electrospray ionization (ESI) method can be used. In the case of ionization by the ESI method, if the analysis device 1 is also equipped with a liquid chromatograph and the ionization unit 21 ionizes the substance in the sample separated by the liquid chromatograph, a high separation ability can be obtained, so it is preferred.

[0040] The ionization unit 21 includes an ion source including a sample plate holder (not shown) for supporting the sample plate and a laser device (not shown) for irradiating the sample plate with laser light. The analyst first places a mixed solution of a sample and a matrix solution on the sample plate.

[0041] Specifically, for example, the analyst premixes the sample with a matrix solution and then drips the matrix solution into a well formed on the sample plate. The well is a circle drawn or engraved on the sample plate (see Figure 4 On the other hand, the matrix solution may be added dropwise to the sample plate and then mixed with the sample on the sample plate.

[0042] The matrix solution contains a matrix substance that easily absorbs laser light and is easily ionized by the laser light. In this specification, the matrix substance is also abbreviated as "matrix". The matrix is ​​used to analyze substances that are difficult to absorb laser light and / or substances that are easily damaged by laser light (proteins, etc.).

[0043] The sample mixed with the matrix solution can be liquid or solid. More specifically, the sample can be, for example, a liquid containing a culture solution of bacteria, or a sample obtained by wiping a colony of bacteria cultured on a solid medium with a toothpick or the like. The culture solution of the bacteria contains bacteria and a liquid culture medium for culturing bacteria. However, the amount of the liquid culture medium contained in the sample preferably mixed with the matrix solution is less. Therefore, it is preferred that the sample is centrifuged as a pre-treatment, and the sample (precipitate) remaining after the supernatant is removed is mixed with the matrix solution. In addition, it is more preferred that the precipitate is further mixed with the matrix solution after centrifugal cleaning with ultrapure water or the like.

[0044] Alternatively, the sample may be a purified sample of cell constituents such as ribosomal proteins, instead of the cells of the microorganisms themselves as described above.

[0045] The mixing ratio of the matrix solution and the sample is not limited within a range that exhibits the effects of the invention. For example, the sample may be mixed in an amount ranging from a fraction to several hundredths of the volume ratio of the matrix solution.

[0046] The analyst dries the matrix solution mixed with the sample as described above on the sample plate to obtain a dried product. Thereafter, the sample plate is placed on a sample plate holder in the vacuum container of the ionization unit 21 .

[0047] In addition, the dried product of a mixed solution of a sample and a matrix solution is also generally referred to as "crystals", and more specifically, it is referred to as "crystals", "mixed crystals", "sample crystals", "matrix crystals", "sample / matrix crystals" and other names.

[0048] In the following description, the matrix solution at the stage of being placed on the sample plate and forming a dried product also includes the case where the matrix solution is mixed with the sample as described above (ie, "mixed solution") unless otherwise specified.

[0049] After the vacuum container provided with the sample plate is depressurized, the ionization unit 21 irradiates the dried material on the sample plate with laser light to ionize the target substance in the dried material. As long as the light absorbed by the matrix solution used can be oscillated, the type of laser device for irradiating the laser light is not particularly limited. For example, when the matrix solution contains CHCA, N2 laser (wavelength 337nm) can be preferably used. The ions S of the target substance ionized by the ionization unit 21 are extracted by an electric field formed by an extraction electrode, etc., which is not shown in the figure, and are introduced into the ion acceleration unit 22.

[0050] The ion acceleration unit 22 includes an acceleration electrode 221 , and accelerates 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 .

[0051] The mass separation unit 23 includes a flight tube 231, and separates the ions S according to the difference in flight time when the ions S fly inside the flight tube 231. Figure 1 A linear flight tube 231 is shown in the figure, but a reflectron type or a multi-turn type may also be used. The mass analysis method is not particularly limited as long as the ions S contained in the sample can be separated and detected.

[0052] The detection unit 24 includes a multi-channel plate plasma detector, detects the ions S separated by the mass separation unit 23, and outputs a detection signal of intensity corresponding to the number of ions incident on the detection unit 24. The detection signal output from the detection unit 24 is input to the processing unit 11 of the control unit 10. Figure 1 , the flow of the detection signal of the ions S from the detection unit 24 of the measurement unit 20 is schematically shown by the arrow A2.

[0053] The control unit 10 includes a processing unit 11 , a storage unit 12 , and an input / output unit 13 .

[0054] The processing unit 11 is configured to include a processor such as a CPU, and functions as a main body for controlling the operation of the analysis device 1. The processing unit 11 performs various processes by executing a program stored in the storage unit 12 or the like.

[0055] The processing unit 11 includes an apparatus control unit 111 , a mass spectrum generating unit 112 , and a mass spectrum analyzing unit 113 . The mass spectrum analyzing unit 113 includes a determining unit 114 .

[0056] The device control unit 111 controls the operation of the measurement unit 20 based on data related to analysis conditions input from the input unit 131 described later. Figure 1 In FIG. 1 , the control of the measuring unit 20 by the device control unit 111 is schematically shown by arrow A3.

[0057] The mass spectrum generating unit 112 converts the flight time into m / z based on the measurement data including the detected amount of ions detected by the detecting unit 24 and the flight time of the ions, and generates a mass spectrum showing the detected amount corresponding to each m / z.

[0058] The number of detection signals of the target substance detected by the detection unit 24 and the intensity of the detection signals are related to the number of peaks corresponding to the target substance in the mass spectrum and the intensity of the peaks. In addition, the ratio of the intensity of the detection signal of the target substance detected by the detection unit 24 to the intensity of the noise (S / N ratio in the detection signal) is related to the ratio of the intensity of the peak of the target substance in the mass spectrum to the intensity of the noise (S / N ratio in the mass spectrum). Therefore, in this specification, a value in the mass spectrum that is related to at least one of the following matters is referred to as "sensitivity": the number of peaks corresponding to the target substance, the intensity of the peak, and the ratio of the intensity of the peak in the mass spectrum to the intensity of the noise (S / N ratio). Moreover, at least one of the state in which the number of peaks corresponding to the target substance is large, the state in which the intensity of the peak is high, and the state in which the S / N ratio of the mass spectrum is large is referred to as "high sensitivity".

[0059] The mass spectrometer 113 detects the peak of the mass spectrum in the mass spectrum. The m / z corresponding to the detected peak is calculated. The mass spectrometer 113 can identify the substance corresponding to the m / z shown by the peak on the mass spectrum based on a protein database, etc. That is, the mass spectrometer 113 can calculate the m / z of a specific or non-specific substance contained in the sample. The mass spectrometer 113 can also determine whether a specific substance is contained in the sample based on the m / z (component identification in the sample).

[0060] The mass spectrometer 113 includes a discriminating unit 114. In one embodiment, the discriminating unit 114 generates a database including mass spectra. The database includes one or more, preferably a plurality of, mass spectra of known microorganisms. The discriminating unit 114 discriminates the type of microorganism using the database of mass spectra.

[0061] In one embodiment, the identification unit 114 identifies the microorganism by fingerprinting. Specifically, the identification unit 114 compares the mass spectrum pattern of the unknown microorganism with the mass spectrum pattern of the known microorganism stored in the above database, thereby identifying the microorganism. The identification of the microorganism is performed, for example, by referring to the peak of the substance, i.e., the biomarker, which shows a characteristic expression pattern in each microorganism. The identification of the microorganism based on the peak pattern of the biomarker will be described in detail later.

[0062] The storage unit 12 has a non-volatile storage medium. The storage unit 12 stores the mass spectrum generated by the mass spectrum generation unit 112, the measurement data output from the measurement unit 20, and the program for the processing unit 11 to perform processing. The storage unit 12 corresponds to an embodiment of the "memory" in the present disclosure. The storage unit 12 may also include a storage medium that can be removed from the analysis device 1. The storage medium can be a CD (Compact Disc), a DVD (Digital Versatile Disc), and a USB (Universal Serial Bus) memory, etc., as long as it can store various data. The storage unit 12 stores a database of mass spectra.

[0063] The input / output unit 13 is an interface for inputting and outputting information between the analysis device 1 and the outside. The input / output unit 13 includes an input unit 131 , an output unit 132 , and a communication unit 133 .

[0064] The input unit 131 is configured to include input devices such as a mouse, a keyboard, various buttons, and / or a touch panel. The input unit 131 receives information required for controlling the operation of the measurement unit 20 and information required for processing by the processing unit 11 from the analyst.

[0065] The output unit 132 includes a display device such as a liquid crystal monitor, a printer, etc. The output unit 132 displays information related to the measurement of the measuring unit 20, the result of the processing of the processing unit 11, etc. on the display device or prints on a paper medium.

[0066] The communication unit 133 includes a communication device capable of communicating via wireless or wired connection such as the Internet, etc. The communication unit 133 receives data required for processing by the processing unit 11, transmits data processed by the processing unit 11 such as determination results, or transmits and receives required data as appropriate.

[0067] Part or all of the functions of the above-mentioned control unit 10 may also be configured on an electronic computer, server, etc. that is physically separated from the measuring unit 20. In addition, the control unit 10 may also be composed of multiple computers. For example, it may also be composed of a first computer that generates a database of mass spectra and a second computer that uses the database of mass spectra to identify microorganisms as follows. In this case, for example, the first computer generates a database of mass spectra based on the detection signal of ions S output from the measuring unit 20, and stores it in a storage medium. The second computer obtains the database of mass spectra from the storage medium and uses the database to identify the type of microorganism. The transmission and handover of the database from the first computer to the second computer can use a wired or wireless network.

[0068] In addition, as the analysis device 1 used in the present embodiment, there is no particular limitation as long as the analysis device is combined with a MALDI (matrix-assisted laser desorption ionization) ion source. For example, it can also be a MALDI-IT (matrix-assisted laser desorption ionization-ion trap) type mass spectrometer, a MALDI-IT-TOF (matrix-assisted laser desorption ionization-ion trap-time of flight) type mass spectrometer or a MALDI-FTICR (matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance) type mass spectrometer. In addition, the analysis conditions in the analysis device 1 are usually set within the range set by those skilled in the art.

[0069] [2. Conventional quality analysis methods]

[0070] In the past, there was a method of distinguishing microorganisms by mass spectrometry. In mass spectrometry, a micro-amount of microorganism samples is used, and a mass spectrum as an analysis result can be obtained simply and in a short time. In addition, by using automatic analysis, rapid and simple multi-sample analysis can be performed.

[0071] Among such mass spectrometry methods, analysis of microorganisms based on MALDI-MS, which is one of the soft ionization methods for ionizing biopolymers such as proteins without substantially decomposing them, is particularly widely used.

[0072] In microbial analysis based on MALDI-MS, a matrix solution mixed with a sample is dried on a sample plate, and the solvent in the matrix solution is vaporized to form a dried object containing the target substance. By irradiating the dried object on the sample plate with laser light, the matrix substance absorbs the energy of the laser light and is heated and vaporized. At this time, the target substance is also vaporized together with the matrix substance, and the target substance is ionized in this process.

[0073] The sample plates used for forming and quality analysis of dried products are usually disposable sample plates corresponding to the use of clinical microbiological analysis and food hygiene inspection. However, in microbiological research, sample plates that can be used repeatedly are sometimes used. The sample plates are made of, for example, stainless steel or conductive resin.

[0074] From the viewpoint of efficiently ionizing a protein sample, the matrix used is, for example, CHCA, sinapinic acid, 2,5-dihydroxybenzoic acid, etc. Usually, the matrix is ​​dissolved in a solvent and mixed with the sample as a matrix solution.

[0075] Various compositions have been proposed for the composition of the matrix solution. For example, various compositions of matrix solvents for CHCA are disclosed in non-patent documents 1 to 4. Table 1 shows the composition of matrix solvents containing acetonitrile (ACN), ethanol (EtOH), water, trifluoroacetic acid (TFA) and the like at a predetermined content ratio disclosed in non-patent documents 1 to 4. Using these solvents, a matrix solution containing 5 to 15 mg / mL of CHCA was prepared.

[0076] [Table 1]

[0077]

[0078] However, when MALDI-MS is performed on dried products using these matrix solutions, there is a problem that sufficient sensitivity cannot be obtained, especially in the high-mass region. In addition, the high-mass region refers to a region with a relatively large m / z, for example, a region above m / z 10,000, and more specifically, a region above m / z 11,000.

[0079] In the discrimination of microorganisms, it is crucial to obtain sufficient sensitivity. In particular, it is crucial to obtain sufficient sensitivity in the high-quality region. The reason is that the amino acid sequence of a protein with a large molecular weight is long, and the possibility of mutation becomes higher accordingly. Therefore, peak detection in the high-quality region is crucial, especially in the case of discriminating microorganisms with high similarity. The discrimination of microorganisms with high similarity is, for example, the discrimination of subspecies or strains within the same species.

[0080] Therefore, the inventors studied the experimental conditions necessary to obtain sufficient sensitivity even in the high mass region.

[0081] [3. Study on the hypothesis of forming a dried product using a conventional matrix solution]

[0082] In the process of studying the experimental conditions, the inventors presumed that there was a problem in the formation process of the dried product in the conventional mass analysis method, and explored a solution thereto.

[0083] The inventors first focus on that, in the process of forming a dried object by dropping a previous matrix solution into a hole formed in a sample plate, the matrix solution is mostly biased in a narrower range. In this case, if a sample crystal is formed at the end of the hole, the laser is not properly irradiated to the sample when automatically measuring sometimes. In such a case, the sensitivity in the mass spectrum obtained by the analysis of the sample may be insufficient.

[0084] Next, the inventors studied the formation of dried matter in the entire hole by dripping an increased amount of matrix solution in order to suppress the formation of dried matter at the end of the hole. However, if the amount of matrix solution dripped into the hole is increased, it is possible that the time taken to dry the matrix solution becomes longer and / or the dried matter becomes thicker, making it impossible to obtain a good mass spectrum.

[0085] Therefore, the inventors studied the composition of the matrix solution and investigated how to form a dried product in the entire well without increasing the amount of the matrix solution added dropwise to the well.

[0086] [4. Study on the composition of the desired matrix solution]

[0087] First, in order to diffuse the matrix solution into the whole hole, the inventors etc. studied the surface tension reduction by adding a large amount of organic solvents. However, when the organic solvent concentration of the matrix solution is too high, the possibility of lipid dissolving in a large amount from the thalline and hindering the ionization of the protein may increase. In addition, it is believed that when the organic solvent concentration of the matrix solution is too high, the drying speed becomes faster, so the smaller dried product that is not suitable for the ionization of the protein is formed, and the peak intensity of the mass spectrum may weaken. Therefore, it is believed that the organic solvent can not be simply increased, but it is necessary to compile the composition of the matrix reagent of the MALDI-MS measurement of the cell constituent component centered on the protein of the microorganism by other methods.

[0088] Based on the above research results, the inventors have found that by forming a dried product that satisfies the first to third conditions described below, a mass spectrum with high sensitivity can be obtained even in a high mass region.

[0089] The first condition is that the dried matter is formed widely, and more preferably formed in the entire hole. In addition, in this specification, the dried matter is formed widely means that the area of ​​the upper surface (the surface on the side irradiated by the laser) of the dried matter is formed widely. By forming the dried matter widely, preferably forming it in the entire hole, in mass analysis, the detection signal of the target substance can be obtained from a large area. Therefore, the sensitivity is directly improved.

[0090] The second condition is that the uniformity of the dried product is high. An example of a high uniformity of the dried product is that the shape of the upper surface of the dried product is high. An example of a high uniformity of the shape of the upper surface of the dried product is that the thickness of the dried product in the vertical direction varies little due to different positions, and the upper surface of the dried product has few irregularities and is nearly flat.

[0091] Another example of high uniformity of the dried object is high uniformity of the internal structure of the dried object. High uniformity of the internal structure of the dried object means, for example, that the ratio of the target substance and the matrix in the dried object and / or the bonding method have small differences due to different positions in at least the upper surface of the dried object. In addition, for example, the dried object in the hole is gathered into one, and does not form a plurality of small dried objects.

[0092] Since the dried product has high uniformity, the protein is appropriately ionized, the signal intensity increases, and thus the sensitivity is improved.

[0093] The third condition is that the dried product is derived from a mixed solution from which the constituent components of the microorganisms are well extracted. The so-called good extraction of the constituent components of the microorganisms means, for example, efficient extraction of the constituent components of the microorganisms, and more specifically, a large amount of the target substance extracted per microorganism. As a result, the concentration of the target substance in the dried product increases, and a stronger detection signal of the target substance can be obtained, thereby improving the sensitivity.

[0094] That is, the inventors believe that as long as a matrix solution that forms a dried product that satisfies the first to third conditions can be obtained, a highly sensitive mass spectrum can be obtained. In addition, the inventors believe that as a result of the improved sensitivity in the entire mass spectrum, sufficient sensitivity can be obtained even in a high mass region where sufficient sensitivity has not been obtained in the past.

[0095] Based on the above observations and examination results, the inventors invented the composition of a matrix solution for forming a dried product capable of obtaining a highly sensitive mass spectrum.

[0096] In particular, the inventors have studied the composition of the matrix solvent and found that a dried product can be obtained that can detect peaks with high sensitivity in the high mass region. In this specification, the composition of the matrix solvent refers to the types of reagents and substances contained in the matrix solvent, and the proportion and / or concentration of the reagents and substances.

[0097] [5. Mass Analysis Using the Matrix Solution According to the Embodiment]

[0098] (5-1. Composition of the substrate solution according to the embodiment)

[0099] Figure 2 : is a diagram illustrating the composition of the base solvent involved in the comparative example and the present embodiment. Figure 2In the above, the volume ratio of acetonitrile, ethanol, trifluoroacetic acid and water in each matrix solvent is expressed as a percentage when the volume of the entire matrix solvent is set to 100. The above acetonitrile, ethanol, trifluoroacetic acid and water can be produced by known methods, or commercial products can be obtained.

[0100] Reference Figure 2 The base solvent according to the present embodiment is characterized in that the ratio of acetonitrile and ethanol as organic solvents is different from that of the base solvents in Comparative Examples 1 and 2.

[0101] The specific composition is described below: The organic solvent contained in the base solvent of Comparative Example 1 is 50% acetonitrile, and the organic solvent contained in the base solvent of Comparative Example 2 is 35% acetonitrile and 25% ethanol.

[0102] On the other hand, the organic solvent included in the matrix solvent involved in the present embodiment is 30-40% acetonitrile, 10-20% ethanol, more preferably 33-37% acetonitrile, 13-17% ethanol, and further preferably about 35% acetonitrile, about 15% ethanol. The matrix solvent involved in the present embodiment also includes 1-3% trifluoroacetic acid, more preferably 1-2% trifluoroacetic acid. In addition, of course, the remainder of the matrix solvent is water.

[0103] In one aspect of the present embodiment, the content of acetonitrile in the base solvent is 30 to 40% by volume, preferably 33 to 37%, and more preferably about 35%.

[0104] In one aspect of the present embodiment, the content ratio of ethanol contained in the base solvent is 10 to 20% by volume, preferably 13 to 17%, and more preferably about 15%.

[0105] Each matrix solvent is prepared in a manner where the sum of the ratios of acetonitrile, ethanol, trifluoroacetic acid, and water is 100%. The preparation method is not particularly limited, and for example, acetonitrile, ethanol, and trifluoroacetic acid can be added to water in a test tube, or acetonitrile, ethanol, and trifluoroacetic acid can be added to water on an industrial scale in a chemical plant or the like.

[0106] The substrate solvent according to the present embodiment is further prepared to contain 5 to 20 mg / ml, preferably 5 to 15 mg / ml, and more preferably 8 to 10 mg / ml of the substrate, and is used as a substrate solution.

[0107] In one embodiment, the matrix is ​​α-cyano-4-hydroxycinnamic acid (4-CHCA, also referred to as CHCA in this specification), which is one of the most commonly used matrices in mass analysis. The matrix may also be 2,5-dihydroxybenzoic acid or sinapinic acid, which are also the most commonly used matrices in mass analysis. Other examples of the matrix are not limited thereto, and may be 3-hydroxy-4-nitrobenzoic acid (3H4NBA), α-cyano-3-hydroxycinnamic acid (3-CHCA), ferulic acid, or 1,5-diaminonaphthalene, etc.

[0108] In addition, Figures 4 to 6 In the example of the matrix solution involved in the embodiment, an example of the experimental results of a solution containing 10 mg / mL of CHCA in a matrix solvent containing 35% acetonitrile, 15% ethanol, 1% trifluoroacetic acid, and 39% water is shown. In addition, as an example of the matrix solution involved in Comparative Example 1, a solution containing 10 mg / mL of CHCA in a matrix solvent containing 50% acetonitrile, 1% trifluoroacetic acid, and 49% water is used. As an example of the matrix solution involved in Comparative Example 2, a solution containing 10 mg / mL of CHCA in a matrix solvent containing 35% acetonitrile, 25% ethanol, 1% trifluoroacetic acid, and 39% water is used.

[0109] (5-2. Formation of a dried product using the matrix solution according to the embodiment)

[0110] The matrix solution according to the embodiment is mixed with a sample and dried on a sample plate to form a dried product.

[0111] Figure 3 This is a diagram for explaining the method of forming the dried material on the sample plate. Figure 3 , a state where a matrix solution M is disposed on a well W in a sample plate P by a pipette 5 is shown.

[0112] The sample plate P is a general MALDI plate, and is a plate made of, for example, stainless steel and / or a conductive resin.

[0113] The sample is a sample derived from a microorganism, and according to one embodiment, is a sample derived from Escherichia coli. The analyst adds 9 μL of the matrix solution to a microtube, and then adds 1 μL of the culture solution of Escherichia coli, mixes them thoroughly, and drops 1 μL of the mixed solution onto the sample plate P.

[0114] The matrix solution placed on the well W of the sample plate P is dried to form a dried product.

[0115] According to the observations made by the naked eyes and the use of a photographing device by the inventors, in the dried product formed by the matrix solution of Comparative Example 1 (hereinafter, also referred to as the dried product of Comparative Example 1) and the dried product formed by the matrix solution of Comparative Example 2 (hereinafter, also referred to as the dried product of Comparative Example 2), the dried product was mostly formed in a narrow range of the plate. In addition, there was also a case where the dried product was formed at the end of the plate in association. On the other hand, the dried product formed by the matrix solution of the present embodiment (hereinafter, also referred to as the dried product of the embodiment) was uniformly spread over the entire plate. Figure 4 An example of the outer appearance of the dried product of Comparative Example 1, the dried product of Comparative Example 2, and the dried product of the embodiment is shown.

[0116] Figure 4 The figure shows the photographed image of the dried material on the sample plate. Figure 4 This is a black-and-white image of the dried object captured by a camera (CCD camera) including a CCD (Charge Coupled Device) as an image sensor in the ionization unit 21 in the analysis device 1 .

[0117] like Figure 4 As shown, the dried product of Comparative Example 1 is formed eccentrically at the lower right portion of the hole W. The dried product has bright and dense particle-like irregularities (not shown). In addition, a portion of the dried product protrudes from the outer frame of the hole W.

[0118] The central portion of the dried product of Comparative Example 2 was slightly bright, and particle-like irregularities (not shown) were observed. The outer edge portion of the dried product of Comparative Example 2 appeared darker than the central portion and spread out in a planar manner.

[0119] The dried product of the embodiment widely exists in the entire pore and spreads evenly, so the overall thickness is thin, and it becomes dark and difficult to distinguish from the base of the sample plate in the CCD black-and-white image.

[0120] Thus, the dried product of the embodiment showed different states from the dried product of Comparative Example 1 and the dried product of Comparative Example 2. The difference in the state of the dried product is considered to reflect that at least one of the strength of the surface tension, the drying speed, the difference in the types and / or amounts of the bacterial cell constituent components contained in the dried product, and the difference in the uniformity of the dried product has changed by changing the concentrations of acetonitrile and ethanol.

[0121] More specifically, the dried product of the embodiment has a wider and more uniform thickness than the dried products of Comparative Examples 1 and 2. The inventors have confirmed that this feature of the dried product of the embodiment has high reproducibility regardless of the sample.

[0122] The inventors and others have confirmed that, compared with each of the matrix solutions of Comparative Examples 1 and 2, the matrix solution involved in the embodiment has a fast drying speed and forms a dried product in a short time. It can be considered that the speed of the drying speed mainly reflects the difference in the vaporization speed caused by the composition (especially the concentration of acetonitrile and ethanol) itself, the difference in the surface area of ​​the matrix solution dripped onto the sample plate and the contact with the air (especially the difference in the area of ​​the surface of the matrix solution). It can also be considered that the speed difference of the drying speed may reflect at least one of the difference in the amount and / or type of the bacterial cell constituents contained in the mixed solution and the uniformity of the mixed solution (the uniformity of the ratio or bonding method of the target substance and the matrix). According to the speed of the drying speed, the above-mentioned high uniformity dried product can be formed in a short time.

[0123] It is considered that the characteristic crystal state of the dried product of such an embodiment is reflected, and the following Figure 5 Highly sensitive mass spectrum as shown.

[0124] (5-3. Results of mass analysis of dried product using matrix solution according to the embodiment)

[0125] Figure 5 2 is a diagram showing the mass spectrum of Escherichia coli, including, from the top, the mass spectrum of the dried product of Comparative Example 1 (hereinafter also referred to as “the mass spectrum of Comparative Example 1”), the mass spectrum of the dried product of Comparative Example 2 (hereinafter also referred to as “the mass spectrum of Comparative Example 2”), and the mass spectrum of the dried product of the embodiment (hereinafter also referred to as “the mass spectrum of the embodiment”). Figure 5 The horizontal axis represents m / z, and the vertical axis represents the intensity of the detection signal. The region with m / z of 11,000 or more shows a signal intensity that is 10 times greater.

[0126] Reference Figure 5 , the mass spectrum involved in the embodiment is compared with each mass spectrum of the mass spectrum involved in Comparative Example 1 and Comparative Example 2, and the intensity of the peak is improved as a whole, and the sensitivity is improved. In particular, in the high-quality region (for example, the region above about m / z11,000), almost no peak is detected in each mass spectrum of the mass spectrum involved in Comparative Example 1 and Comparative Example 2, and the intensity of the very few peaks detected is also very small. On the contrary, a large number of high-intensity peaks are detected in the mass spectrum of the present embodiment. In this way, the sensitivity of the entire mass spectrum of the present embodiment is improved, and the sensitivity is significantly improved in the high-quality region. Thus, even in the high-quality region, sufficient sensitivity can be obtained. That is, by using the matrix solution of the embodiment, the peak can be detected with high sensitivity.

[0127] In addition, since a large number of peaks were detected with high intensity in the mass spectrum of the embodiment, it can be seen that the extraction of bacterial cell constituents from microorganisms is also well performed. That is, the matrix solution of the present embodiment can efficiently extract bacterial cell constituents with an efficiency at least equal to or higher than that of the matrix solutions of Comparative Examples 1 and 2.

[0128] Figure 6 is a graph showing the amount of ribosomal proteins detected.

[0129] In more detail, Figure 6 It is aimed at Figure 5 A peak list including the corresponding m / z and peak intensity is shown in FIG. Figure 6 In FIG, the number of ribosomal proteins contained in the peak list is shown, which is calculated by comparing the m / z of the peak list with the theoretical m / z calculated from the molecular weight of the known ribosomal proteins expressed in E. coli.

[0130] For the dried product using the matrix solution involved in the embodiment, an average of 25 types of ribosomal proteins were detected in 16 samples. On the other hand, for the dried product using the matrix solution involved in Comparative Examples 1 and 2, the average number of ribosomal proteins was 14 and 20. That is, the dried product of the present embodiment can detect more ribosomal proteins than those of Comparative Examples 1 and 2. That is, by using the matrix solution of the embodiment, more biomarkers can be used to distinguish microorganisms than when using the matrix solution involved in Comparative Examples 1 and 2.

[0131] according to Figure 5 , Figure 6 If the matrix solution involved in the embodiment is used, the sensitivity in the entire mass spectrum is improved, and sufficient sensitivity can be obtained in the high mass region in particular. As a result, the accuracy of microbial identification based on the mass spectrum is improved. As a result, as described later Figure 8 As shown, microorganisms with high similarity can also be classified.

[0132] (5-4. Effects of Matrix Solution and Mass Spectrometry Method According to Embodiment)

[0133] like Figure 4 As shown, according to the matrix solution involved in this embodiment, a dried product that is widely and uniformly diffused in the pores can be prepared. Therefore, a wide and uniform signal can be obtained from the upper surface of the dried product, so a highly sensitive mass spectrum can be obtained efficiently.

[0134] In this connection, the mixed solution diffuses widely in the pores, resulting in an increase in the drying speed. As a result, a uniform dried product can be prepared in a short time, and thus a good sample can be prepared efficiently.

[0135] Since the components constituting the bacterial cell are extracted well, more proteins derived from microorganisms can be detected by MALDI-MS. That is, the number of peaks detected in the mass spectrum increases. Such an increase in the amount of information also contributes to the improvement of the accuracy of microbial identification.

[0136] Due to the above effects, the accuracy of microorganism identification by mass spectrometry using the matrix solution according to the present embodiment is improved.

[0137] [6. Quality analysis and microbial identification]

[0138] Figure 7 It is a flowchart showing the mass analysis process and the microorganism identification process. Figure 7 The steps shown are performed by manual operation of an analyst using an experimental device used for general microbial culture and quality analysis. In addition, in the figure, "S" is used as an abbreviation for "STEP".

[0139] In S1 , the analyst mixes the matrix solution according to the present embodiment with a sample.

[0140] In S2, the analyst dries the matrix solution mixed with the sample on the sample plate to form a dried product.

[0141] In S3, the analyst acquires a mass spectrum by performing matrix-assisted laser desorption ionization mass analysis on the dried material.

[0142] In S4 , the analyst creates a database including mass spectra and stores the database in the storage unit 12 .

[0143] In S5, the analyst uses the mass spectra contained in the database to identify the type of microorganism.

[0144] according to Figure 7 By using the matrix solution of this embodiment, a highly sensitive mass spectrum can be obtained, and in particular, a large number of peaks with sufficient intensity can be detected even in the high mass region. As a result, the accuracy of microbial identification can be improved.

[0145] In the above example, it is shown that if the matrix solution involved in this embodiment is used, high-sensitivity mass spectrometry can be performed in MALDI-MS, resulting in improved accuracy in distinguishing samples derived from microorganisms, but it is obvious that the matrix solution of this embodiment can also be used for biological samples derived from organisms other than microorganisms.

[0146] In addition, the kind of material included in the matrix solution involved in the present embodiment, containing ratio and / or concentration are an example. Especially, the kind of material (for example trifluoroacetic acid, water, matrix) other than acetonitrile and ethanol, containing ratio and / or concentration can be changed within the scope that those skilled in the art usually consider and adjustable range, in this case, it is also believed that the effect equivalent to the above-mentioned embodiment can be obtained. For example, as long as it is the scope of the effect of the present application, it is also possible to use the matrix solution prepared in the mode of 8~20mg / ml by containing a variety of matrixes and making its total concentration. In addition, the kind of material (for example acetonitrile, ethanol) included in the matrix solution involved in the present embodiment, containing ratio and / or concentration can be measured by gas chromatograph, for example.

[0147] [7. Experimental example]

[0148] The following shows the results of analyzing microorganisms by the mass spectrometry method using the substrate solution according to the present embodiment.

[0149] Figure 8 This is a table showing the peaks of ribosomal proteins obtained as a result of mass analysis of Propionibacterium acnes (C. acnes: Cutibacterium acnes) by the mass spectrometry method using the substrate solution according to the present embodiment.

[0150] Figure 8 In the left column 31 of FIG. 1 , strains of Propionibacterium acnes are shown for each phylotype by the management number in the Japan Collection of Microorganisms (JCM) of the RIKEN Institute of Physical and Chemical Research.

[0151] The detection results of the biomarker proteins are shown in the right column 32. The names of the ribosomal proteins used as biomarkers are shown in row 33. The molecular weight of each ribosomal protein is shown in row 34 below row 33. In row 35, whether a peak is detected in the above molecular weight is indicated for each strain with 1 (detected) or 0 (not detected).

[0152] Regarding the ribosomal proteins L7 / L12 to S19 shown in the region 36, the molecular weight of the specific ribosomal protein does not change depending on the strain. Therefore, even if the peaks of L7 / L12 to S19 are used, it is impossible to distinguish the strains within Propionibacterium acnes.

[0153] On the other hand, regarding the ribosomal proteins shown by L6, L13, L15, and L23 shown in area 37, the molecular weights differ depending on the strain. More specifically, regarding the ribosomal proteins shown by L6, L13, L15, and L23, the molecular weights differ by about 10 to 30 depending on the strain. It can be seen that based on the peaks of L6, L13, L15, and L23, strains within Propionibacterium acnes can be distinguished. For example, the three strains with sorting numbers 6425, 18918, and 18920 can be distinguished based on the peak of L13. In this way, it can be seen that L6, L13, L15, and L23 can be used as biomarkers for distinguishing strains within Propionibacterium acnes.

[0154] The molecular weight changes of the ribosomal proteins of L6, L13, L15, and L23 were 19679 / 19707, 16154 / 16168 / 16182, 15358 / 15385, and 11181 / 11200, respectively, all of which were included in the high-mass region of molecular weight above 11,000. Therefore, strains within Propionibacterium acnes can be distinguished based on high-mass ribosomal proteins.

[0155] As described above, by obtaining the peak in the high-quality region, microorganisms in Propionibacterium acnes can be identified. It is also known that peak detection in the high-quality region is important for identifying strains in Propionibacterium acnes.

[0156] Thus, by obtaining sufficient sensitivity even in the high-quality region, the accuracy of microorganism identification is improved. As a result, it is possible to identify highly similar microorganisms that have similar genetic information and are difficult to identify. Therefore, in the identification of microorganisms, especially in the identification of highly similar microorganisms, it is important to obtain sufficient sensitivity even in the high-quality region.

[0157] [plan]

[0158] Those skilled in the art will appreciate that the above-mentioned multiple exemplary embodiments are specific examples of the following schemes.

[0159] (Item 1) The matrix solution involved in one scheme is a matrix solution for mixing with a sample in matrix-assisted laser desorption ionization mass spectrometry. The matrix solution is an aqueous solution containing acetonitrile, ethanol, trifluoroacetic acid and water. The content of acetonitrile is 30 to 40% by volume based on the matrix solution. The content of ethanol is 10 to 20% by volume based on the matrix solution. The content of trifluoroacetic acid is 1 to 3% by volume based on the matrix solution. The matrix solution contains 5 to 20 mg / ml of α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid or sinapinic acid.

[0160] According to the matrix solution described in Item 1, it is possible to provide a matrix solution capable of performing highly sensitive mass spectrometry in MALDI-MS. Furthermore, when the sample is a sample derived from microorganisms, it is possible to improve the accuracy of microorganism identification using mass spectrometry.

[0161] (Item 2) In the matrix solution described in Item 1, it is more preferred that the content of acetonitrile is 33 to 37% by volume based on the matrix solution, and the content of ethanol is 13 to 17% by volume based on the matrix solution.

[0162] (Item 3) The base solution described in Item 1 or 2 more preferably contains 8 to 10 mg / ml of α-cyano-4-hydroxycinnamic acid.

[0163] (Item 4) In the base solution according to any one of Items 1 to 3, the sample is a sample derived from a microorganism.

[0164] As described in Item 4, when the sample is a sample derived from microorganisms, the accuracy of distinguishing microorganisms using mass spectrometry can be improved.

[0165] (Item 5) A mass spectrometry method according to one scheme comprises: a step of mixing a sample with a matrix solution; a step of drying the matrix solution mixed with the sample on a sample plate to form a dried product; and a step of acquiring a mass spectrum by performing matrix-assisted laser desorption ionization mass analysis on the dried product. The matrix solution is an aqueous solution containing acetonitrile, ethanol, trifluoroacetic acid and water. The content of acetonitrile is 30 to 40% by volume based on the matrix solution. The content of ethanol is 10 to 20% by volume based on the matrix solution. The content of trifluoroacetic acid is 1 to 3% by volume based on the matrix solution. The matrix solution contains 5 to 20 mg / ml of α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid or sinapinic acid.

[0166] According to the mass spectrometry method described in Item 5, a matrix solution capable of performing highly sensitive mass spectrometry can be provided. Furthermore, when the sample is a sample derived from microorganisms, the accuracy of microorganism identification using mass spectrometry can be improved.

[0167] (Item 6) In the mass spectrometry method described in Item 5, the sample is a sample derived from microorganisms.

[0168] As described in Item 6, when the sample is a sample derived from microorganisms, the accuracy of distinguishing microorganisms using mass spectrometry can be improved.

[0169] (Item 7) A storage medium storing a database including mass spectra acquired by the mass analysis method described in Item 5 or 6.

[0170] According to the storage medium described in Item 7, a database including a highly sensitive mass spectrum can be stored and taken out to the outside.

[0171] (Item 8) A method for distinguishing the type of a microorganism using a database including a mass spectrum obtained by the mass analysis method described in Item 6.

[0172] According to the identification method described in Item 8, microorganisms can be identified with high accuracy using a database including highly sensitive mass spectra.

[0173] (Item 9) In the identification method described in Item 8, the type of microorganism includes at least one systematic taxonomic level of the genotype, strain, subspecies, species, genus and family of the microorganism.

[0174] According to the discrimination method described in Item 9, it is possible to improve the discrimination accuracy for the type of microorganism described in Item 9. In particular, microorganisms with high similarity can be discriminated.

[0175] (Item 10) The identification of the type of the microorganism according to Item 8 or Item 9 includes at least one of classification, identification, and recognition related to the type of the microorganism.

[0176] In the discrimination method described in Item 10, it is considered that the accuracy of classification, identification, and recognition related to the types of microorganisms is improved.

[0177] The embodiments disclosed this time should be considered as illustrative in all aspects and not restrictive. The scope of the present invention is indicated by the claims rather than the above description, and is intended to include all modifications within the meaning and scope equivalent to the claims.

[0178] Description of Reference Numerals

[0179] 1 Analysis device

[0180] 5. Pipette

[0181] M Matrix Solution

[0182] 10. Control Unit

[0183] 11 Processing Department

[0184] 12 Storage

[0185] 13 Input and output section

[0186] 20 Measurement Department

[0187] 21. Ionization unit

[0188] 22 Ion Acceleration Department

[0189] 23. Mass separation section

[0190] 24. Inspection Department

[0191] 111 Device control unit

[0192] 112 Mass Spectrum Generation Unit

[0193] 113 Mass Spectrometry Department

[0194] 114 Discrimination Department

[0195] 131 Input section

[0196] 132 Output

[0197] 133 Ministry of Communications

[0198] 221 Accelerating Electrode

[0199] 231 Flight Tube

[0200] P Sample Plate

[0201] S ion

[0202] W hole.

Claims

1. A matrix solution for mixing with a sample in matrix-assisted laser desorption ionization mass spectrometry, characterized in that: The matrix solution is an aqueous solution comprising acetonitrile, ethanol, trifluoroacetic acid and water, Based on the matrix solution, the acetonitrile content is 30-40% by volume. Based on the matrix solution, the ethanol content is 10-20% by volume. Based on the matrix solution, the content of trifluoroacetic acid is 1 to 3% by volume. The matrix solution contains 5-20 mg / ml of α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid or sinapinic acid.

2. The matrix solution according to claim 1, characterized in that Based on the matrix solution, the acetonitrile content is 33-37% by volume. The content ratio of the ethanol is 13 to 17 volume % based on the base solution.

3. The matrix solution according to claim 1 or 2, characterized in that The α-cyano-4-hydroxycinnamic acid is contained in an amount of 8 to 10 mg / ml.

4. The matrix solution according to claim 1 or 2, characterized in that The sample is a sample derived from microorganisms.

5. A quality analysis method, characterized in that have: The step of mixing the sample with the matrix solution; The step of drying the matrix solution mixed with the sample on a sample plate to form a dried product; The step of obtaining a mass spectrum by performing matrix-assisted laser desorption ionization mass analysis on the dried product, The matrix solution is an aqueous solution comprising acetonitrile, ethanol, trifluoroacetic acid and water, Based on the matrix solution, the acetonitrile content is 30-40% by volume. Based on the matrix solution, the ethanol content is 10-20% by volume. Based on the matrix solution, the content of trifluoroacetic acid is 1 to 3% by volume. The matrix solution contains 5-20 mg / ml of α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid or sinapinic acid.

6. The mass analysis method according to claim 5, characterized in that The sample is a sample derived from microorganisms.

7. A storage medium, characterized in that: A database containing mass spectra acquired by the mass analysis method according to claim 5 or 6 is stored.

8. A discrimination method, characterized in that: The type of microorganism is identified using a database including the mass spectrum acquired by the mass analysis method according to claim 6.

9. The identification method according to claim 8, characterized in that: The type of microorganism includes at least one systematic taxonomic level of the genotype, strain, subspecies, species, genus and family of the microorganism.

10. The identification method according to claim 8 or 9, characterized in that: The determination of the type of the microorganism includes at least one of classification, identification and recognition related to the type of the microorganism.

Images