JP7686961B2 - Method for preparing analytical samples and method for mass spectrometry - Google Patents

Description

The present invention relates to a method for preparing a sample to be subjected to analysis such as mass spectrometry, and a mass spectrometry method using said method.

In recent years, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (hereafter referred to as "MALDI mass spectrometry") has rapidly become popular in the field of microbial identification, which identifies various microorganisms such as bacteria and fungi. One of the main reasons for this is that, compared to the conventional culture method, it does not require skilled techniques and can be performed quickly and at low cost.

Conventionally, a method for preparing samples for MALDI to identify microorganisms is known in which a portion of cultured microorganisms is taken and directly applied to a sample plate, and a matrix is then dropped onto the sample plate and allowed to dry. Another known sample preparation method involves extracting proteins from microorganisms using an appropriate reagent, and dropping the extract and matrix onto a sample plate and allowing them to dry.

For more reliable microbial identification using MALDI mass spectrometry, it is important to prepare the sample so that it contains an adequate amount of analyte.
For example, according to Non-Patent Document 1, if an excessive amount of bacterial cells is loaded onto a sample plate for MALDI, problems such as ineffective extraction of proteins from the bacterial cells when a matrix is added arise. Also, Non-Patent Document 2 reports that when identifying gram-negative bacilli of the Enterobacteriaceae family, a good mass spectrum cannot be obtained if an excessive amount of bacteria is picked up, and in order to solve this, it is necessary to accumulate experience and acquire the skill of finely adjusting the amount of bacteria picked up.

Shimadzu Corporation, Analytical Measurement Division, "AXIMA Microbial Identification System Operation Guide 2.4.1 Bacterial Mass per Well", Shimadzu Corporation, December 2011 Takuya Hattori and 7 others, "Comparative study of clinical isolate identification by mass spectrometry (VITEK MS) and biochemical properties," Medical Testing, Japan Association of Medical Technologists, 2014, Vol. 63, No. 5, pp. 573-578 "ZTC04S960 Millipore Zip Tip 0.6 μL C4 resin filled", [online], [searched September 2, 2020], Merck, Germany, Internet <URL: https://www.merckmillipore.com/JP/ja/product/ZipTip-with-0.6-L-C4-resin,MM_NF-ZTC04S960>

Thus, when identifying microorganisms using MALDI mass spectrometry, not only can the amount of microbial molecules contained in the sample be too small, but accurate identification can also be hindered if the amount is too large. However, with the conventional sample preparation methods described above, it is difficult to accurately control the amount of target molecules contained in the sample, and the accuracy of such work is largely dependent on the experience and skill of the operator. Note that the target molecule referred to here refers to both a single type and multiple types. This also applies to the following explanations.

The present invention has been made to solve the above problems, and its purpose is to provide a method for preparing an analytical sample that can prepare an analytical sample so that the target molecule is present in an appropriate amount with high precision, without relying on the experience or skill of the operator, and a mass spectrometry method using said method.

In order to solve the above problems, one aspect of the method for preparing an analytical sample according to the present invention is to
an adsorption step of adsorbing a target substance to a predetermined amount of solid phase extraction support in an amount that is saturated with the support;
a desorption step of desorbing the target substance from the solid-phase extraction support after the adsorption step into the elution solution using a predetermined amount of the elution solution;
a preparation step of preparing a sample using the eluate obtained after the desorption step;
The following is to be implemented.

The sample prepared by the analytical sample preparation method according to the present invention is, for example, a sample to be subjected to MALDI mass spectrometry, in which case the sample is usually formed on a sample plate. The present invention can also be used to prepare a sample to be subjected to a mass spectrometer equipped with an atmospheric pressure ionization source such as electrospray ionization or atmospheric pressure chemical ionization, in which case the sample is a liquid sample.

The solid-phase extraction support can be a material generally used for solid-phase extraction for purposes such as desalting and concentration.

In the above-mentioned aspect of the method for preparing an analytical sample according to the present invention, in the adsorption step, for example, a liquid containing an excess of the target substance (at least the saturation amount of the solid-phase extraction carrier or more) is brought into contact with the solid-phase extraction carrier, and the target substance is adsorbed onto the solid-phase extraction carrier. If the amount of solid-phase extraction carrier is constant, the amount of the adsorbed target substance is constant. In the desorption step, a predetermined amount of elution liquid is brought into contact with the solid-phase extraction carrier, and the entire amount of the target substance adsorbed onto the solid-phase extraction carrier is desorbed into the elution liquid. If the saturation amount of the target substance in the solid-phase extraction carrier is constant and the amount of the elution liquid is constant, the amount of the target substance contained in the elution liquid after the desorption step, i.e., the concentration, is constant.

In this way, according to the above-mentioned aspect of the method for preparing an analytical sample according to the present invention, a sample containing an appropriate amount of target molecules at all times, that is, a sample in which the concentration of the target molecules is accurately controlled, can be easily prepared without relying on the experience or skill of the operator. In addition, a process for removing impurities such as desalting can be performed at the same time as adjusting the concentration of the target molecules, that is, the purification of the sample can also be performed.

1 is a flowchart showing the procedure of a sample preparation method according to one embodiment of the present invention. FIG. 13 shows an example of a mass spectrum actually measured for a four-stage diluted sample when the sample preparation method of the present embodiment is not applied. FIG. 13 shows an example of the mass spectrum of a four-stage diluted sample when the sample preparation method of the present embodiment is applied. 1 is a graph plotting the intensity of a specific peak in a mass spectrum when the sample preparation method of the present embodiment is not applied. 1 is a graph plotting the intensity of a specific peak in a mass spectrum when the sample preparation method of the present embodiment is applied.

One embodiment of the method for preparing an analytical sample according to the present invention will be described with reference to the attached drawings.

[Sample preparation method description]
FIG. 1 is a flow chart showing the procedure of the sample preparation method of the present embodiment.
This sample preparation method uses a solid-phase extraction support that is capable of adsorbing a target molecular species under specific conditions and desorbing the adsorbed molecular species under specific conditions.

The maximum amount of target molecules that can be adsorbed by a unit amount of solid-phase extraction carrier, that is, the saturation amount, is determined by the type and structure of the solid-phase extraction carrier and can be measured in advance. Therefore, if the amount of solid-phase extraction carrier is constant, the amount of target molecules that can be adsorbed by it will be constant and will not exceed the saturation amount of the solid-phase extraction carrier. Therefore, if the target molecules are adsorbed onto the solid-phase extraction carrier until the saturation amount is reached and the amount of elution solution that desorbs the adsorbed molecules is kept constant, the concentration of the desorbed target molecules in the elution solution will be constant. This is the basic principle of adjusting the amount of substance (concentration) in the sample preparation method of this embodiment.

Specifically, first, the operator prepares a sample solution containing the target molecule at a concentration that exceeds the saturation amount V of the target molecule in the solid-phase extraction support being used (step S1).

Next, the sample solution prepared in step S1 is brought into sufficient contact with a predetermined amount of solid-phase extraction carrier, and the target molecules in the sample solution are adsorbed onto the solid-phase extraction carrier (step S2). A certain amount of target molecules (i.e., the saturation amount V) corresponding to the amount of solid-phase extraction carrier is adsorbed onto the solid-phase extraction carrier. The solid-phase extraction carrier has molecular selectivity, and the target molecular species is adsorbed onto the solid-phase extraction carrier, but other impurities are not adsorbed onto the solid-phase extraction carrier.

Then, using a specific elution solution that has the effect of releasing the target molecules from the solid-phase extraction support, all of the target molecules adsorbed to the solid-phase extraction support are eluted into a certain amount C of elution solution (step S3). As described above, the amount of target molecules adsorbed to the solid-phase extraction support is a constant saturation amount V, so the concentration of the target molecules in the certain amount C of elution solution is also constant.

Since the concentration of the target molecule in the eluate obtained in step S3 is fixed, the concentration may be adjusted by diluting the eluate appropriately, if necessary (step S4).

Then, the eluate obtained in step S3 or the diluted eluate obtained in step S4 is used to prepare a sample for mass spectrometry (step S5).
For example, when preparing a sample for MALDI mass spectrometry, a predetermined amount of elution solution 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 ion source such as electrospray ionization, the sample can be prepared as it is, or by carrying out pretreatment such as adding an appropriate reagent to a sample solution appropriately diluted as necessary.

In this way, the sample preparation method of this embodiment makes it possible to prepare samples that always contain the same concentration (i.e., the same amount) of target molecules without requiring skilled or experienced work.

In the above steps S2 and S3, the solid-phase extraction carrier needs to be brought into contact with the sample solution and the eluate, respectively. For this purpose, for example, a device in which the solid-phase extraction carrier is filled in a pipette tip can be used. As an example, ZipTip 0.6 μL C4 resin-filled pipette tip (catalog number: ZTC04S960) of the Merck Millipore brand by Merck, 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 a chemically bonded porous spherical silica gel whose surface is modified with an octadecylsilyl group (C ₁₈ H ₃₇ Si), which is also used as the stationary phase of a liquid chromatography column.

In steps S2 and S3, a centrifuge can be used to bring the solid-phase extraction carrier into contact with the eluate. In this case, for example, a centrifuge tip filled with a solid-phase extraction carrier, such as Thermo Fisher Scientific's Pierce C18 Spin Tip (catalog number: 89870), can be used. In steps S2 and S3, a liquid chromatograph column filled with the above-mentioned solid-phase extraction carrier can also be used.

[Experimental Example]
An experimental example using the above-mentioned sample preparation method will be described. In this example, a sample is prepared for microbial identification by MALDI mass spectrometry, and the target substance (molecule) to be analyzed is a protein extracted from a microorganism. The above-mentioned Merck Millipore brand ZipTip 0.6 μL C4 resin-filled pipette tip was used as the device filled with the solid-phase extraction carrier.
Specific procedures for preparing a sample for MALDI are as follows (A) to (J).

(A) Escherichia coli K12 strain was cultured in 10 mL of liquid medium (1% polypeptone, 0.2% yeast extract, 0.1% MgSO ₄ /7H ₂ O) for 24 hours to increase the number of bacteria.

(B) The entire amount of the culture medium after the above (A) culture was centrifuged at 10,000 G for 5 minutes, the supernatant was discarded, and the remainder was resuspended in 3 mL of purified water. A portion of the suspension was diluted with purified water, and the turbidity was measured to calculate the bacterial concentration. As a result, the turbidity was equivalent to OD = 5.

(C) 1 mL of the solution after resuspension in (B) above was taken and centrifuged at 10,000 G for 5 minutes. The supernatant was then discarded and the solution was resuspended in 1 mL of 1% trifluoroacetic acid and 50% acetonitrile water. By carrying out this procedure, protein molecules and other substances present within the E. coli cells were extracted into the solution.

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

(E) 100 μL of 0.1% trifluoroacetic acid water was added to the dried recovered material in (D) above to dissolve it, and this solution was used as a 1x diluted sample.

(F) 50 μL of the solution obtained in (E) above (i.e., the 1-fold diluted sample) was diluted with 150 μL of 0.1% trifluoroacetic acid water to prepare a 4-fold diluted sample. The same dilution was further repeated to prepare a 16-fold diluted sample and a 64-fold diluted sample.

(G) The 1-fold diluted sample, 4-fold diluted sample, 16-fold diluted sample, and 64-fold diluted sample obtained in (E) and (F) above were used as 4-stage diluted samples to which the sample preparation method of the present embodiment described above was not applied. Hereinafter, these 4-stage diluted samples are referred to as non-concentration-adjusted 4-stage diluted samples.

(H) 10 μL of solution was taken from each of the 1-fold diluted sample, 4-fold diluted sample, 16-fold diluted sample, and 64-fold diluted sample contained in the above-mentioned non-concentration-adjusted 4-stage diluted samples, and these were processed using the above-mentioned solid-phase extraction carrier-filled pipette tip in the procedure of steps S2 and S3, and the target protein molecules were eluted into 10 μL of elution solution (0.1% trifluoroacetic acid, 50% acetonitrile water). These four types of elution solution were used as 4-stage diluted samples in a state in which the sample preparation method of this embodiment was applied. Hereinafter, these 4-stage diluted samples are referred to as concentration-adjusted 4-stage diluted samples.

(I) 1 μL of each of the four types of samples contained in the non-adjusted 4-step diluted sample obtained in (G) above was taken, mixed with 1 μL of acetonitrile, and further mixed with 2 μL of α-cyano-4-hydroxycinnamic acid solution (solvent: 0.1% trifluoroacetic acid, 50% acetonitrile water, concentration: 10 mg/mL) as a matrix for MALDI. 2 μL of the mixed solution was dropped onto a sample plate and dried to prepare a sample for MALDI. In other words, this is the analytical sample corresponding to the non-adjusted 4-step diluted sample.

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

The four types of MALDI samples obtained in (I) above and the four types of MALDI samples obtained in (J) above were each measured using a time-of-flight mass spectrometer to obtain mass spectra. A Shimadzu MALDI-8020 was used for the measurements.

Mass spectra, which are the measurement results for the MALDI samples corresponding to the four-stage non-concentration diluted samples obtained in (I) above, are shown in Figure 2. From the top of Figure 2, the measurement results are for a 1-fold diluted sample, a 4-fold diluted sample, a 16-fold diluted sample, and a 64-fold diluted sample.
Mass spectra, which are the measurement results for the MALDI samples corresponding to the concentration-adjusted 4-step diluted samples obtained in (J) above, are shown in Figure 3. Figure 3 also shows, from the top, the measurement results for the 1-fold diluted sample, the 4-fold diluted sample, the 16-fold diluted sample, and the 64-fold diluted sample.

Figures 4 and 5 show the relationship between peak intensity and concentration for 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 Figures 2 and 3, respectively. In Figures 4 and 5, 1/1 on the horizontal axis indicates a 1-fold diluted sample, and 1/64 indicates a 64-fold diluted sample. In other words, the concentration increases from left to right on the horizontal axis. Figures 4 and 5 also show two results (first and second runs) for each mass-to-charge ratio value.

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

On the other hand, looking at the graph in Figure 5, which applies the sample preparation method of this embodiment, the peak intensity also increases as the concentration increases from 64-fold dilution to 16-fold dilution to 4-fold dilution. Also, when comparing 4-fold dilution with 1-fold dilution, there are some peaks where the intensity is slightly lower or the same, but overall the peak intensity is higher for the higher concentration 1-fold dilution. This is in contrast to the results in Figure 4.

This means that by using the sample preparation method of this embodiment, even if the total amount of protein in the original sample is large, only the amount of protein equivalent to the saturation amount of the solid-phase extraction carrier is eluted into the eluate, so the protein concentration in the eluate is appropriate (i.e., no ion suppression effect due to excess protein occurs). In addition, since the concentration of impurities that do not adsorb to the solid-phase extraction carrier should be relatively low in the eluate, it is fully expected that the ion suppression effect due to a large amount of impurities in the sample is suppressed.

From these experimental results, it can be seen that the concentration of target molecules in a mass spectrometry sample can be easily and accurately controlled using a solid-phase extraction carrier that is generally used for desalting and concentration. As a result, the amount of sample used during ionization in mass spectrometry is appropriate, and a clear mass spectrum can be obtained in which peaks corresponding to trace components can be easily observed.

In the above embodiments and experimental examples, 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. Similarly, by using a solid-phase extraction carrier that can selectively adsorb the substance under specified conditions (and desorb it under specified conditions), 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 living organisms, is used as the target substance, a carbon chip using graphite carbon, carbon nanotubes, or the like can be used as the solid-phase extraction support.

In addition, in the above-mentioned sample preparation method, 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, if the amount of the target molecule is small to begin with, it is advisable to reduce the amount of solid-phase extraction support accordingly to lower the saturation amount.

As already mentioned, the sample preparation method of the present invention can be used to prepare samples for mass spectrometry using various ionization methods, such as MALDI, laser desorption ionization (LDI), surface-assisted laser desorption ionization (SALDI), ionization methods used in secondary ion analysis, direct analysis in real time (DART), electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and probe electrospray ionization (PESI). Furthermore, samples prepared by the sample preparation method of the present invention can be subjected to analysis not only by mass spectrometry devices, but also by various other analytical devices.

Furthermore, the above-described embodiment and the variations described above are merely examples of the present invention, and any appropriate modifications, amendments, additions, etc. made within the spirit of the present invention will naturally be included within the scope of the claims of this application.

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

(Item 1) One aspect of the method for preparing an analytical sample according to the present invention comprises:
an adsorption step of adsorbing a target substance to a predetermined amount of solid phase extraction support in an amount that is saturated with the support;
a desorption step of desorbing the target substance from the solid-phase extraction support after the adsorption step into the elution solution using a predetermined amount of the elution solution;
a preparation step of preparing a sample using the eluate obtained after the desorption step;
The following is to be implemented.

(Paragraphs 2 to 4) In the method for preparing an analytical sample described in paragraph 1, the solid-phase extraction carrier may be packed in a pipette tip, a centrifuge column, or a liquid chromatography column.

According to the method for preparing an analytical sample described in paragraphs 1 to 4, a sample containing an appropriate amount of a target molecule at all times, that is, a sample in which the concentration of the target molecule is precisely controlled, can be easily prepared without relying on the experience or skill of the operator. As a result, when the target substance is, for example, a protein derived from a microorganism, the microorganism can be more accurately identified based on the analytical results obtained by mass spectrometry of the prepared sample.

(5) In the method for preparing an analytical sample described in any one of paragraphs 1 to 4, the preparation step may include a step of forming a sample on a plate for ionization by matrix-assisted laser desorption/ionization (MALDI) method.

(6) In addition, one aspect of the mass spectrometry method according to the present invention can be a method for irradiating a sample formed on the plate in the method for preparing an analytical sample according to claim 5 with laser light to ionize a target substance in the sample, and subjecting the generated ions or ions derived therefrom to mass spectrometry.

As described above, in the MALDI method, when the sample contains too much of the target substance or contains a large amount of impurities other than the target substance (especially substances that are more easily ionized than the target substance), the amount of ions generated from the target substance may be reduced due to the ion suppression effect. In contrast, the method for preparing an analytical sample described in paragraph 5 makes it possible to appropriately control the amount of target substance, including the sample for MALDI, and also to reduce the amount of impurities. Therefore, the method for preparing an analytical sample described in paragraph 5 and the mass spectrometry method described in paragraph 6 make it possible to efficiently generate ions derived from the target substance and obtain a mass spectrum with high sensitivity.

(7) In the mass spectrometry method described in 6, the target substance is a protein derived from a microorganism, and the microorganism can be identified using the results of ionizing the protein and performing mass spectrometry.

The mass spectrometry method described in paragraph 7 allows accurate identification of microorganisms without relying on the experience or skill of the operator.

Claims (7)

an adsorption step of adsorbing a target substance to a predetermined amount of solid phase extraction support in an amount that is saturated with the support;
a desorption step in which a predetermined amount of elution solution is used to desorb all of the target substance from the solid-phase extraction support after the adsorption step into the elution solution;
A preparation step of preparing a sample using an eluate containing the target substance at a concentration determined by the amount of the solid-phase extraction carrier, the saturation amount, and the amount of the eluate used in the desorption step, after the desorption step;
A method for preparing an analytical sample, comprising: The method for preparing an analytical sample according to claim 1, wherein the solid-phase extraction carrier is packed in a pipette tip. The method for preparing an analytical sample according to claim 1, wherein the solid-phase extraction carrier is packed in a centrifugal column. The method for preparing an analytical sample according to claim 1, wherein the solid-phase extraction carrier is packed in a liquid chromatography column. 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 on a plate. A mass spectrometry method in which a sample formed on the plate in 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 ions generated or ions derived therefrom are subjected to mass spectrometry. 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 results of ionizing and mass spectrometry of the protein.

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