Method for identifying acid shock proteins, and method for identifying microorganisms

Abstract

This method for identifying acid shock proteins comprises: (i) a step for preparing a first solution containing an acid shock protein produced by a predetermined type of microorganism at a first concentration and a second solution containing the acid shock protein produced by the predetermined type of microorganism at a second concentration lower than the first concentration; (ii) a step for measuring a first mass spectrum and a second mass spectrum corresponding respectively to the first solution and the second solution; and (iii) a step for comparing the first mass spectrum and the second mass spectrum to determine a specific peak corresponding to the acid shock protein in each mass spectrum in accordance with the degree to which the peak intensity is maintained in the second mass spectrum.

Description

Method for identifying acid shock proteins and microorganisms

[0001] The present invention relates to a method for identifying an acid shock protein and a method for identifying a microorganism, and more particularly to a method for detecting a mass spectrum peak corresponding to an acid shock protein and a method for identifying a microorganism based on the mass spectrum peak.

[0002] Among the classifications of microorganisms, the order Enterobacteriale is known to include pathogenic bacteria such as enterohemorrhagic Escherichia coli, Salmonella, Shigella, and Yersinia pestis. The order Enterobacteriale also includes bacteria that are the subject of epidemiological investigations of food poisoning. Classifying and analyzing microorganisms in the order Enterobacteriale, which includes these important bacterial groups, is important in microbial research and medical settings.

[0003] As one of the classification and analysis methods for microorganisms belonging to the order Enterobacteriaceae, International Publication No. 2020 / 202861 (Patent Document 1) shows that when some strains belonging to the order Enterobacteriaceae are cultured under appropriate conditions, they produce acid shock protein (Acid shock protein, hereinafter also referred to as "Asr"). It has also been disclosed that Asr peaks showing different m / z were found in the mass spectra of different strains. Thus, a method for identifying microorganisms based on the peak corresponding to Asr is considered promising.

[0004] International Publication No. 2020 / 202861

[0005] In Patent Document 1, a mass spectrum obtained by culturing a specific type of microorganism under conditions that produce Asr is compared with a mass spectrum obtained by culturing a specific type of microorganism under conditions that do not produce Asr, thereby identifying peaks derived from Asr. However, culturing and analyzing the specific type of microorganism under the two conditions described above requires twice the effort compared to culturing and analyzing the specific type of microorganism under one condition. Therefore, a method for more easily identifying peaks derived from Asr has been desired.

[0006] The present disclosure has been made to solve such problems, and its purpose is to simply identify the mass spectrum peaks of Asr and / or fragments thereof produced by microorganisms.

[0007] A method for identifying an acid shock protein according to a first aspect of the present disclosure includes the steps of: (i) preparing a first solution containing an acid shock protein produced by a predetermined type of microorganism at a first concentration; and (ii) preparing a second solution containing the acid shock protein produced by the predetermined type of microorganism at a second concentration lower than the first concentration; (ii) measuring a first mass spectrum and a second mass spectrum corresponding to the first solution and the second solution, respectively; and (iii) comparing the first mass spectrum with the second mass spectrum and determining specific peaks in each mass spectrum corresponding to the acid shock protein depending on the degree of maintenance of peak intensity in the second mass spectrum.

[0008] According to the method for identifying Asr of the present disclosure, the mass spectrum peak of Asr and / or its fragments produced by microorganisms can be easily identified.

[0009] Fig. 1 is a schematic diagram showing the configuration of an analytical device; Fig. 2 is a flowchart showing an example of an experimental method according to an embodiment; Fig. 3 is a diagram showing an example of a first mass spectrum and a second mass spectrum; Fig. 4 is a diagram showing the amino acid sequence and theoretical m / z of an Asr fragment of Escherichia coli K12 strain; and Fig. 5 is a table showing the intensities of peaks for common m / z in the first mass spectrum and the second mass spectrum.

[0010] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the following, the same or corresponding parts in the drawings are denoted by the same reference numerals, and their description will not be repeated in principle.

[0011] [1. Configuration of the Analytical Apparatus] First, an example of an analytical apparatus 1 that analyzes microorganisms using Asr will be shown. Fig. 1 is a schematic diagram showing the configuration of the analytical apparatus 1. The analytical apparatus 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).

[0012] In this embodiment, the sample is a sample derived from a microorganism. The sample includes a target substance, which is a molecule to be analyzed. The sample may also include a standard substance (calibrant), which is a molecule used to calibrate a mass spectrum. Furthermore, 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, and the target substance is Asr and / or a fragment thereof. Analysis by the analyzer 1 includes determining whether a specific substance is contained in the sample based on the m / z indicated by the peak in the mass spectrum (hereinafter also referred to as "measured m / z"), and identifying microorganisms contained in the sample. In this specification, identifying a microorganism includes determining the classification of the microorganism at at least one level, such as family, genus, species, or strain. Furthermore, if a microorganism can be identified, it is possible to classify the microorganism. Classifying a microorganism includes determining whether the microorganism belongs to a specific classification. Furthermore, classifying a microorganism includes identifying whether a microorganism belongs to a different classification from other microorganisms.

[0013] 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.

[0014] 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. After a sample is placed on the sample plate, a matrix is ​​added to the sample and the sample is dried. The sample plate is then placed in the sample plate holder within the vacuum chamber of the ionization unit 21. The type of matrix is ​​not particularly limited, but examples include sinapinic acid, α-cyano-4-hydroxycinnamic acid (CHCA), and 2,5-dihydroxybenzoic acid. The matrix is ​​added to a concentration of, for example, 5 to 10 mg / ml.

[0015] The ionization unit 21 depressurizes the vacuum chamber containing the sample plate, and then irradiates each sample on the sample plate with laser light to sequentially ionize them. 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 selected matrix. For example, when the matrix contains sinapinic acid or CHCA, an N2 laser (wavelength 337 nm) or the like can be suitably used. The ions S ionized in 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] The control unit 10 includes a processing unit 11, a storage unit 12, and an input / output unit 13. 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, etc. The processing unit 11 corresponds to an example of a "processor" in this disclosure.

[0020] The processing unit 11 includes an apparatus control unit 111 , a mass spectrum creation unit 112 , a mass spectrum analysis unit 113 , and a calibration unit 114 .

[0021] 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.

[0022] The mass spectrum creation unit 112 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.

[0023] The mass spectrum analysis unit 113 detects mass spectrum peaks (also simply referred to as "peaks" in this specification) in the mass spectrum. It calculates the m / z (measured m / z) corresponding to the detected peak. The mass spectrum analysis unit 113 may determine the substance to which the measured 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 measured m / z of a specific or non-specific substance contained in the sample. For example, the mass spectrum analysis unit 113 determines whether a specific substance (e.g., a protein) is contained in the sample based on the measured m / z, and identifies the organism contained in the sample. A method for identifying an organism based on the measured m / z will be described later.

[0024] The calibration unit 114 calibrates the mass spectrum based on the measured m / z and theoretical m / z of the standard material. The theoretical m / z is generally referred to as a theoretical value or theoretical m / z, and is a theoretical mass-to-charge ratio calculated taking into account the molecular weight, the added ions, and the number of charges. Calibration in mass spectrometry involves correcting the measured m / z of the standard material so that it approaches the theoretical m / z, and applying this correction to the entire spectrum.

[0025] The storage unit 12 includes a non-volatile storage medium and stores the mass spectrum created by the mass spectrum creation unit 112, 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.

[0026] 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.

[0027] The input unit 131 is configured to include input devices such as a mouse, a keyboard, various buttons, and / or a touch panel, etc. The input unit 131 receives information necessary for controlling the operation of the measurement unit 20, information necessary for the processing performed by the processing unit 11, etc. from the user.

[0028] 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.

[0029] 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.

[0030] 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 .

[0031] By using the analytical device 1 described above, it is possible to detect peaks of Asr and / or fragments thereof and identify microorganisms based on the peaks, as will be described below.

[0032] [2. Conventional Methods for Identifying Asr] Identification and / or classification of microorganisms using Asr has been carried out, for example, as follows: First, a sample containing Asr is prepared from cultured bacterial cells, and the sample is measured by mass spectrometry to obtain a mass spectrum. Peaks derived from Asr are identified from the numerous peaks contained in the mass spectrum. Next, the measured m / z values ​​of the identified peaks are sequentially compared with theoretical m / z values ​​calculated based on the amino acid sequences of Asr of various bacteria (which are known from DNA sequence information for many bacteria) to find matches, and the bacteria are identified and / or classified.

[0033] The problem with this is that identifying and / or classifying bacteria means that the classification of the bacteria is unknown, and it is impossible to know in advance the mass spectrum peaks attributable to Asr of bacteria of unknown classification. For this reason, as disclosed in Patent Document 1, the mass spectrum under conditions in which Asr is not produced is compared with the mass spectrum under conditions in which Asr is produced, and peaks seen in both are excluded to identify peaks attributable to Asr.

[0034] However, the conventional method described above required culturing under two conditions, one in which Asr is produced and the other in which it is not produced, and the work involved in preparing the culture medium, culturing, preparing samples for mass spectrometry, and measuring required approximately twice as much effort as when culturing and measuring under a single condition.

[0035] [3. Method for Identifying Asr According to an Embodiment] The inventors have discovered a natural phenomenon underlying the method for identifying Asr according to an embodiment: Asr fragments are generated by cleavage of Asr at specific amino acid sequences within the cells of microorganisms belonging to the Enterobacteriaceae, Eriuniaceae, Pectobacteriaceae, Yersinaceae, and Hafininiaceae families. Herein, "Asr fragments" refer to fragments generated by cleavage of Asr within the cells. The inventors have also discovered a phenomenon in which peaks corresponding to Asr and its fragments (hereinafter also referred to as "peaks derived from Asr") are not attenuated by dilution, compared to other peaks derived from ribosomal proteins and the like. According to the inventors' research, this phenomenon is thought to reflect the characteristics of Asr, which is a protein with a large number of molecules in cells but is susceptible to the effects of ion suppression. Taking advantage of this phenomenon, the inventors have developed the following method for easily identifying peaks derived from Asr. Hereinafter, when simply referring to a "method for identifying Asr," this includes the "method for identifying peaks derived from Asr."

[0036] 2 is a flowchart showing a method for specifying Asr and a method for identifying microorganisms according to an embodiment. Some of the steps shown in FIG. 2 are performed manually by an analyst using laboratory equipment and devices commonly used for culturing microorganisms and mass spectrometry. Other parts of the steps shown in FIG. 2 are performed by the control unit 10. In the figure, "S" is used as an abbreviation for "STEP."

[0037] In steps S1 to S3, the analyst prepares a first solution containing Asr produced by a predetermined type of microorganism at a first concentration and a second solution containing Asr produced by the predetermined type of microorganism at a second concentration lower than the first concentration. Note that the Asr described in the above description of steps S1 to S3 includes fragmented Asr. In one embodiment, this process is performed as follows.

[0038] In S1, the analyst cultures a predetermined type of microorganism under conditions (hereinafter also referred to as "specific conditions") that allow the predetermined type of microorganism to produce Asr.

[0039] In S2, the analyst lyses the cultured microorganism to prepare a lysate, and in S3, the analyst prepares a first solution and a second solution from the lysate.

[0040] In S4, the analyst uses the analytical device 1 to measure a first mass spectrum and a second mass spectrum corresponding to the first solution and the second solution, respectively.

[0041] In steps S5 to S7, the control unit 10 compares the first mass spectrum with the second mass spectrum and determines specific peaks corresponding to Asr and / or fragments thereof in each mass spectrum. More specifically, the control unit 10 determines the specific peaks depending on the degree to which the peak intensities in the second mass spectrum are maintained.

[0042] In S5, the control unit 10 identifies a first peak and a second peak that correspond to a common m / z in the first mass spectrum and the second mass spectrum, respectively.

[0043] In S6, the control unit 10 obtains the first intensity of the first peak and the second intensity of the second peak.

[0044] In S7, the control unit 10 determines that the first peak and the second peak are specific peaks if the ratio of the second intensity to the first intensity is equal to or greater than a specific threshold value.

[0045] In S8, the control unit 10 searches for microorganisms that produce proteins corresponding to the m / z of the specific peak, and identifies the predetermined type of microorganism as the microorganism obtained as a result of the search.

[0046] According to the process shown in Figure 2, in order to identify peaks corresponding to Asr and / or its fragments, culturing under two or more conditions can be simplified to culturing under one condition. Therefore, peaks corresponding to Asr and / or its fragments produced by microorganisms can be easily identified. Furthermore, microorganisms can be easily identified based on the peaks corresponding to Asr and / or its fragments.

[0047] Each step in FIG. 2 will be described in detail below. In S1, the predetermined type of microorganism is, for example, one type (one strain) of microorganisms among types of microorganisms expected to have the Asr gene, and a more specific example is one of the microbial strains belonging to the order Enterobacteriaceae. It has been revealed that many microbial strains in the order Enterobacteriaceae have the Asr gene. However, the Asr specification method and microbial identification method according to this embodiment may also be performed on microorganisms whose presence or absence of the Asr gene is unknown.

[0048] In step S1, the specific conditions include at least one of the following: a condition in which a reagent for acidifying the culture medium is added to the culture medium for culturing the organism; a condition in which sugar is added to the culture medium for culturing the microorganism; a condition in which the microorganism is over-cultured; and a condition in which the microorganism is cultured under anaerobic conditions. The culture medium may be a liquid or solid medium. The specific conditions for producing Asr are culture conditions that the inventors have confirmed through many years of research, and that allow microorganisms to easily produce Asr. The condition of a culture medium containing added sugars can be satisfied, for example, by adding sugars when preparing the culture medium in step S1. The sugars added to the culture medium are not particularly limited, but monosaccharides, disaccharides, trisaccharides, and tetrasaccharides are preferred. However, sugars obtained by combining five or more monosaccharides may also be used. The monosaccharides added to the medium include, for example, at least one of allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fucose, fuculose, rhamnose, psicose (also called allulose), fructose, sorbose, tagatose, ribose, arabinose, xylose, lyxose, ribulose, xylulose, deoxyribose, sedoheptulose, ketotetrose, erythrulose, aldotetrose, erythrose, threose, ketotriose (dihydroxyacetone), and aldotriose (glyceraldehyde). Erythrulose is preferred as the ketotetrose. Erythrose or threose is preferred as the aldotetrose. One of these monosaccharides may be added, or multiple types of sugars may be used in combination. The disaccharide added to the medium is, for example, at least one of sucrose, lactose, maltose, trehalose, turanose, and cellobiose, preferably lactose. The trisaccharide added to the medium is, for example, at least one of raffinose, melezitose, and maltotriose. The tetrasaccharide added to the medium is, for example, at least one of acarbose and stachyose. Since sugar-metabolizing microorganisms metabolize glucose, it is more preferable that the sugar is glucose.More specifically, among sugar-metabolizing microorganisms, there are microorganisms that metabolize only glucose and not other sugars, but no microorganisms are known that metabolize sugars other than glucose but not glucose. Therefore, by using a glucose-containing medium when culturing an unknown microorganism, if the unknown microorganism is capable of metabolizing sugars to produce Asr, Asr can be produced almost reliably. This eliminates the need to determine the type of sugar metabolized by the unknown microorganism. Furthermore, the range of sugar concentration added to the medium is preferably 0.1 wt% or more, more preferably 0.5 wt% or more. As used herein, "overculture" refers to culturing for a specific period longer than the period typically used for culturing microorganisms in experiments. However, the normal culture period and the specific period for overculture may vary depending on the microorganism. Therefore, the condition for overculture may vary depending on the microorganism, but for example, the condition can be met by culturing the microorganism used in the experiment for two nights, after culturing it overnight. More specifically, the condition can be met by culturing the microorganism used in the experiment for approximately 36 to 42 hours, after culturing it for approximately 12 to 18 hours. The condition of an anaerobic state is usually satisfied by culturing the bacteria at an oxygen concentration of 5% or less (preferably 1% or less), for example.

[0049] In S2, the analyst disrupts the bacterial cells, for example, by adding an acid. In one embodiment, the analyst centrifuges the bacteria cultured in a liquid medium, washes them, and then adds trifluoroacetic acid (TFA) to lyse them. The supernatant obtained by centrifugation after lysis is used as the lysate. The lysate prepared in this manner contains proteins such as Asr and ribosomal proteins. The method for preparing the lysate is not limited to the above example; for example, the analyst may lyse the bacteria using a reagent that disrupts cell walls, such as lysozyme.

[0050] In S3, for example, the first solution is a low-dilution sample of a lysate solution, which is an undiluted sample, and the second solution is a high-dilution sample of the lysate solution. The diluent used to dilute the lysate solution can be, for example, water or 50% acetonitrile. For example, the first solution is a solution obtained by diluting a lysate solution extracted from a culture solution in which bacteria have grown to a turbidity of about 1 using the method described in the Experimental Examples below at a ratio of 1 / 1 to 1 / 2. The first solution may be the undiluted lysate solution. The second solution is, for example, a solution obtained by diluting a lysate solution extracted from a culture solution in which bacteria have grown to a turbidity of about 1 using the method described in the Experimental Examples below at a ratio of 1 / 100 to 1 / 500. In one embodiment, each of the first solution and the second solution is prepared so that the second concentration is 1 / 50 to 1 / 500 of the first concentration. By utilizing the concentration difference between the first solution and the second solution as described above, peaks of Asr and its fragments can be clearly detected. Furthermore, when the first and second solutions are prepared by diluting a single lysate solution as described above, the time and effort required for preparing the culture medium and the steps from culturing the bacteria to preparing the lysate solution can be halved compared to when the lysate solutions are prepared by culturing the bacteria under two different conditions. In one embodiment, the second solution may be prepared by diluting the first solution.

[0051] However, it is of course possible to prepare separate lysates for bacteria cultured under specific conditions in one medium. In this case, the effort required for preparing the medium and culturing the bacteria is halved compared to culturing bacteria in two media under different conditions. It is also possible to prepare a first solution and a second solution for each of the bacteria cultured under specific conditions in two media. In this case, it is also possible to control the culture conditions less time-consuming than culturing two media under different conditions. Thus, the methods for preparing the first solution and the second solution are not particularly limited and may be changed as long as the effects of the Asr identification method and the microbial identification method according to this embodiment are achieved.

[0052] In S4, the analyzer 1 measures the first mass spectrum and the second mass spectrum, for example, by matrix-assisted laser desorption / ionization (MALDI). As an ionization method, any soft ionization method, such as electrospray ionization (ESI), can be used in addition to MALDI. However, MALDI is superior to ESI in that it allows measurement with a simpler sample preparation method. For example, because the supernatant obtained by lysing bacteria with acid as described above contains impurities, the supernatant is unsuitable for ionization by ESI directly, and it is more preferable to ionize the supernatant by MALDI. When ionization by ESI is performed, it is preferable to further include a liquid chromatograph in the analyzer 1, and to configure the ionization unit 21 to ionize substances in the sample separated by the liquid chromatograph, since this can achieve high separation ability.

[0053] In S5 to S7, the degree of peak intensity maintenance is a degree that is appropriately set to determine a specific peak depending on the experimental conditions (such as the dilution ratios of the first solution and the second solution) and the calculation method for the degree of peak intensity maintenance. An example of a calculation method and numerical examples for the degree of peak intensity maintenance are shown below, but these are merely examples, and the calculation method and numerical values ​​for the degree of peak intensity maintenance are not limited to the following example calculation method and numerical example.

[0054] In S6 and S7, the first intensity and the second intensity may be absolute intensities or relative intensities, as long as the rate of attenuation in the second mass spectrum can be calculated using the same standard for multiple peaks included in the first mass spectrum. For example, the first intensity and the second intensity may be absolute intensities, and the control unit 10 may determine whether the ratio of the absolute intensities is equal to or greater than a specific threshold. The specific threshold for the ratio of the absolute intensities is, for example, a single numerical value between 0.072 and 0.12. Alternatively, the first intensity may be a relative intensity when the maximum peak in the first mass spectrum is set to 1, and the second intensity may be a relative intensity when the maximum peak in the second mass spectrum is set to 1, and the control unit 10 may determine whether the ratio of the relative intensities is equal to or greater than a specific threshold. Alternatively, as shown in an experimental example described below, the first intensity and the second intensity may each be a relative intensity when the maximum peak in the first mass spectrum is set to 1, and the control unit 10 may determine whether the ratio of the relative intensities is equal to or greater than a specific threshold. The specific threshold value for the ratio of the relative intensities is, for example, a single numerical value determined between 0.072 and 0.12. As described above, the control unit 10 can automatically and quantitatively detect the specific peak based on the ratio between the first intensity and the second intensity.

[0055] As another example, in the second mass spectrum, if the maximum peak in the second mass spectrum is defined as 1, a peak whose relative intensity is equal to or greater than "a single value between 0.06 and 0.38" may be detected as a specific peak. This allows peaks that maintain their peak intensity even at high dilutions to be detected as specific peaks. In this case, it is preferable to confirm that the specific peak is also detected in the first mass spectrum. This confirms that the specific peak is a peak that is present even in the low dilution solution, and therefore is due to a cell-derived component, rather than due to a foreign substance introduced during the preparation of the highly dilution solution.

[0056] The determination of specific peaks in steps S5 to S7 based on the degree of attenuation of the peaks in the second mass spectrum may be performed visually by an analyst. For example, the first mass spectrum and the second mass spectrum may be displayed on the output unit 132, and the analyst may visually determine peaks in the second mass spectrum that are less attenuated as specific peaks.

[0057] In S8, the control unit 10 refers to the theoretical m / z corresponding to the amino acid sequence of Asr and / or its fragments produced by microorganisms belonging to a specified classification, and identifies microorganisms whose theoretical m / z corresponds to the m / z of a specific peak.

[0058] In the first example of S8, the control unit 10 accesses a database containing theoretical m / z values ​​of Asr and / or its fragments produced by microorganisms belonging to the order Enterobacteriaceae, and identifies the microorganisms by searching the database for microorganisms whose theoretical m / z values ​​of Asr and / or its fragments correspond to the m / z values ​​of specific peaks.

[0059] In a second example of S8, the control unit 10 first determines a specific classification of a predetermined type of microorganism based on the m / z of a peak other than the specific peak using the first mass spectrum. Then, the control unit 10 searches for microorganisms in the specific classification that produce proteins corresponding to the m / z of the specific peak. Peaks other than the specific peak are, for example, peaks derived from proteins other than Asr. The specific classification is, for example, at the genus level. For example, the control unit 10 determines the genus level classification using fingerprinting, using ribosomal protein peaks in the first mass spectrum (see Figure 3, described below). Note that fingerprinting is a method for determining the classification of microorganisms based on the pattern of peaks in a mass spectrum. Next, the control unit 10 searches for microorganisms in the determined genus level classification that produce proteins corresponding to the m / z of the specific peak, which is a peak derived from Asr, and identifies the microorganisms at, for example, the species level (or a more detailed level). In this specification, this two-stage identification method, in which a rough classification is determined by the conventional fingerprint method and a detailed classification is determined based on the m / z of a specific peak derived from Asr, is referred to as a two-stage identification method.

[0060] It has been reported that conventional fingerprinting methods can distinguish microorganisms up to the genus level but not at the species level. Even in such cases, the use of a two-stage identification method may enable identification at the species level. As mentioned above, Asr fragments in many microorganisms, which has the advantage of having a small molecular weight and making it easy to measure by mass spectrometry. Specifically, while the molecular weight of ribosomal proteins is approximately 4,000 or more, Asr fragments have a molecular weight of approximately 1,000 to 4,000. Therefore, Asr fragment peaks are likely to be clearly detected in mass spectra (in other words, have sufficient intensity for identification). Therefore, compared to identifying microorganisms using only fingerprinting methods based on peaks of ribosomal proteins, etc., the use of a two-stage identification method is likely to enable more accurate identification down to a more detailed classification and / or with higher accuracy.

[0061] Furthermore, the identification of microorganisms based solely on peaks derived from Asr, as shown in the first example of S8, can only be applied to Enterobacteriaceae that have the Asr gene. Therefore, although the two-stage identification method is more general, it has the advantage of being more widely applicable.

[0062] Furthermore, the above-mentioned example of using only the conventional fingerprinting method or the first example of S8 uses information on a single protein, ribosomal protein or Asr, respectively, whereas the two-stage identification method uses multiple pieces of information, ribosomal protein and Asr, and is therefore considered to be more accurate.

[0063] 2 may be mechanized or computer-controlled as appropriate. In this case, known techniques required for each operation may be used as appropriate.

[0064] As described above, the method for identifying Asr according to the present embodiment allows for identification of peaks derived from Asr with fewer steps than conventional methods in which microorganisms are cultured separately under conditions in which Asr is cultured and conditions in which Asr is not cultured, and peaks derived from Asr are identified. Therefore, mass spectrum peaks of Asr and / or its fragments produced by microorganisms can be easily identified.

[0065] [4. Experimental Example] An experimental example using the Asr specification method and microorganism identification method according to the embodiment will be described below. In this experimental example, Escherichia coli K12 strain was used as the sample strain. However, since the classification of the sample strain is unknown in the actual identification, it will be referred to as microorganism A in the following description.

[0066] To produce Asr, microorganism A was inoculated into 10 mL of IFO804 medium (0.5% glucose, 0.5% hypopeptone, 0.5% yeast extract, and 0.1% magnesium sulfate heptahydrate) and cultured overnight at 37°C under aerobic conditions.

[0067] Next, 1 mL of the bacterial suspension obtained as a result of the culture was centrifuged (15,000 g, 5 minutes) to obtain a sediment. This sediment was resuspended in 1 mL of purified water and centrifuged again to obtain a sediment. Purified water was added to this sediment so that the turbidity became 1, and the resuspended sediment was then centrifuged in 0.5 mL. 50 μL of 1% TFA was added to the obtained sediment, which was then resuspended and centrifuged. The supernatant obtained after centrifugation was used as a lysate.

[0068] Next, 10 μL of the resulting lysate was mixed with 10 μL of acetonitrile to prepare a first solution (low dilution) at a dilution ratio of 1 / 2, and 1 μL of the first solution was mixed with 199 μL of 50% acetonitrile aqueous solution to prepare a second solution (high dilution) at a dilution ratio of 1 / 400.

[0069] Next, 1 μL of 5 mg / mL CHCA was dropped into two wells of a MALDI target plate (sample plate) and dried to a solid. Then, 1 μL each of the first solution and the second solution was dropped into each of the two wells and dried to a solid, and these were used as samples for MALDI measurement. These samples were measured using a mass spectrometer (MALDI-8020, manufactured by Shimadzu Corporation). The first mass spectrum corresponding to the first solution and the second mass spectrum corresponding to the second solution obtained as a result of the measurement are shown in FIG. 3.

[0070] Comparing the first and second mass spectra in FIG. 3 , numerous distinct peaks were observed in the first mass spectrum. More quantitatively, 11 peaks with relative intensities of 10% or more were detected in the first mass spectrum, with the maximum peak intensity in the first mass spectrum taken as 100%. On the other hand, only three distinct peaks were observed in the second mass spectrum: m / z 2372.5, m / z 2402.9, and m / z 3824.1. In the second mass spectrum, these three peaks were the only peaks with relative intensities of 10% or more, with the maximum peak intensity in the second mass spectrum taken as 100%. Even with high dilution, these three peaks maintained their relative intensities and were clearly distinguishable from other peaks. Therefore, it was possible to determine that these three peaks were specific peaks derived from Asr.

[0071] Next, we verified whether these peaks were actually specific peaks derived from Asr. Figure 4 is a table showing the amino acid sequences of the Asr fragments of the E. coli K12 strain and the theoretical m / z values ​​calculated based on the sequences.

[0072] The m / z values ​​(measured m / z values) of the three peaks clearly observed in the second mass spectrum of Figure 3 agreed with the theoretical m / z values ​​of the Asr fragments of the E. coli K12 strain shown in Figure 4, with a small error of within 500 ppm. Therefore, the microorganism A was identified as the E. coli K12 strain. Furthermore, with reference to Figure 3, it was confirmed that the determination that the above three peaks were specific peaks derived from Asr was correct.

[0073] Next, we demonstrated that a specific peak originating from Asr can be quantitatively determined. Figure 5 is a table showing the intensities of peaks corresponding to a common m / z in the first and second mass spectra. Figure 5 shows the m / z and relative intensities of 11 peaks in the first mass spectrum whose relative intensities are 0.1 (10%) or greater. It also shows the m / z and relative intensities of peaks in the second mass spectrum corresponding to these 11 peaks. Note that in Figure 5, the relative intensities of peaks in the first mass spectrum and the second mass spectrum are both relative intensities when the intensity of the highest peak (m / z 3823.3) in the first mass spectrum is set to 1.0. Figure 5 also shows the ratio of the relative intensities of peaks in the second mass spectrum to the relative intensities of peaks in the first mass spectrum corresponding to the common m / z. As a result, the relative intensity ratios of the peaks originating from Asr were all 0.120 or greater, while the relative intensity ratios of peaks originating from other proteins (e.g., ribosomal proteins) were 0.072 or less. Therefore, in the method for identifying Asr according to the embodiment, when the ratio of the relative intensities is equal to or greater than a predetermined threshold value (e.g., 0.1) set between 0.072 and 0.12, the peak is determined to be derived from Asr, and it has been shown that the peak of Asr can be easily detected.

[0074] In the above example, the Asr peak is detected based on the ratio of relative intensities, but it is also possible to calculate the ratio of the absolute intensity of the peak in the second mass spectrum to the absolute intensity of the peak in the first mass spectrum for a given m / z, and detect the Asr peak based on this absolute intensity ratio. In other words, the Asr peak can be simply detected by determining that the peak is derived from Asr when this absolute intensity ratio is equal to or greater than a given threshold value (a single number between 0.072 and 0.12).

[0075] Furthermore, in the above, referring to FIG. 3 , peaks having a relative intensity of 10% or more when the maximum peak intensity in the second mass spectrum is taken as 100% were determined to be specific peaks derived from Asr. However, since the relative intensity of the smallest Asr peak (m / z 3823.3) in the second mass spectrum is 38.1% or more and the relative intensity of the largest ribosome peak (m / z 4365.3) is 5.8% or less, it is also possible to determine a peak having a relative intensity of "a single numerical value between 6% and 38%" when the maximum peak intensity in the second mass spectrum is taken as 100% as a specific peak derived from Asr.

[0076] As described above, in this experimental example, two types of diluted solutions with different dilution rates were prepared from a lysate prepared from a microorganism cultured under specific conditions. It was then visually and numerically demonstrated that, unlike other proteins such as other ribosomes, Asr maintains a relatively high peak intensity even at high dilutions. In other words, the specific peak derived from Asr could be distinguished from peaks derived from other proteins based on its intensity. Therefore, it was demonstrated that the Asr identification method according to this embodiment is a method that can easily identify Asr peaks.

[0077] Aspects It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0078] (Item 1) A method for identifying an acid shock protein according to one embodiment includes the steps of: (i) preparing a first solution containing an acid shock protein produced by a predetermined type of microorganism at a first concentration; and (ii) preparing a second solution containing the acid shock protein produced by the predetermined type of microorganism at a second concentration lower than the first concentration; (ii) measuring a first mass spectrum and a second mass spectrum corresponding to the first solution and the second solution, respectively; and (iii) comparing the first mass spectrum with the second mass spectrum and determining a specific peak in each mass spectrum corresponding to the acid shock protein depending on the degree of decrease in the peak in the second mass spectrum.

[0079] According to the method for identifying an acid shock protein described in paragraph 1, peaks derived from an acid shock protein can be identified with less work than conventional methods in which microorganisms are cultured separately under conditions for culturing the acid shock protein and conditions for not culturing the acid shock protein, and peaks derived from the acid shock protein are identified. Therefore, mass spectrum peaks of the acid shock protein and / or fragments thereof produced by the microorganism can be easily identified.

[0080] (Item 2) In the method for identifying an acid shock protein described in item 1, the step of determining a specific peak includes the steps of identifying a first peak and a second peak corresponding to a common m / z in each of the first mass spectrum and the second mass spectrum, acquiring a first intensity of the first peak and a second intensity of the second peak, and determining that the first peak and the second peak are specific peaks if the ratio of the second intensity to the first intensity is equal to or greater than a specific threshold.

[0081] According to the method for identifying acid shock proteins described in Section 2, peaks derived from acid shock proteins can be identified automatically.

[0082] (Item 3) In the method for identifying an acid shock protein according to Items 1 or 2, the preparing step includes culturing a predetermined type of microorganism under specific conditions, preparing a lysate by lysing the cultured microorganism, and preparing a first solution and a second solution from the lysate. The specific conditions are at least one of a condition in which a reagent for acidifying the medium is added to the medium in which the predetermined type of microorganism is cultured, a condition in which sugar is added to the medium in which the predetermined type of microorganism is cultured, a condition in which the microorganism is overcultured, and a condition in which the microorganism is cultured under anaerobic conditions.

[0083] According to the method for identifying an acid shock protein described in item 3, it is possible to easily induce a microorganism to produce an acid shock protein, and therefore, it is possible to more easily identify a peak derived from the acid shock protein.

[0084] (Item 4) In the method for identifying an acid shock protein according to item 3, the sugar is glucose.

[0085] Microorganisms that metabolize sugars metabolize glucose. Therefore, the method for identifying acid shock proteins described in Section 4 can eliminate the need to investigate the types of sugars metabolized by unknown microorganisms.

[0086] (Item 5) In the method for identifying an acid shock protein according to any one of items 1 to 4, the second concentration is 1 / 50 to 1 / 500 of the first concentration.

[0087] According to the method for identifying acid shock proteins described in item 5, by utilizing the difference in concentration between the first solution and the second solution as described above, peaks derived from acid shock proteins can be clearly detected.

[0088] (Item 6) In the method for identifying an acid shock protein according to any one of items 1 to 5, the measuring step includes measuring a first mass spectrum and a second mass spectrum by matrix-assisted laser desorption / ionization mass spectrometry.

[0089] According to the method for identifying acid shock proteins described in item 6, measurement can be performed using a simpler sample preparation method than when the ESI method is used.

[0090] (Item 7) A method for identifying microorganisms, which comprises searching for microorganisms that produce proteins corresponding to the m / z of a specific peak determined by the method for identifying acid shock proteins described in any one of Items 1 to 6, and identifying a specific type of microorganism as the microorganism obtained as a result of the search.

[0091] According to the method for identifying a microorganism described in item 7, it is possible to easily identify a microorganism based on a peak derived from an acid shock protein.

[0092] (Item 8) In the method for identifying microorganisms described in Item 7, the identifying step includes a step of determining a specific classification of a predetermined type of microorganism based on the m / z of peaks other than the specific peak using the first mass spectrum, and a step of searching for a microorganism that produces a protein corresponding to the m / z of the specific peak from among the microorganisms in the specific classification.

[0093] According to the method for identifying microorganisms described in paragraph 8, it is highly likely that microorganisms can be identified accurately down to a more detailed classification and / or with higher accuracy than when identifying microorganisms using only fingerprinting methods based on peaks of ribosomal proteins, etc.

[0094] 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.

[0095] 1 Analysis 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, 100 Analysis system, 111 Device control unit, 112 Mass spectrum creation unit, 113 Mass spectrum analysis unit, 114 Calibration unit, 131 Input unit, 132 Output unit, 133 Communication unit, 221 Acceleration electrode, 231 Flight tube, S ion.

Claims

1. A method for identifying an acid shock protein, comprising the steps of: preparing a first solution containing an acid shock protein produced by a predetermined type of microorganism at a first concentration; and a second solution containing the acid shock protein produced by the predetermined type of microorganism at a second concentration lower than the first concentration; measuring a first mass spectrum and a second mass spectrum corresponding to the first solution and the second solution, respectively; and comparing the first mass spectrum with the second mass spectrum to determine a specific peak in each mass spectrum corresponding to the acid shock protein, depending on the degree to which the peak intensity in the second mass spectrum is maintained.

2. The method for identifying an acid shock protein according to claim 1, wherein the step of determining a specific peak comprises the steps of: identifying a first peak and a second peak corresponding to a common m / z in the first mass spectrum and the second mass spectrum, respectively; acquiring a first intensity of the first peak and a second intensity of the second peak; and determining that the first peak and the second peak are specific peaks if a ratio of the second intensity to the first intensity is equal to or greater than a specific threshold.

3. The method for identifying an acid shock protein according to claim 1 or 2, wherein the preparing step includes the steps of culturing the predetermined type of microorganism under specific conditions, lysing the cultured microorganism to prepare a lysate, and preparing the first solution and the second solution from the lysate, and the specific conditions are at least one of a condition in which a reagent that acidifies the medium is added to the medium in which the predetermined type of microorganism is cultured, a condition in which sugar is added to the medium in which the predetermined type of microorganism is cultured, a condition in which the microorganism is overcultured, and a condition in which the microorganism is cultured under anaerobic conditions.

4. The method for identifying an acid shock protein according to claim 3, wherein the sugar is glucose.

5. A method for identifying an acid shock protein according to claim 1 or 2, wherein the second concentration is 1 / 50 to 1 / 500 of the first concentration.

6. The method for identifying an acid shock protein according to claim 1 or 2, wherein the measuring step includes measuring the first mass spectrum and the second mass spectrum by matrix-assisted laser desorption / ionization mass spectrometry.

7. A method for identifying microorganisms, which comprises searching for microorganisms that produce proteins corresponding to the m / z of a specific peak determined by the method for identifying acid shock proteins described in claim 1 or 2, and identifying the microorganisms of the specified type as the microorganisms obtained as a result of the search.

8. A method for identifying microorganisms as described in claim 7, wherein the identifying step includes: using the first mass spectrum to determine a specific classification of the predetermined type of microorganism based on the m / z of peaks other than the specific peak; and searching for microorganisms in the specific classification that produce proteins corresponding to the m / z of the specific peak.

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