Method for analyzing microorganisms

Abstract

In a method for analyzing a microorganism using a matrix assisted laser desorption/ionization mass spectrometer, a matrix-and-additive mixture solution prepared by mixing one or both of an alkylphosphonic acid and a surfactant with a matrix substance is used for matrix assisted laser desorption/ionization. Either an alkylphosphonic acid or a surfactant, or both of them are used as matrix additives and are mixed with the matrix substance beforehand to prepare a matrix-and-additive mixture solution. After a solution which contains a microorganism to be analyzed has been dropped onto a sample plate, the matrix-and-additive mixture solution is dropped onto that solution and dried to form a mixed crystal which contains both the constituents of the microorganism and the matrix substance. This crystal is used as a sample for MALDI-MS analysis. The sensitivity of analysis is thereby improved, without increasing the amount of time and labor required for sample preparation.

Claims

1. A method for analyzing a microorganism using a matrix assisted laser desorption/ionization mass spectrometer, wherein the method comprises forming a mixture of a matrix-and-additive mixture solution and a test microorganism, wherein the matrix-and-additive mixture solution comprises a) an alkylphosphonic acid and/or a surfactant, and b) a matrix substance, wherein the mixture is formed by preparing a matrix-microorganism mixture comprising the matrix-and-additive mixture solution and the test microorganism, and then dropping the matrix-microorganism mixture on a sample plate, and analyzing the mixture comprising the test microorganism in a matrix assisted laser desorption/ionization spectrometer.

2. The method for analyzing a microorganism according to claim 1, wherein the alkylphosphonic acid is methylenediphosphonic acid.

3. The method for analyzing a microorganism according to claim 2, wherein the surfactant is decyl-β-D-maltopyranoside.

4. The method for analyzing a microorganism according to claim 3, wherein the matrix substance is sinapinic acid.

5. The method for analyzing a microorganism according to claim 2, wherein the matrix substance is sinapinic acid.

6. The method for analyzing a microorganism according to claim 1, wherein the surfactant is decyl-β-D-maltopyranoside.

7. The method for analyzing a microorganism according to claim 6, wherein the matrix substance is sinapinic acid.

8. The method for analyzing a microorganism according to claim 1, wherein the matrix substance is sinapinic acid.

9. The method for analyzing a microorganism according to claim 8, wherein sinapinic acid is present in an amount of 10 to 30 mg/mL, the alkylphosphonic acid is 0.1 to 5% of methylenediphosphonic acid and the surfactant is 0.1 to 10 mM of decyl-β-D-maltopyranoside in an aqueous solution containing 30 to 70% of acetonitrile and 0.1 to 3% of trifluoroacetic acid.

10. The method for analyzing a microorganism according to claim 1, wherein the matrix substance is present in an amount of 10 to 30 mg/mL, the alkylphosphonic acid is 0.1 to 5% of methylenediphosphonic acid and the surfactant is 0.1 to 10 mM of decyl-β-D-maltopyranoside in an aqueous solution containing 30 to 70% of acetonitrile and 0.1 to 3% of trifluoroacetic acid.

11. The method for analyzing a test microorganism according to claim 1, further comprising steps of: obtaining a mass spectrum for a sample containing the test microorganism, the mass spectrum covering a mass range including a high-mass range of m/z 10000 or higher in mass-to-charge ratio; comparing the mass spectrum with mass-spectrum patterns stored in a database; and identifying the test microorganism based on a result obtained by comparing the mass spectrum with the mass-spectrum patterns.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a chart showing a mass-to-charge-ratio range of m/z 7000 to 20000 in a first set of mass spectra obtained in the first example in which Escherichia coli (E. coli DH5α Electro-Cells) was used as the test microorganism.

(2) FIG. 2 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 40000 in the first set of mass spectra obtained in the first example in which Escherichia coli (E. coli DH5α Electro-Cells) was used as the test microorganism.

(3) FIG. 3 is a chart showing a mass-to-charge-ratio range of m/z 7000 to 20000 in a second set mass spectra obtained in the first example in which Escherichia coli (E. coli DH5α Electro-Cells) was used as the test microorganism.

(4) FIG. 4A is a chart showing a mass-to-charge-ratio range of m/z 20000 to 33000 in the second set of mass spectra obtained in the first example in which Escherichia coli (E. coli DH5α Electro-Cells) was used as the test microorganism, with all mass spectra having the same intensity scale on their vertical axes.

(5) FIG. 4B is a chart showing a mass-to-charge-ratio range of m/z 20000 to 33000 in the second set of mass specta obtained in the first example in which Escherichia coli (E. coli DH5α Electro-Cells) was used as the test microorganism, with each mass spectrum having a vertical axis which indicates relative intensities.

(6) FIG. 5 is a chart showing a mass-to-charge-ratio range of m/z 9000 to 20000 in mass spectra obtained in the second example in which Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) was used as the test microorganism.

(7) FIG. 6A is a chart showing a mass-to-charge-ratio range of m/z 20000 to 34000 in the mass spectra obtained in the second example in which Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) was used as the test microorganism, with all mass spectra having the same intensity scale on their vertical axes.

(8) FIG. 6B is a chart showing a mass-to-charge-ratio range of m/z 20000 to 34000 in the mass spectra obtained in the second example in which Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) was used as the test microorganism, with each mass spectrum having a vertical axis which indicates relative intensities.

(9) FIG. 6C is a chart showing a mass-to-charge-ratio range of m/z 40000 to 90000 in the mass spectra obtained in the second example in which Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) was used as the test microorganism, with each mass spectrum having a vertical axis which indicates relative intensities.

(10) FIG. 7 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 40000 in the mass spectra obtained in the second example in which Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) was used as the test microorganism.

(11) FIG. 8 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 34000 in the mass spectra obtained in the second example in which Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) was used as the test microorganism.

(12) FIG. 9 is a chart showing a mass-to-charge-ratio range of m/z 40000 to 90000 in the mass spectra obtained in the second example in which Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) was used as the test microorganism.

(13) FIG. 10 is a chart showing a mass-to-charge-ratio range of m/z 10000 to 20000 in mass spectra obtained in the third example in which Salmonella enterica (serotype: Typhimurium, NBRC 13245) was used as the test microorganism.

(14) FIG. 11 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 40000 in the mass spectra obtained in the third example in which Salmonella enterica (serotype: Typhimurium, NBRC 13245) was used as the test microorganism.

(15) FIG. 12 is a chart showing a mass-to-charge-ratio range of m/z 40000 to 90000 in the mass spectra obtained in the third example in which Salmonella enterica (serotype: Typhimurium, NBRC 13245) was used as the test microorganism.

(16) FIG. 13 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 40000 in the mass spectra obtained in the third example in which Salmonella enterica (serotype: Typhimurium, NBRC 13245) was used as the test microorganism.

(17) FIG. 14 is a chart showing a mass-to-charge-ratio range of m/z 40000 to 90000 in the mass spectra obtained in the third example in which Salmonella enterica (serotype: Typhimurium, NBRC 13245) was used as the test microorganism.

(18) FIG. 15 is a chart showing a mass-to-charge-ratio range of m/z 9000 to 20000 in mass spectra obtained in the fourth example in which Salmonella enterica (serotype: Orion, jfrlSe 1402-15) was used as the test microorganism.

(19) FIG. 16 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 40000 in the mass spectra obtained in the fourth example in which Salmonella enterica (serotype: Orion, jfrlSe 1402-15) was used as the test microorganism.

(20) FIG. 17 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 34000 in the mass spectra obtained in the fourth example in which Salmonella enterica (serotype: Orion, jfrlSe 1402-15) was used as the test microorganism.

(21) FIG. 18 is a chart showing a mass-to-charge-ratio range of m/z 38000 to 90000 in the mass spectra obtained in the fourth example in which Salmonella enterica (serotype: Orion, jfrlSe 1402-15) was used as the test microorganism.

(22) FIG. 19 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 40000 in the mass spectra obtained in the fourth example in which Salmonella enterica (serotype: Orion, jfrlSe 1402-15) was used as the test microorganism.

(23) FIG. 20 is a chart showing a mass-to-charge-ratio range of m/z 20000 to 34000 in the mass spectra obtained in the fourth example in which Salmonella enterica (serotype: Orion, jfrlSe 1402-15) was used as the test microorganism.

(24) FIG. 21 is a chart showing a mass-to-charge-ratio range of m/z 40000 to 90000 in the mass spectra obtained in the fourth example in which Salmonella enterica (serotype: Orion, jfrlSe 1402-15) was used as the test microorganism.

DESCRIPTION OF EMBODIMENTS

(25) In the method for analyzing a microorganism according to the present invention, a sample which contains constituents of a microorganism to be analyzed (which is hereinafter called the “test microorganism”) is prepared. With this sample set in a MALDI-MS, a mass spectrometric analysis is performed to obtain a mass spectrum. The obtained mass spectrum is compared with mass-spectrum patterns stored in a database, whereby the kind of test microorganism (i.e. family, genus, species, subspecies, pathotype, serotype, strain or similar group to which the microorganism belongs) can be identified. As will be described later in detail, the method for analyzing a microorganism according to the present invention particularly allows for high-sensitivity acquisition of information concerning the molecular weight for a microorganism within a high-mass range of m/z 10000 or higher in mass-to-charge ratio in the obtained mass spectrum. This is suitable for identifying a microorganism for which a useful marker peak for the identification of the test microorganism is present within the high-mass range.

(26) As the MALDI-MS, a MALDI time-of-flight mass spectrometer (MALDI-TOFMS) may preferably be used. MALDI-TOFMS can perform measurements over an extremely wide range of mass-to-charge ratios and yield mass spectra which are suitable for an analysis of high-mass molecules, such as proteins which constitute a microorganism.

(27) The constituents of the test microorganism may be a cell extract or purified cell constituents, such as ribosomal proteins, obtained from the cell extract. Fungus bodies or cell suspension may also be directly used in their original form.

(28) In the method for analyzing a microorganism according to the present invention, a matrix-and-additive mixture solution is prepared by mixing an alkylphosphonic acid or/and a surfactant as matrix additives with the matrix substance beforehand. This matrix-and-additive mixture solution is used for the preparation of samples for MALDI-MS analysis.

(29) The matrix-and-additive mixture solution is obtained by dissolving one or both of the alkylphosphonic acid and the surfactant, and the matrix substance, in a solvent so that the concentrations of these substances become their respective specified values. It is also possible to initially prepare a matrix solution (by dissolving a matrix substance in a solvent) and a matrix additive solution (by dissolving a matrix additive in a solvent), and subsequently mix these two solutions to obtain the matrix-and-additive mixture solution.

(30) The use of an alkylphosphonic acid as the matrix additive added to the matrix-and-additive mixture solution has the effect of suppressing the formation of alkaline metal adduct ions. This suppresses the detection of the peaks of the alkaline metal adduct ions which form background noise in the mass spectrum. Consequently, it is expected the peaks originating from the constituents of the microorganism will be more easily observed, and the detection sensitivity for the peaks originating from the constituents of the microorganism will be improved.

(31) The use of a surfactant as the matrix additive added to the matrix-and-additive mixture solution is likely to improve the solubility of the test microorganism when a solution containing the constituents of the test microorganism (sample solution) is mixed with the matrix-and-additive mixture solution. Therefore, it is expected that the peaks of a sample which is barely soluble and has been difficult to analyze will be more satisfactorily detected.

(32) Examples of the solvents that can be used for the matrix-and-additive mixture solution include a mixed solution of water and acetonitrile (ACN) as well as a mixed solution obtained by adding an organic acid, such as a trifluoroacetic acid (TFA), trichloroacetic acid or acetic acid, to the mixed solution of water and ACN. It is expected that the addition of the organic acid to the solvent of the matrix-and-additive mixture solution will lead to high-sensitivity detection of the peak of a protonated molecule of the test microorganism ([M+H].sup.+) or related ion.

(33) After a test microorganism is applied to a sample plate, or after a solution containing constituents of the test microorganism is dropped onto the sample plate, the matrix-and-additive mixture solution is additionally dropped onto it and dried. Consequently, a mixed crystal of the constituents of the test microorganism and the matrix is obtained. This mixed crystal is used as the sample for the MALDI-MS analysis. Unlike the conventional method in which a matrix solution and an additive solution are mixed together by being sequentially and individually dropped onto a sample plate, the mixed solution of the matrix substance and the additive in the present invention is prepared beforehand, so that fewer process steps are required to drop solutions onto a sample plate. It is also possible to mix the matrix-and-additive mixture solution with the constituents of the test microorganism beforehand, drop the mixture onto the sample plate, and dry the dropped solution to obtain the mixed crystal. In this case, the number of process steps for sample preparation on the sample plate will be even further decreased.

(34) Hereinafter, the method for analyzing a microorganism according to the present invention is described by means of examples. It should be noted that the following examples are merely illustrative and should not be construed as limiting the present invention. In particular, the kinds of substances contained in the various solutions used in the following examples, such as the matrix solution, matrix-and-additive mixture solution and additive solution, as well as the mixture ratio and concentration of each substance are mere examples and can be changed or varied within controllable ranges based on the common knowledge of a person skilled in the art. It is expected that any change or variation which falls within such a range will produce similar effects to those described in the following examples.

(35) In the following examples, samples which had been prepared by a method for analyzing a microorganism according to the present invention were analyzed with a MALDI-MS. In order to confirm the effects of those samples, additional samples were also prepared by two methods different from the present invention and analyzed with the MALDI-MS.

(36) As will be described later in detail, in the present invention, a matrix solution in which a matrix additive is mixed beforehand (“matrix-and-additive mixture solution”) is used for sample preparation. For example, after a matrix-and-additive mixture solution has been prepared, the matrix-and-additive mixture solution is mixed with a test microorganism beforehand, and the obtained solution is dropped onto a sample plate to obtain a sample. This method is hereinafter called the “additive pre-mix method”.

(37) One of the two methods different from the present invention is a method in which a mixed solution of the matrix substance and the test microorganism is dropped onto a sample plate, and a matrix additive solution is additionally dropped onto the mixed solution to obtain a sample. This method is hereinafter called the “additive post-placement method”.

(38) The other one of the two different methods is a method in which no matrix additive is used, and only the matrix solution is used for sample preparation. This method is hereinafter called the “no-additive method”.

FIRST EXAMPLE

(39) <1. Test Microorganism>

(40) As the test microorganism, a commercially available Escherichia coli (E. coli DH5α Electro-Cells, Product Code: TKR 9027, manufactured by Takara Bio Inc.) was used. E. coli DH5α Electro-Cells is a solution containing E. coli DH5α strain. This solution is hereinafter called the “test microorganism solution”.

(41) <2-1. Preparation of Matrix Solution>

(42) Sinapinic acid (SA, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in an aqueous solution containing 50% acetonitrile (ACN) and 0.6% trifluoroacetic acid (TFA) solution to obtain a matrix solution containing 25 mg/mL sinapinic acid. This matrix solution is hereinafter labeled “SA-2”.

(43) <2-2. Preparation of Matrix-and-Additive Mixture Solutions>

(44) In the present example, three kinds of matrix-and-additive mixture solution were prepared as follows:

(45) (1) SA and methylenediphosphonic acid (MDPNA) were dissolved in an aqueous solution containing 50% ACN and 0.6% TFA solution to obtain a matrix-and-additive mixture solution containing 25 mg/mL SA and 1% (percent by weight; the same applies to the following descriptions) MDPNA. This matrix-and-additive mixture solution is hereinafter labeled “SA-3”.

(46) (2) SA and decyl-β-D-maltopyranoside (DMP) were dissolved in an aqueous solution containing 50% ACN and 0.6% TFA solution to obtain a matrix-and-additive mixture solution containing 25 mg/mL SA and 1 mM DMP. This matrix-and-additive mixture solution is hereinafter labeled “SA-4”.

(47) (3) SA, MDPNA and DMP were dissolved in an aqueous solution containing 50% ACN and 0.6% TFA solution to obtain a matrix-and-additive mixture solution containing 25 mg/mL SA, 1% MDPNA and 1 mM DMP. This matrix-and-additive mixture solution is hereinafter labeled “SA-5”.

(48) <2-3. Preparation of Additive Solution>

(49) In the present example, two kinds of additive solutions were prepared as follows:

(50) (1) MDPNA was dissolved in an aqueous solution containing 50% ACN and 0.6% TFA solution to obtain an additive solution containing 1% MDPNA. This additive solution is hereinafter labeled “A-1”.

(51) (2) DMP was dissolved in an aqueous solution containing 50% ACN and 0.6% TFA solution to obtain an additive solution containing 1 mM DMP. This additive solution is hereinafter labeled “A-2”.

(52) <3. Mass Spectrometric Analysis>

(53) A MALDI time-of-flight mass spectrometer (MALDI-TOFMS, manufactured by Shimadzu Corporation under the trade name of AXIMA-Performance) was used for the analysis of the samples. The measurement was performed in the linear mode (positive ion mode). All data were acquired by “raster analysis”. Raster analysis is an automatic measurement function provided in the aforementioned mass spectrometer, in which mass spectrum data is acquired for each of the samples in the wells on a sample plate by delivering a specified number of shots of laser beam onto each of the specified number of points on the sample. In the present example, the data-collecting task including the steps of delivering the laser beam onto four wells (n=4) on the sample plate and acquiring mass spectrum data was performed multiple times for each of a plurality of test microorganisms.

(54) <4. Preparation of Samples>

(55) <4-1. Preparation of Sample by Additive Pre-mix Method>

(56) (1) Initially, the test microorganism solution was 50-fold diluted with each of the three matrix-and-additive mixture solutions SA-3, SA-4 and SA-5 to obtain three kinds of matrix-additive-microorganism mixture solutions.

(57) (2) Next, for each of the three matrix-additive-microorganism mixture solutions, 1 μL of the solution was dropped onto the wells on one sample plate.

(58) (3) Subsequently, the matrix-additive-microorganism mixture solution in the wells of each sample plate was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(59) <4-2. Preparation of Samples by Additive Post-Placement Method>

(60) (1) Initially, the test microorganism solution was 50-fold diluted with the matrix solution SA-2 to obtain a matrix-microorganism mixture solution.

(61) (2) Next, 1 μL of the matrix-microorganism mixture solution was dropped onto each of the wells on a sample plate and dried.

(62) (3) Subsequently, 1 μL of the additive solution A-1 was dropped onto each of the dried matrix-microorganism mixture solutions in the wells on one sample plate, while 1 μL of the additive solution A-2 was dropped onto each of the dried matrix-microorganism mixtures in the wells on another sample plate. Then, the dropped solutions on both sample plates were dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(63) <4-3. Preparation of Samples by No-Additive Method>

(64) (1) Initially, the test microorganism solution was 50-fold diluted with the matrix solution SA-2 to obtain a matrix-microorganism mixture solution.

(65) (2) Next, 1 μL of the matrix-microorganism mixture solution was dropped onto each of the wells on the sample plate.

(66) (3) Subsequently, the matrix-microorganism mixture solution in the wells was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(67) The previously described sample-preparing tasks may be manually performed by an operator, or an automatic dropping device may be used. The solution dropped into the wells on the sample plate may be naturally dried, or warm air may be supplied to dry it.

(68) <5. Results>

(69) FIGS. 1, 2, 3, 4A and 4B show mass spectra obtained in the first example.

(70) Charts (A) and (B) in each of FIGS. 1 and 2 are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-3 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 1 and 2 is a mass spectrum obtained for a sample prepared by the additive post-placement method in which the matrix additive solution A-1 was used. Chart (D) in each of FIGS. 1 and 2 is a mass spectrum obtained for a sample prepared by the no-additive method. FIG. 1 shows the mass spectra within a mass-to-charge-ratio range of m/z 7000 to 20000. FIG. 2 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 40000.

(71) As shown in FIG. 1, within the mass-to-charge-ratio range of m/z 7000 to 20000, the mass spectrum (C) obtained by the additive post-placement method exhibited a slightly different peak profile from the mass spectrum (D) obtained by the no-additive method. By comparison, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited almost the same peak profile as the mass spectrum (D) obtained by the no-additive method.

(72) Furthermore, as shown in FIG. 2, within the mass-to-charge-ratio range of m/z 20000 to 40000, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectrum (C) obtained by the additive post-placement method as well as the mass spectrum (A) and (B) obtained by the additive pre-mix methods as compared to the mass spectrum (D) obtained by the no-additive method. In particular, in the mass spectrum (A) of the sample prepared by the additive pre-mix method using the matrix-and-additive mixture solution SA-5 in which both MDPNA and DMP as the matrix additives were mixed with the matrix substance, the background noise within the high-mass range was reduced, and the peaks were detected with a high level of sensitivity.

(73) Charts (A) and (B) in each of FIGS. 3, 4A and 4B are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-4 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 3, 4A and 4B is a mass spectrum obtained for a sample prepared by the additive post-placement method in which the matrix additive solution A-2 was used. Chart (D) in each of FIGS. 3, 4A and 4B is a mass spectrum obtained for a sample prepared by the no-additive method. FIG. 3 shows the mass spectra within a mass-to-charge-ratio range of m/z 7000 to 20000. FIGS. 4A and 4B each show the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 33000. It should be noted that all charts (A) to (D) in FIG. 4A have the same intensity scale on the vertical axis, whereas the scales on the vertical axes of charts (A) to (D) in FIG. 4B are adjusted to show the peak heights in relative intensity in a similar manner to the other data so as to allow for the comparison of peak profiles.

(74) As shown in FIG. 3, within the mass-to-charge-ratio range of m/z 7000 to 20000, the mass spectrum (C) obtained by the additive post-placement method exhibited a slightly different peak profile from the mass spectrum (D) obtained by the no-additive method. By comparison, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited almost the same peak profile as the mass spectrum (D) obtained by the no-additive method, regardless of whether SA-5 or SA-4 was used as the matrix-and-additive mixture solution.

(75) Furthermore, as shown in FIGS. 4A and 4B, within the mass-to-charge-ratio range of m/z 20000 to 33000, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited almost the same peak profile as the mass spectrum (D) obtained by the no-additive method. Additionally, some peaks which did not appear on the mass spectrum (D) obtained by the no-additive method were observed in the mass spectra (A) and (B). Thus, an improvement in the peak intensity or signal-to-noise ratio was confirmed.

(76) The results described thus far demonstrate that the sample preparation by the additive pre-mix method is particularly effective for improving the sensitivity within a high-mass range. Since the improvement of the sensitivity is achieved without any change in the peak profile, there is no loss of peaks due to the suppression effect caused by a change in the order of peak intensity or other factors. This consequently produces favorable effects. For example, the self-calibration peaks which have been used before the addition of the additive can be continuously used as they are, and the target peaks designated before the addition of the additive can be certainly detected with the improved sensitivity. Furthermore, as compared to the additive post-placement method, the additive pre-mix method expedites an analysis of a sample since the number of process steps for dropping solutions onto a sample plate is the same as in the no-additive method.

SECOND EXAMPLE

(77) <1. Test Microorganism>

(78) Salmonella enterica (serotype: Infantis, jfrlSe 1402-4) isolated from a field isolate and cultured on LB agar at 37 degrees Celsius for 20 hours was used as the test microorganism.

(79) <2-1. Preparation of Matrix Solution>

(80) A (saturated) matrix solution was prepared by dissolving sinapinic acid (SA) in an ethanol solution to a content of 25 mg/mL This matrix solution is hereinafter labeled “SA-1”.

(81) Additionally, the matrix solution SA-2 was prepared using the same solvent and matrix substance as in the first example.

(82) <2-2. Preparation of Matrix-and-Additive Mixture Solutions>

(83) The three kinds of matrix-and-additive mixture solutions SA-3, SA-4 and SA-5 were prepared using the same combinations of the solvent and matrix substances as in the first example.

(84) <2-3. Preparation of Additive Solution>

(85) The additive solution A-1 was prepared using the same combination of the solvent and matrix additive as in the first example.

(86) <3. Mass Spectrometric Analysis>

(87) For the analysis of the samples, as in the first example, the MALDI time-of-flight mass spectrometer (MALDI-TOFMS, manufactured by Shimadzu Corporation under the trade name of AXIMA-Performance) operated in the linear mode (positive ion mode) was used, and the same method was used to acquire data.

(88) <4. Preparation of Samples>

(89) <4-1. Preparation of Sample by Additive Pre-mix Method>

(90) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of each of the three kinds of matrix-and-additive mixture solutions SA-3 to SA-5 to obtain sample suspensions of SA-3 to SA-5.

(91) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped in each well on a sample plate and dried. Then, one of the three sample suspensions of SA-3 to SA-5 (1.2 μL) was dropped onto the dried substance.

(92) (3) Subsequently, the sample suspension of SA-3, SA-4 or SA-5 in the wells on the sample plate was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(93) <4-2. Preparation of Samples by Additive Post-Placement Method>

(94) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of the matrix solution SA-2 to obtain a sample suspension of SA-2.

(95) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped onto each well on a sample plate and dried. Then, 1.2 μL of the sample suspension of SA-2 was additionally dropped onto the dried substance.

(96) (3) Subsequently, the sample suspension of SA-2 in the wells on the sample plate was dried. Then, 1 μL of the additive solution A-1 was dropped onto the dried substance and dried.

(97) (4) The crystal of the matrix-microorganism mixture obtained in (3) was used as a sample.

(98) <4-3. Preparation of Samples by No-Additive Method>

(99) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of the matrix solution SA-2 to obtain a sample suspension of SA-2.

(100) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped into each well on a sample plate and dried. Then, 1.2 μL of the sample suspension of SA-2 was additionally dropped onto the dried substance.

(101) (3) Subsequently, the sample suspension of SA-2 in the wells was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(102) <5. Results>

(103) FIGS. 5 to 9 show mass spectra obtained in the second example.

(104) Charts (A) and (B) in each of FIGS. 5, 6A, 6B and 6C are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-3 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 5, 6A, 6B and 6C is a mass spectrum obtained for a sample prepared by the additive post-placement method. Chart (D) in each of FIGS. 5, 6A, 6B and 6C is a mass spectrum obtained for a sample prepared by the no-additive method. FIG. 5 shows the mass spectra within a mass-to-charge-ratio range of m/z 9000 to 20000. FIGS. 6A and 6B each show the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 34000. FIG. 6C shows the mass spectra within a mass-to-charge-ratio range of m/z 40000 to 90000.

(105) As shown in FIG. 5, within the mass-to-charge-ratio range of m/z 9000 to 20000, the mass spectrum (C) obtained by the additive post-placement method exhibited a slightly different peak profile from the mass spectrum (D) obtained by the no-additive method. By comparison, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited almost the same peak profile as the mass spectrum (D) obtained by the no-additive method.

(106) Within the mass-to-charge-ratio range of m/z 20000 to 34000 shown in FIGS. 6A and 6B, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectra (A) to (C) obtained by the additive pre-mix methods and the additive post-placement method as compared to the mass spectrum (D) obtained by the no-additive method.

(107) Similarly, within the high-mass range of m/z 40000 to 90000 in mass-to-charge ratio shown in FIG. 6C, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectra (A) to (C) obtained by the additive pre-mix methods and the additive post-placement method as compared to the mass spectrum (D) obtained by the no-additive method. In particular, within a high-mass range of m/z 40000 or higher in mass-to-charge-ratio, the peaks were detected with high sensitivity by the additive post-placement method. It should be noted that the number of process steps for dropping in the preparation of samples by the additive pre-mix method is the same as in the no-additive method.

(108) Charts (A) and (B) in each of FIGS. 7 to 9 are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-4 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 7 to 9 is a mass spectrum obtained for a sample prepared by the no-additive method. FIG. 7 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 40000. FIG. 8 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 34000. FIG. 9 shows the mass spectra within a mass-to-charge-ratio range of m/z 40000 to 90000.

(109) As shown in FIG. 7, within the mass-to-charge-ratio range of m/z 20000 to 40000, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited the same peak profile as the mass spectrum (C) obtained by the no-additive method.

(110) Furthermore, within the mass-to-charge-ratio range of m/z 20000 to 34000 shown in FIG. 8 as well as the mass-to-charge-ratio range of m/z 40000 to 90000 shown in FIG. 9, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectra (A) and (B) obtained by the additive pre-mix method as compared to the mass spectrum (C) obtained by the no-additive method. In particular, within a high-mass range of m/z 20000 or higher in mass-to-charge ratio, the peaks were detected with high sensitivity by the additive pre-mix method.

(111) The results described thus far demonstrate that the sample preparation by the additive pre-mix method is particularly effective for improving the sensitivity within a high-mass range. Since the improvement of the sensitivity is achieved without any change in the peak profile, there is no loss of peaks due to the suppression effect caused by a change in the order of peak intensity or other factors. This consequently produces favorable effects. For example, the self-calibration peaks which have been used before the addition of the additive can be continuously used as they are, and the target peaks designated before the addition of the additive can be certainly detected with the improved sensitivity. Furthermore, as compared to the additive post-placement method, the additive pre-mix method expedites an analysis of a sample since the number of process steps for dropping solutions onto a sample plate is the same as in the no-additive method.

THIRD EXAMPLE

(112) <1. Test Microorganism>

(113) Salmonella enterica (serotype: Typhimurium, NBRC 13245) cultured on LB agar at 37 degrees Celsius for 20 hours was used as the test microorganism.

(114) <2-1. Preparation of Matrix Solutions>

(115) The matrix-and-additive mixture solutions SA-1 and SA-2 were prepared using the same combinations of the solvent and matrix substances as in the second example.

(116) <2-2. Preparation of Matrix-and-Additive Mixture Solutions>

(117) The three kinds of matrix-and-additive mixture solutions SA-3, SA-4 and SA-5 were prepared using the same combinations of the solvent and matrix substances as in the first and second examples.

(118) <2-3. Preparation of Additive Solution>

(119) The additive solution A-1 was prepared using the same combination of the solvent and matrix additive as in the second example.

(120) <3. Mass Spectrometric Analysis>

(121) For the analysis of the samples, as in the first example, the MALDI time-of-flight mass spectrometer (MALDI-TOFMS, manufactured by Shimadzu Corporation under the trade name of AXIMA-Performance) operated in the linear mode (positive ion mode) was used, and the same method was used to acquire data.

(122) <4. Preparation of Samples>

(123) <4-1. Preparation of Sample by Additive Pre-mix Method>

(124) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of each of the three kinds of matrix-and-additive mixture solutions SA-3 to SA-5 to obtain microorganism suspensions of SA-3 to SA-5.

(125) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped in each well on a sample plate and dried. Then, one of the three microorganism suspensions of SA-3 to SA-5 (1.2 μL) was dropped onto the dried substance.

(126) (3) Subsequently, the microorganism suspensions of SA-3 to SA-5 in the wells on the sample plate was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(127) <4-2. Preparation of Samples by Additive Post-Placement Method>

(128) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of the matrix solution SA-2 to obtain a microorganism suspension of SA-2.

(129) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped onto each well on a sample plate and dried. Then, 1.2 μL of the microorganism suspension of SA-2 was additionally dropped onto the dried substance.

(130) (3) Subsequently, the microorganism suspension of SA-2 in the wells on the sample plate was dried. Then, 1 μL of the additive solution A-1 was dropped onto the dried substance and dried.

(131) (4) The crystal of the matrix-microorganism mixture obtained in (3) was used as a sample.

(132) <4-3. Preparation of Samples by No-Additive Method>

(133) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of the matrix solution SA-2 to obtain a microorganism suspension of SA-2.

(134) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped into each well on a sample plate and dried. Then, 1.2 μL of the microorganism suspension of SA-2 was additionally dropped onto the dried substance.

(135) (3) Subsequently, the microorganism suspension of SA-2 in the wells was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(136) <5. Results>

(137) FIGS. 10 to 14 show mass spectra obtained in the third example.

(138) Charts (A) and (B) in each of FIGS. 10 to 12 are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-3 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 10 to 12 is a mass spectrum obtained for a sample prepared by the additive post-placement method using the additive solution A-1. Chart (D) in each of FIGS. 10 to 12 is a mass spectrum obtained for a sample prepared by the no-additive method.

(139) FIG. 10 shows the mass spectra within a mass-to-charge-ratio range of m/z 10000 to 20000. FIG. 11 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 40000. FIG. 12 shows the mass spectra within a mass-to-charge-ratio range of m/z 40000 to 90000.

(140) As shown in FIG. 10, within the mass-to-charge-ratio range of m/z 10000 to 20000, the mass spectrum (C) obtained by the additive post-placement method exhibited a slightly different peak profile from the mass spectrum (D) obtained by the no-additive method. By comparison, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited almost the same peak profile as the mass spectrum (D) obtained by the no-additive method.

(141) Within the mass-to-charge-ratio range of m/z 20000 to 40000 shown FIG. 11 as well as the mass-to-charge-ratio range of m/z 40000 to 90000 in shown FIG. 12, an improvement in the peak intensity and signal-to-noise ratio was confirmed in the mass spectra (A), (B) and (C) obtained by the additive pre-mix methods and the additive post-placement method as compared to the mass spectrum (D) obtained by the no-additive method.

(142) In particular, within a mass-to-charge-ratio range of m/z 40000 or higher, high-intensity peaks were observed in the mass spectrum obtained by the additive post-placement method (C).

(143) Charts (A) and (B) in each of FIGS. 13 and 14 are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-4 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 13 and 14 is a mass spectrum obtained for a sample prepared by the no-additive method. FIG. 13 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 40000. FIG. 14 shows the mass spectra within a mass-to-charge-ratio range of m/z 40000 to 90000.

(144) As shown in FIG. 13, within the mass-to-charge-ratio range of m/z 20000 to 40000, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited the same peak profile as the mass spectrum (C) obtained by the no-additive method. Furthermore, an improvement in the peak intensity and signal-to-noise ratio was confirmed in the mass spectra (A) and (B) obtained by the additive pre-mix methods as compared to the mass spectrum (C) obtained by the no-additive method.

(145) Additionally, within the mass-to-charge-ratio range of m/z 40000 to 90000 shown in FIG. 14, a slight yet certain improvement in the signal-to-noise ratio was confirmed in the mass spectrum (A) obtained by the additive pre-mix method as compared to the mass spectrum (C) obtained by the no-additive method.

(146) The results described thus far demonstrate that the sample preparation by the additive pre-mix method is particularly effective for improving the sensitivity within a high-mass range. Since the improvement of the sensitivity is achieved with no change in the peak profile, there is no loss of peaks due to the suppression effect caused by a change in the order of peak intensity or other factors. This consequently produces favorable effects. For example, the self-calibration peaks which have been used before the addition of the additive can be continuously used as they are, and the target peaks designated before the addition of the additive can be certainly detected with the improved sensitivity. Furthermore, as compared to the additive post-placement method, the additive pre-mix method expedites an analysis of a sample since the number of process steps for dropping solutions onto a sample plate is the same as in the no-additive method.

FOURTH EXAMPLE

(147) <1. Test Microorganism>

(148) Salmonella enterica (serotype: Orion, jfrlSe 1402-15) cultured on LB agar at 37 degrees Celsius for 20 hours was used as the test microorganism.

(149) <2-1. Preparation of Matrix Solutions>

(150) The matrix-and-additive mixture solutions SA-1 and SA-2 were prepared using the same combinations of the solvent and matrix substances as in the second and third examples.

(151) <2-2. Preparation of Matrix-and-Additive Mixture Solutions>

(152) The three kinds of matrix-and-additive mixture solutions SA-3, SA-4 and SA-5 were prepared using the same combinations of the solvent and matrix substances as in the first and second examples.

(153) <2-3. Preparation of Additive Solution>

(154) The additive solution A-1 was prepared using the same combination of the solvent and matrix additive as in the second example.

(155) <3. Mass Spectrometric Analysis>

(156) For the analysis of the samples, as in the first example, the MALDI time-of-flight mass spectrometer (MALDI-TOFMS, manufactured by Shimadzu Corporation under the trade name of AXIMA-Performance) operated in the linear mode (positive ion mode) was used, and the same method was used to acquire data.

(157) <4. Preparation of Samples>

(158) <4-1. Preparation of Sample by Additive Pre-mix Method>

(159) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of each of the three kinds of matrix-and-additive mixture solutions SA-3 to SA-5 to obtain microorganism suspensions of SA-3 to SA-5.

(160) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped in each well on a sample plate and dried. Then, one of the three microorganism suspensions of SA-3 to SA-5 (1.2 μL) was dropped onto the dried substance.

(161) (3) Subsequently, the microorganism suspensions of SA-3 to SA-5 in the wells on the sample plate was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(162) <4-2. Preparation of Samples by Additive Post-Placement Method>

(163) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of the matrix solution SA-2 to obtain a microorganism suspension of SA-2.

(164) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped onto each well on a sample plate and dried. Then, 1.2 μL of the microorganism suspension of SA-2 was additionally dropped onto the dried substance.

(165) (3) Subsequently, the microorganism suspension of SA-2 in the wells on the sample plate was dried. Then, 1 μL of the additive solution A-1 orA-2 was dropped onto the dried substance and dried.

(166) (4) The crystal of the matrix-microorganism mixture obtained in (3) was used as a sample.

(167) <4-3. Preparation of Samples by No-Additive Method>

(168) (1) Initially, an amount of test microorganism corresponding to a few colonies was put in 10 μL of the matrix solution SA-2 to obtain a microorganism suspension of SA-2.

(169) (2) Next, 0.5 μL of the matrix solution SA-1 was dropped into each well on a sample plate and dried. Then, 1.2 μL of the microorganism suspension of SA-2 was additionally dropped onto the dried substance.

(170) (3) Subsequently, the microorganism suspension of SA-2 in the wells was dried, and the obtained crystal of the matrix-microorganism mixture was used as a sample.

(171) <5. Results>

(172) FIGS. 15 to 21 show mass spectra obtained in the fourth example. Charts (A) and (B) in each of FIGS. 15 to 18 are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-3 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 15 to 18 is a mass spectrum obtained for a sample prepared by the additive post-placement method in which the additive solution A-1 was used. Chart (D) in each of FIGS. 15 to 18 is a mass spectrum obtained for a sample prepared by the no-additive method. FIG. 15 shows the mass spectra within a mass-to-charge-ratio range of m/z 9000 to 20000. FIG. 16 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 40000. FIG. 17 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 34000. FIG. 18 shows the mass spectra within a mass-to-charge-ratio range of m/z 37000 to 90000.

(173) As shown in FIG. 15, within the mass-to-charge-ratio range of m/z 9000 to 20000, the mass spectrum (C) obtained by the additive post-placement method exhibited a slightly different peak profile from the mass spectrum (D) obtained by the no-additive method. By comparison, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited almost the same peak profile as the mass spectrum (D) obtained by the no-additive method.

(174) Within the mass-to-charge-ratio range of m/z 20000 to 40000 shown in FIG. 16 as well as the mass-to-charge-ratio range of m/z 20000 to 34000 shown in FIG. 17, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectra (A), (B) and (C) obtained by the additive pre-mix methods and the additive post-placement method as compared to the mass spectrum (D) obtained by the no-additive method.

(175) Within the mass-to-charge-ratio range of m/z 37000 to 90000 shown in FIG. 18, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectra (A), (B) and (C) obtained by the additive pre-mix methods and the additive post-placement method as compared to the mass spectrum (D) obtained by the no-additive method. In particular, within a mass-to-charge-ratio range of m/z 40000 or higher, the peaks were detected with high sensitivity by the additive post-placement method (C).

(176) Charts (A) and (B) in each of FIGS. 19 to 21 are mass spectra obtained for samples prepared by the additive pre-mix methods in which SA-5 and SA-4 were respectively used as the matrix-and-additive mixture solution. Chart (C) in each of FIGS. 19 to 21 is a mass spectrum obtained for a sample prepared by the no-additive method. FIG. 19 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 40000. FIG. 20 shows the mass spectra within a mass-to-charge-ratio range of m/z 20000 to 34000. FIG. 21 shows the mass spectra within a mass-to-charge-ratio range of m/z 40000 to 90000.

(177) Within the mass-to-charge-ratio range of m/z 20000 to 40000 shown in FIG. 19 as well as the mass-to-charge-ratio range of m/z 20000 to 34000 shown in FIG. 20, the mass spectra (A) and (B) obtained by the additive pre-mix methods both exhibited the same peak profile as the mass spectrum (C) obtained by the no-additive method. Furthermore, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectra (A) and (B) obtained by the additive pre-mix methods as compared to the mass spectrum (C) obtained by the no-additive method.

(178) Furthermore, within the mass-to-charge-ratio range of m/z 40000 to 90000 shown in FIG. 21, an improvement in the peak intensity or signal-to-noise ratio was confirmed in the mass spectra (A) and (B) obtained by the additive pre-mix methods as compared to the mass spectrum (C) obtained by the no-additive method.

(179) The results described thus far demonstrate that the sample preparation by the additive pre-mix method is particularly effective for improving the sensitivity within a high-mass range. Since the improvement of the sensitivity is achieved with no change in the peak profile, there is no loss of peaks due to the suppression effect caused by a change in the order of peak intensity or other factors. This consequently produces favorable effects. For example, the self-calibration peaks which have been used before the addition of the additive can be continuously used as they are, and the target peaks designated before the addition of the additive can be certainly detected with the improved sensitivity. Furthermore, as compared to the additive post-placement method, the additive pre-mix method expedites an analysis of a sample since the number of process steps for dropping solutions onto a sample plate is the same as in the no-additive method.