Method for Preparing Sample for Analysis and Mass Spectrometry Method

Abstract

To easily prepare a sample always containing the right amount of target molecule without relying on the experience and skill of a worker.SOLUTION: A preparation method of sample for analysis in an embodiment includes: an adsorption step (S1, S2) in which a predetermined amount of a solid-phase extraction carrier is allowed to adsorb a target substance of a saturated amount of the carrier; a desorption step (S3) of using a predetermined amount of eluate to allow the target substance to desorb from the solid-phase extraction carrier into the eluate after the adsorption step; and a preparation step (S4, S5) of preparing the sample using the eluate after the desorption step.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for preparing a sample to be used for analysis such as mass spectrometry, and a mass spectrometry method using the method.

Background Art

[0002] In recent years, in the field of microorganism identification for identifying various microorganisms such as bacteria and fungi, the Matrix Assisted Laser Desorption / Ionization (MALDI) mass spectrometry method (hereinafter referred to as the "MALDI mass spectrometry method") has been rapidly spreading. One of the major reasons is that, compared with the conventionally common culturing method and the like, it does not require skilled techniques, and the identification work can be performed at low cost and quickly.

[0003] Conventionally, as a method for preparing a MALDI sample for microorganism identification, a method is known in which a part of the cultured microorganism is collected and directly applied onto a sample plate, and a matrix is dropped thereon and dried. Further, as another sample preparation method, a method is also known in which proteins are extracted from microorganisms using an appropriate reagent, and the extract and the matrix are dropped onto a sample plate and dried.

[0004] In order to perform more reliable microorganism identification using the MALDI mass spectrometry method, it is important to prepare a sample so that an appropriate amount of the analyte is contained. For example, according to Non-Patent Document 1, when an excessive amount of bacterial cells is loaded onto a MALDI sample plate, problems such as ineffective extraction of proteins from the bacterial cells when a matrix is added occur. Further, Non-Patent Document 2 reports that when identifying Gram-negative bacilli of the Enterobacteriaceae family, if the amount of inoculated bacteria is excessive, a good mass spectrum cannot be obtained, and in order to solve this, it is necessary to accumulate experience and acquire the skill of finely adjusting the amount of inoculated bacteria.

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] Shimadzu Corporation, Analytical and Measuring Business Division, "AXIMA Microbial Identification System Operation Guide 2.4.1 Amount of Bacterial Cells per Well", Shimadzu Corporation, December 2011 [Non-Patent Document 2] Takuya Hattori, et al., "Comparative Study of Identification of Clinical Isolates by Mass Spectrometry (VITEK MS) and Biochemical Properties", Medical Laboratory, Japanese Society for Clinical Laboratory Technology, 2014, Vol. 63, No. 5, p. 573-578 [Non-Patent Document 3] "ZTC04S960 Millipore Zip Tip 0.6 μL C4 Resin Filled", [online], [searched on September 2, 2020], Merck KGaA, Internet <URL: https: / / www.merckmillipore.com / JP / ja / product / ZipTip-with-0.6-L-C4-resin,MM_NF-ZTC04S960> [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] Thus, when performing microbial identification by MALDI mass spectrometry, not only when the amount of molecules derived from microorganisms contained in the sample is too small, but also when the amount is too large, there is a risk of hindering accurate identification. However, with the conventional sample preparation methods as described above, it is difficult to accurately control the amount of target molecules contained in the sample, and the accuracy of such operations largely depends on the experience and skill of the operator. Here, the target molecule means both the case of a single species and the case of multiple species. This is the same in the following description.

[0007] The present invention has been made to solve the above problems, and an object thereof is to provide a method for preparing an analytical sample that can prepare an analytical sample so that a target molecule has an appropriate amount with high accuracy without depending on the experience and skill of an operator, and a mass spectrometry method using the method.

Means for Solving the Problems

[0008] One aspect of the method for preparing an analytical sample according to the present invention made to solve the above problems is as follows: An adsorption step of adsorbing a target substance in a saturated amount of the carrier to a predetermined amount of a solid-phase extraction carrier; A desorption step of desorbing the target substance from the solid-phase extraction carrier after the adsorption step into the eluent using a predetermined amount of the eluent; A preparation step of preparing a sample using the eluent after the desorption step; is carried out.

[0009] The sample prepared by the method for preparing an analytical sample according to the present invention is, for example, a sample to be subjected to MALDI mass spectrometry. In that case, the sample is usually formed on a sample plate. Further, using the present invention, it is also possible to prepare a sample to be subjected to a mass spectrometer equipped with an atmospheric pressure ionization source such as an electrospray ionization method or an atmospheric pressure chemical ionization method. In that case, the sample is a liquid sample.

[0010] As the solid-phase extraction carrier, generally, a member for solid-phase extraction used for purposes such as desalting and concentration can be used.

Effects of the Invention

[0011] In the above aspect of the method for preparing an analytical sample according to the present invention, in the adsorption step, for example, a liquid containing an excessive amount of the target substance (at least equal to or more than the saturation amount of the solid-phase extraction carrier) is brought into contact with the solid-phase extraction carrier to adsorb the target substance onto the solid-phase extraction carrier. If the amount of the solid-phase extraction carrier is constant, the amount of the target substance adsorbed is constant. In the desorption step, a predetermined amount of an eluent is brought into contact with the solid-phase extraction carrier to desorb the entire amount of the target substance adsorbed on the solid-phase extraction carrier into the eluent. If the saturation amount of the target substance in the solid-phase extraction carrier is constant and the volume of the eluent is constant, the amount, that is, the concentration, of the target substance contained in the eluent after the desorption step becomes constant.

[0012] In this way, according to the above aspect of the method for preparing an analytical sample according to the present invention, a sample containing an appropriate amount of the target molecule, that is, a sample in which the concentration of the target molecule is accurately controlled, can be easily prepared without relying on the experience and skills of the operator. Further, simultaneously with the adjustment of the concentration of the target molecule, a treatment for removing impurities such as desalting, that is, purification of the sample can also be performed.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

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Figure 5

Embodiments for Carrying Out the Invention

[0014] An embodiment of a method for preparing a sample for analysis according to the present invention will be described with reference to the accompanying drawings.

[0015] [Explanation of the sample preparation method] FIG. 1 is a flowchart showing the working procedure in the sample preparation method of this embodiment. In this sample preparation method, a solid-phase extraction carrier that can adsorb a target molecular species under specific conditions and desorb the adsorbed molecular species under specific conditions is used.

[0016] The maximum amount of the target molecule that can be adsorbed on a unit amount of the solid-phase extraction carrier, that is, the saturation amount, is determined by the type and structure of the solid-phase extraction carrier, and it can be measured in advance. Therefore, if the amount of the solid-phase extraction carrier is constant, the amount of the target molecule that can be adsorbed on it will not exceed the saturation amount of the solid-phase extraction carrier and will be constant. Therefore, if the target molecule is adsorbed on the solid-phase extraction carrier until the saturation amount is reached and the liquid volume of the eluent for desorbing the adsorbed molecule is made constant, the concentration of the target molecule desorbed in the eluent will be constant. This is the basic principle of the adjustment of the amount of substance (concentration) in the sample preparation method of this embodiment.

[0017] Specifically, first, the operator prepares a sample solution containing the target molecule at a concentration exceeding the saturation amount V of the target molecule in the solid-phase extraction carrier to be used (step S1).

[0018] Next, the sample solution prepared in step S1 is brought into sufficient contact with a predetermined amount of the solid-phase extraction carrier to adsorb the target molecule in the sample solution onto the solid-phase extraction carrier (step S2). A certain amount (that is, the above saturation amount V) of the target molecule corresponding to the amount of the solid-phase extraction carrier is adsorbed on the solid-phase extraction carrier. The solid-phase extraction carrier has molecular selectivity, and the target molecular species is adsorbed on the solid-phase extraction carrier, while other impurities are not adsorbed on the solid-phase extraction carrier.

[0019] Thereafter, a predetermined eluent having an action of detaching the target molecule from the solid-phase extraction carrier is used to elute all of the target molecules adsorbed on the solid-phase extraction carrier into a certain amount C of the eluent (step S3). As described above, since the amount of the target molecule adsorbed on the solid-phase extraction carrier is a constant saturation amount V, the concentration of the target molecule in a certain amount C of the eluent also becomes constant.

[0020] Since the concentration of the target molecule in the eluent obtained in step S3 is determined, the eluent may be appropriately diluted to adjust the concentration as needed (step S4).

[0021] Then, using the eluent obtained in step S3 or the diluted eluent obtained in step S4, a sample for mass spectrometry is prepared (step S5). For example, when preparing a sample for MALDI mass spectrometry, a predetermined amount of the eluent and a predetermined matrix are dropped onto a sample plate and dried to prepare a sample on the plate. On the other hand, when preparing a sample for a mass spectrometer equipped with an atmospheric pressure ionization source such as an electrospray ionization method, the sample can be prepared by using the sample solution appropriately diluted as it is, or by performing pretreatment such as adding an appropriate reagent.

[0022] As described above, in the sample preparation method of the present embodiment, a sample containing target molecules with always the same concentration (i.e., the same amount) can be prepared without performing operations that require skill or experience.

[0023] In the above steps S2 and S3, it is necessary to bring the solid-phase extraction carrier into contact with the sample solution and the eluent, respectively. For this purpose, for example, a device in which a pipette tip is filled with a solid-phase extraction carrier can be used. As an example, a ZipTip 0.6 μL C4 resin-filled pipette tip (catalog number: ZTC04S960) of the Merck millipore brand of Merck KGaA, Germany, which is commercially available for the purpose of desalting and concentrating proteins, can be used. The solid-phase extraction carrier in this pipette tip is chemically bonded porous spherical silica gel whose surface is modified with an octadecylsilyl (C 18 H 37 Si) group, which is also used as the stationary phase of a column for liquid chromatography.

[0024] Also, in steps S2 and S3, it is also possible to bring the solid-phase extraction carrier into contact with the eluent using a centrifuge. In that case, for example, a centrifuge tip filled with a solid-phase extraction carrier, such as the Pierce C18 Spin Tip (catalog number: 89870) of Thermo Fisher Scientific, USA, can be used. Also, in steps S2 and S3, a column for liquid chromatography filled with the solid-phase extraction carrier as described above can also be used.

[0025] [Experimental Example] An experimental example using the above-described sample preparation method will be described. This example prepares a sample for identifying microorganisms by MALDI mass spectrometry, and the target substance (molecule) to be analyzed is a protein extracted from microorganisms. As the device filled with the solid-phase extraction carrier, the above-described ZipTip 0.6 μL C4 resin-filled pipette tip of the Merck millipore brand was used. The specific preparation procedure for the MALDI sample is as follows (A) to (J).

[0026] (A) The Escherichia coli K12 strain was grown by culturing it in 10 mL of a liquid medium (1% polypeptone, 0.2% yeast extract, 0.1% MgSO 4 / 7H 2 O) for 24 hours.

[0027] (B) The entire volume of the medium after culturing in (A) above was centrifuged at 10,000 G for 5 minutes. The supernatant was discarded, and the residue was resuspended in 3 mL of purified water. A portion of the suspension was diluted with purified water, and the bacterial concentration was calculated by measuring the turbidity. As a result, the turbidity corresponded to OD = 5.

[0028] (C) 1 mL of the solution after resuspension in (B) above was aliquoted and centrifuged at 10,000 G for 5 minutes. Then, the supernatant was discarded, and it was resuspended in 1 mL of 1% trifluoroacetic acid, 50% acetonitrile water. By performing this operation, protein molecules and the like inside the Escherichia coli cells were extracted into the solution.

[0029] (D) The entire volume of the solution after resuspension in (C) above was centrifuged at 10,000 G for 5 minutes. The supernatant was collected and dried using a centrifugal evaporator.

[0030] (E) 100 μL of 0.1% trifluoroacetic acid water was added to the dried residue obtained in (D) above and dissolved. This solution was used as a 1-fold diluted sample.

[0031] (F) 150 μL of 0.1% trifluoroacetic acid water was added to 50 μL of the solution obtained in (E) above (i.e., the 1-fold diluted sample) and diluted. This was used as a 4-fold diluted sample. Further, the same dilution was repeated to prepare a 16-fold diluted sample and a 64-fold diluted sample.

[0032] (G) The 1-fold diluted sample, 4-fold diluted sample, 16-fold diluted sample, and 64-fold diluted samples obtained in (E) and (F) above were used as 4-step diluted samples in a state where the sample preparation method of the present embodiment described above was not applied. Hereinafter, this 4-step diluted sample is referred to as a concentration-unadjusted 4-step diluted sample.

[0033] (H) 10 μL of the solutions were respectively aliquoted from the 1-fold diluted sample, 4-fold diluted sample, 16-fold diluted sample, and 64-fold diluted sample contained in the above-mentioned undiluted-concentration four-step diluted sample, and these were processed by the procedures of steps S2 and S3 using the above-mentioned solid-phase extraction carrier-packed pipette tip, and the protein molecules, which are the target molecules, were eluted into 10 μL of an eluate (0.1% trifluoroacetic acid, 50% acetonitrile water). These four types of eluates were used as four-step diluted samples in the state where the sample preparation method of the present embodiment was applied. Hereinafter, this four-step diluted sample is referred to as a concentration-adjusted four-step diluted sample.

[0034] (I) 1 μL of each of the four types of samples contained in the undiluted-concentration four-step diluted sample obtained in the above (G) was collected, 1 μL of acetonitrile was mixed therewith, and further 2 μL of an α-cyano-4-hydroxycinnamic acid solution (the solvent is 0.1% trifluoroacetic acid, 50% acetonitrile water, and the concentration is 10 mg / mL) as a matrix for MALDI was mixed, and 2 μL of the mixed solution was dropped onto a sample plate and dried to prepare a sample for MALDI. That is, this is the analytical sample corresponding to the undiluted-concentration four-step diluted sample.

[0035] (J) 2 μL of each of the four types of samples contained in the concentration-adjusted four-step diluted sample obtained in the above (H) was collected, and it was mixed with 2 μL of an α-cyano-4-hydroxycinnamic acid solution (the solvent is 0.1% trifluoroacetic acid, 50% acetonitrile water, and the concentration is 10 mg / mL) as a matrix for MALDI, and 2 μL of the mixed solution was dropped onto a sample plate and dried to prepare a sample for MALDI. That is, this is the analytical sample corresponding to the concentration-adjusted four-step diluted sample.

[0036] The four types of MALDI samples obtained in the above (I) and the four types of MALDI samples obtained in the above (J) were respectively measured using a time-of-flight mass spectrometer to obtain mass spectra. For the measurement, MALDI-8020 manufactured by Shimadzu Corporation was used.

[0037] The mass spectrum, which is the measurement result for the MALDI sample corresponding to the non-concentration-adjusted four-step dilution sample obtained in (I) above, is shown in FIG. 2. In FIG. 2, in order from the top, they are the measurement results for the 1-fold dilution sample, 4-fold dilution sample, 16-fold dilution sample, and 64-fold dilution sample. Also, the mass spectrum, which is the measurement result for the MALDI sample corresponding to the concentration-adjusted four-step dilution sample obtained in (J) above, is shown in FIG. 3. Similarly in FIG. 3, in order from the top, they are the measurement results for the 1-fold dilution sample, 4-fold dilution sample, 16-fold dilution sample, and 64-fold dilution sample.

[0038] Also, the relationships between the peak intensities and the concentrations at four representative mass-to-charge ratio values (m / z 4365, m / z 5381, m / z 6255, m / z 7274) observed in the mass spectra shown in FIGS. 2 and 3 are shown in FIGS. 4 and 5, respectively. In FIGS. 4 and 5, 1 / 1 on the horizontal axis represents the 1-fold dilution sample, and 1 / 64 represents the 64-fold dilution sample. That is, the concentration increases from left to right on the horizontal axis. Also, in FIGS. 4 and 5, two results (the first time and the second time) are shown for each mass-to-charge ratio value.

[0039] Looking at the graph of FIG. 4 where the sample preparation method of this embodiment is not applied, as the concentration increases from 64-fold dilution to 16-fold dilution and 4-fold dilution, the peak intensity increases. However, when comparing the 4-fold dilution and the 1-fold dilution, the 1-fold dilution with a higher concentration has lower peak intensities for all peaks. This is presumably due to the ion suppression effect caused by the large total amount of proteins and the like contained in the sample and the ion suppression effect caused by the large amount of impurities present in the sample. Such ion suppression effects are often observed in the MALDI method.

[0040] On the other hand, looking at the graph of Fig. 5 to which the sample preparation method of this embodiment is applied, also in this case, as the concentration increases from 64-fold dilution to 16-fold dilution and 4-fold dilution, the peak intensity increases. Further, when comparing 4-fold dilution and 1-fold dilution, although there are some cases where the peak intensity slightly decreases or is about the same in part, overall, the peak intensity is higher for 1-fold dilution with a higher concentration. This point is in contrast to the results of Fig. 4.

[0041] This means that by using the sample preparation method of this embodiment, even when the total amount of proteins etc. in the original sample is large, only the amount corresponding to the saturation amount of the solid-phase extraction carrier elutes into the eluate, so the protein concentration in the eluate is at an appropriate concentration (that is, the ion suppression effect caused by excessive proteins does not occur). Also, in the eluate, the concentration of contaminants that do not adsorb to the solid-phase extraction carrier should relatively decrease, so it is fully assumed that the ion suppression effect due to a large amount of contaminants in the sample is suppressed.

[0042] From this experimental result as well, it can be understood that generally, the concentration of the target molecule in the sample for mass spectrometry is easily and accurately controlled by using a solid-phase extraction carrier generally used for desalting and concentration. As a result, the sample amount at the time of ionization in mass spectrometry becomes appropriate, and a clear mass spectrum can be obtained in which peaks corresponding to trace components can also be sufficiently observed.

[0043] In the above embodiment and experimental example, proteins derived from microorganisms such as fungi were used as the substances (molecules) to be analyzed, but the target substance is not limited to this. If a solid-phase extraction carrier that can selectively adsorb the substance under predetermined conditions (and can be desorbed under predetermined conditions) is used, similarly, the concentration of the target substance in the sample can be appropriately controlled. For example, when a sugar chain, which is one of the substances derived from a living body, is used as the target substance, a carbon chip using graphite carbon, carbon nanotubes, etc. can be used as the solid-phase extraction carrier.

[0044] In addition, in the sample preparation method described above, it is necessary to first prepare a sample solution in which the amount of the target molecule is equal to or greater than the saturation amount. However, when the amount of the target molecule is small in the first place, it is advisable to reduce the amount of the solid-phase extraction carrier accordingly to lower the saturation amount.

[0045] Also, as described above, the sample preparation method according to the present invention can be used not only for the MALDI method, but also for various ionization methods such as the laser desorption ionization (LDI) method, the surface-assisted laser desorption ionization (SALDI) method, the ionization method used in secondary ion analysis, the direct analysis in real time (DART) method, the electrospray ionization (ESI) method, the atmospheric pressure chemical ionization (APCI) method, and the probe electrospray ionization (PESI) method when preparing samples for mass spectrometry. In addition, the sample prepared by the sample preparation method according to the present invention can be used for analysis not only with a mass spectrometer but also with various other analytical instruments.

[0046] Furthermore, the above-described embodiments and the modified examples described above are merely examples of the present invention, and it is natural that appropriate modifications, corrections, additions, etc. within the scope of the gist of the present invention are also included in the scope of the claims of the present application.

[0047] [Various Aspects] It is understood by those skilled in the art that the exemplary embodiments described above are specific examples of the following aspects.

[0048] (Item 1) One aspect of the method for preparing an analytical sample according to the present invention is an adsorption step of adsorbing a target substance in an amount equal to the saturation amount of the carrier to a predetermined amount of a solid-phase extraction carrier, a desorption step of desorbing the target substance from the solid-phase extraction carrier after the adsorption step into the eluent using a predetermined amount of the eluent, a preparation step of preparing a sample using the eluent after the desorption step, and performing.

[0049] (Items 2 to 4) In the method for preparing an analytical sample according to Item 1, the solid-phase extraction carrier can be any of those filled in a pipette tip, those filled in a centrifugal column, or those filled in a column for liquid chromatography.

[0050] According to the method for preparing an analytical sample described in Items 1 to 4, a sample containing an appropriate amount of a target molecule, that is, a sample in which the concentration of the target molecule is accurately controlled, can be easily prepared without relying on the experience and skill of an operator. Thereby, for example, when a protein derived from a microorganism is the target substance, the microorganism can be more accurately identified based on the analysis results obtained by mass-analyzing the prepared sample.

[0051] (Item 5) In the method for preparing an analytical sample according to any one of Items 1 to 4, the preparation step can include a step of forming a sample for ionization on a plate by matrix-assisted laser desorption ionization method, that is, MALDI method.

[0052] (Item 6) Further, one aspect of the mass spectrometry method according to the present invention can be to irradiate a laser beam on the sample formed on the plate by the method for preparing an analytical sample described in Claim 5 to ionize the target substance in the sample, and mass-analyze the generated ions or ions derived therefrom.

[0053] As described above, in the MALDI method, when there is too much target substance in the sample or when there are many contaminants other than the target substance (especially substances that are more easily ionized than the target substance), the amount of ions derived from the target substance may be suppressed due to the ion suppression effect. On the other hand, according to the method for preparing an analytical sample described in paragraph 5, the amount of the target substance including the sample for MALDI can be appropriately controlled, and the amount of contaminants can also be reduced. Therefore, according to the method for preparing an analytical sample described in paragraph 5 and the mass spectrometry method described in paragraph 6, ions derived from the target substance can be efficiently generated, and a mass spectrum with high sensitivity and good quality can be obtained.

[0054] (Paragraph 7) In the mass spectrometry method described in paragraph 6, the target substance is a protein derived from a microorganism, and the microorganism can be identified using the result of ionizing and mass-analyzing the protein.

[0055] According to the mass spectrometry method described in paragraph 7, a microorganism can be accurately identified without relying on the experience and skill of the operator.

Claims

1. An adsorption step of adsorbing a target substance in a saturated amount of the carrier to a solid-phase extraction carrier in a predetermined amount; A desorption step of using a predetermined amount of an eluent to desorb all of the target substance from the solid-phase extraction carrier after the adsorption step into the eluent; A preparation step of preparing a sample using an eluent containing the target substance at a concentration of the target substance determined by the amount of the solid-phase extraction carrier, the saturated amount, and the amount of the eluent used in the desorption step, after the desorption step; A method for preparing an analytical sample, which performs the above steps.

2. The method for preparing an analytical sample according to claim 1, wherein the solid-phase extraction carrier is filled in a pipette tip.

3. The method for preparing an analytical sample according to claim 1, wherein the solid-phase extraction carrier is filled in a centrifugal column.

4. The method for preparing an analytical sample according to claim 1, wherein the solid-phase extraction carrier is filled in a column for liquid chromatography.

5. The method for preparing an analytical sample according to any one of claims 1 to 4, wherein the preparation step includes a step of forming a sample for ionization by matrix-assisted laser desorption ionization method on a plate.

6. A mass spectrometry method in which a sample formed on the plate by the method for preparing an analytical sample according to claim 5 is irradiated with laser light to ionize a target substance in the sample, and the generated ions or ions derived therefrom are mass-analyzed.

7. The mass spectrometry method according to claim 6, wherein the target substance is a protein derived from a microorganism, and the microorganism is identified using the result of ionizing and mass-analyzing the protein.

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