JP2004184149A - Sample preparing device for maldi-tofms - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To save the labors for sample preparation work for an MALDI-TOFMS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry). <P>SOLUTION: A preparative LC (liquid chromatography) using an LC/MS (mass spectrometry) is used to collect a micro amount of sample liquid into each recessed part of a sample slide 21. That is, one portion of an eluate from a column 4 is analyzed by a mass spectrometer 6, a mass chromatogram is prepared for every objective mass. The branched eluates are fed to a fraction collector 7 equipped with the sample slide 21. A signal processing part 8 issues an indication to a control part 9 to conduct preparative fractionation only during a short time in the vicinity of a peak top, when a peak is appeared in the mass chromatogram, and the eluate of very small amount corresponding to the peak is dropped down into an assigned recessed part, in the fraction collector 7. When all the assigned eluate are collected, an analytical worker takes out the sample slide 21 to conduct dropping of a matrix solution, mixing and air-drying. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a sample preparation apparatus for a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOFMS = Matrix-assisted Laser Desorption Ionization / Time of Flight Mass Spectrometer).
[0002]
[Prior art]
In MALDI-TOFMS, a sample is prepared by mixing a solution called a matrix, such as sinapinic acid, and a trace amount of a substance to be analyzed substantially uniformly, and the sample is irradiated with laser light. Then, the matrix in the sample absorbs the laser beam to generate heat, and a part of the matrix is rapidly heated and vaporized together with the substance to be analyzed, whereby the substance to be analyzed is ionized. The ions thus generated are extracted by an electric field and introduced into a flight space having no electric and magnetic fields. The velocity of each ion flying in the flight space depends on its mass number, and the smaller the mass number, the greater the velocity. Therefore, various ions can be separated and detected for each mass number according to the flight time until reaching the ion detector.
[0003]
Such MALDI-TOFMS can perform analysis up to a high mass region where the molecular weight exceeds 10,000, which was impossible with other mass spectrometers, and the measurement of molecular weight distribution and structural analysis can be performed simultaneously and in a short time. It has been widely used in the analysis of biological macromolecules such as proteins and peptides because it can be performed in a short time and can analyze even substances that are unstable to heat and easily decompose without fragmentation. I have.
[0004]
An example of a method for preparing a sample for MALDI will be described in detail. Usually, a plurality of concave portions are formed in a metal plate called a sample slide. The analysis operator mixes the sample solution containing the substance to be analyzed and the matrix solution in advance, and applies the mixture in the concave portion of the sample slide. Alternatively, the sample solution and the matrix solution are respectively dropped into the concave portions of the sample slide, and the two are mixed therein. Thereafter, the sample slide and the solvent of the matrix solution are evaporated by blowing dry air on the sample slide, and the mixture of the matrix and the substance to be analyzed is left in the recess.
[0005]
[Patent Document 1]
JP-A-10-48110 (FIGS. 1 to 3 and paragraphs 0012 to 0020)
[0006]
[Problems to be solved by the invention]
Heretofore, the above-described sample preparation work has generally been performed manually exclusively by an analysis operator. Therefore, not only the work efficiency was poor, but also mistakes related to the work were likely to occur. In order to reduce the labor of such operations, a special sample preparation device for preparing a sample for MALDI-TOFMS has also been proposed. For example, in the sample preparation device disclosed in Patent Document 1, if a sample solution and a matrix solution are prepared, a movable pipette automatically aspirates the sample solution and the matrix solution, respectively, and drops the sample solution and the matrix solution into the concave portion of the sample slide. Then, a configuration is provided in which drying air is automatically applied to the sample slide to dry the sample slide.
[0007]
However, even if such a sample preparation device is used, it may be necessary to manually perform a considerable part of the preparation of the sample for MALDI-TOFMS. That is, as described above, MALDI-TOFMS is often used for the analysis of biologically related samples such as proteins and peptides, and such biologically related samples usually contain a very large variety of substances such as proteins. ing. Therefore, even if this sample is directly mixed with the matrix and analyzed, various substances other than the target substance to be analyzed become so-called contaminants, and it is practically impossible to perform accurate analysis. Therefore, when analyzing a biologically relevant sample, it is necessary to separate and extract the target analyte to a certain extent, and even if the sample preparation device as described above is used for pretreatment, In order to prepare a sample solution to be mixed, it is necessary to perform an operation of separating and extracting a substance to be analyzed from an original sample. In the end, such work must be performed manually, which is very inefficient and is a factor that hinders improvement in throughput.
[0008]
The present invention has been made to solve such problems, and a main object thereof is to prepare a sample for MALDI-TOFMS, particularly when the original sample contains other types of substances. However, an object of the present invention is to provide a sample preparation apparatus for MALDI-TOFMS that can efficiently prepare a sample while reducing the burden on an analysis operator.
[0009]
[Means for Solving the Problems]
The present invention made to solve the above problem is used for preparing a sample for MALDI-TOFMS in which a mixture of an analyte and a matrix is accommodated in a recess formed in a sample slide. A sample preparation device,
a) a column of a liquid chromatograph for separating a sample component contained in a sample solution in a time direction;
b) a detector for sequentially detecting an eluate eluted from the column;
c) a fraction collector for collecting a part of the eluate and dropping it into the concave portion of the sample slide;
d) control means for controlling the fraction collector so that the eluate is collected at a timing corresponding to a part of the target peak appearing in the chromatogram created based on the detection signal from the detector.
It is characterized by having.
[0010]
Embodiments and effects of the present invention
In the present invention, in general, a preparative liquid chromatograph used to separate and fractionate a sample containing many sample components for each component is used for preparing a sample for MALDI-TOFMS. Used for separation and collection of As one embodiment of the present invention, the detector is a mass spectrometer, for specifying a sample component or a set of sample components to be collected in a concave portion of a sample slide by a mass number or a mass number range. Further comprising input setting means, wherein the control means separates the eluate at a timing corresponding to a part of a peak appearing in a mass chromatogram created for each mass number or mass number range designated by the input setting means. As described above, the configuration may be such that the fraction collector is controlled.
[0011]
In this configuration, the analysis operator previously sets a sample component to be collected or a sample component group that is a set thereof as a mass number of the sample component or a mass number range including the sample component group. However, usually, the upper limit of the mass number that can be measured by LC / MS combining a liquid chromatograph and a mass spectrometer is smaller than the mass number that can be measured by MALDI-TOFMS. Therefore, the mass number of many analytes to be analyzed by MALDI-TOFMS exceeds the upper limit of the mass number that can be measured by LC / MS. Therefore, the mass number or mass number range set by the input setting means may be not the mass number of the molecular ion of the sample component itself to be collected but the mass number of an ion such as a fragment ion that can be estimated for the sample component.
[0012]
After performing the above settings, the sample is subjected to liquid chromatographic analysis, and the eluate eluted from the column is detected by a detector. If the detector is a mass spectrometer, the eluate introduced into the detector will be consumed. Therefore, the eluate is branched before the detector and introduced into the fraction collector in parallel. The control means detects a peak appearing in the mass chromatogram created for each of the set mass numbers or mass number ranges, and at a timing corresponding to a part of the peak, the branched eluate is collected. So that the operation of the fraction collector is controlled. As a result, the fraction collector drops a small amount of the eluate containing the set sample component or sample component group into a concave portion at a predetermined position on a predetermined sample slide. The analysis operator removes the sample slide from which the eluate has been collected in this manner, and drops the matrix solution into the concave portion by using the above-mentioned automated apparatus or manually, mixes the eluate with the eluate, and further performs drying. Just do it.
[0013]
As described above, in the sample preparation apparatus for MALDI-TOFMS according to the present invention, when analyzing a sample containing a large number of components, an operation for separating and extracting one or more sample components to be analyzed is performed. Since it can be performed automatically, the working efficiency can be greatly improved as compared with the related art, and the throughput of analysis is also improved. In addition to reducing the burden on the analysis operator, it is also effective in preventing the occurrence of a mistake in the operation related to sample preparation.
[0014]
【Example】
Hereinafter, a sample preparation apparatus for MALDI-TOFMS which is an embodiment of the present invention will be described with reference to FIGS.
[0015]
FIG. 1 is an overall configuration diagram of the present sample preparation apparatus. The basis of this apparatus is a preparative liquid chromatograph using a high performance liquid chromatograph. That is, the eluent (mobile phase) stored in the eluent tank 1 is sucked by the liquid sending pump 2 and flows into the column 4 via the sample introducing unit 3 at a constant flow rate. The sample solution injected into the mobile phase in the sample introduction unit 3 is introduced into the column 4 on the mobile phase, and is separated and eluted in the time direction while passing through the column 4. At the outlet of the column 4, a flow path branching section 5 is provided. In the flow path branching section 5, one of the flow paths branched at a predetermined branch ratio has a mass spectrometer 6, and the other flow path has a fraction collector. 7 are provided. Usually, a plurality of vials are set in the fraction collector for separating and fractionating the eluate, but the fraction collector 7 of the present apparatus is used for preparing a sample of MALDI-TOFMS. The sample slide 21 is configured to be set.
[0016]
In the mass spectrometer 6, the introduced eluate is sequentially vaporized, and each component molecule contained in the eluate is ionized. Then, these various ions are separated and detected for each mass number (mass / charge) by a mass analyzer such as a quadrupole mass filter. The detection signal of the mass spectrometer 6 is input to the signal processing unit 8. The signal processing unit 8 creates a mass chromatogram representing time-signal intensity focusing on a certain mass number or mass number range based on a detection signal from the mass spectrometer 6, and uses a peak appearing in the mass chromatogram. Thus, a control signal for instructing the sampling timing of the sample component to be sampled is generated. Upon receiving this control signal, the fractionation control unit 9 controls the solenoid valve, the arm, and the like incorporated in the fraction collector 7 to appropriately fractionate the eluate branched and supplied by the flow path branching unit 5. The sample slide 21 is dropped.
[0017]
FIG. 2 is a longitudinal sectional view of a sample slide 21 for explaining a sample preparation operation using the present embodiment. As shown in FIG. 2A, the sample slide 21 has a plurality of recesses 21a. In the fraction collector 7, a sample liquid (eluate from the column 4) 22 is dropped from the lower end hole of the needle 20 moved above the concave portion 21a of the sample slide 21 toward the concave portion 21a. At this time, the amount of the sample liquid dropped per one concave portion 21a is a very small amount of 1 mL or less.
[0018]
FIG. 3 is a simplified timing chart for explaining a sample collecting operation in the sample preparation apparatus. The operation of the present apparatus will be described with reference to FIG. Prior to the sample collection, the analysis operator sets the mass number corresponding to the substance to be analyzed, that is, the substance to be collected in the concave portion 21a, or the mass number range corresponding to the substance group including a plurality of substances from the input setting unit 10 to one. Specify a plurality. Now, consider a case where three substance groups respectively included in the three mass number ranges of m1, m2, and m3 are collected in different recesses 21a of one sample slide 21.
[0019]
As already described above, the molecular weight of proteins, peptides, and the like often analyzed by MALDI-TOFMS is large, and exceeds the upper limit of the mass number range that can be analyzed by LC / MS. Therefore, the mass number range of m1, m2, and m3 is not limited to the mass number of the molecular ion of the substance to be analyzed, but is such a mass that can characterize each substance group among the fragmentation ions generated from the substance. Choose a number. Therefore, the separation of substances based on such a mass number or mass number range is not perfect, and for example, other substances that accidentally enter the same mass number range may be mixed, but they can be eliminated by TOFMS. Practically not a problem.
[0020]
After setting as described above, the analysis operator instructs the start of sampling. Then, the liquid chromatographic analysis is started, the sample is injected into the mobile phase sent to the column 4 at a constant flow rate as described above, and the components temporally separated from the outlet of the column 4 are sequentially eluted. A part of the eluate is introduced into the mass spectrometer 6, and a detection signal is repeatedly obtained for the set mass number range m1, m2, and m3. In response to the detection signal, the signal processing unit 8 creates a mass chromatogram for each of the mass number ranges m1, m2, and m3, as shown in FIG.
[0021]
The signal processing unit 8 detects the start point of the peak in each mass chromatogram, and generates a control signal for performing fractionation for a predetermined time before and after the peak top (FIGS. 3B to 3D). )reference). The length of the flow path from the flow path branching section 5 to the mass spectrometer 6 and the length of the flow path from the flow path branching section 5 to the fraction collector 7 are required until the above-described control signal is generated. It is appropriately determined in consideration of the time and the time required for the eluate to be collected by the control signal to reach the fraction collector 7. Therefore, when the fractionation control unit 9 controls the operation of the fraction collector 7 based on the control signal generated as described above, the eluate corresponding to the vicinity of the peak top from which each control signal is generated is extracted from the needle 20. Each of the three concave portions 21a of the sample slide 21 is dropped (see FIG. 3E).
[0022]
When the target sample component (analyte) is collected in each of the recesses 21a of the sample slide 21 in this way, the analysis operator takes out the sample slide 21 and proceeds to the next operation. That is, the analysis operator aspirates a small amount of the matrix solution using a micropipette, and drops a part of the solution into the concave portion 21a where the eluate 22 is collected, as shown in FIG. 2B. At this time, the amount of the matrix solution 23 is considerably larger than the amount of the eluate 22. Then, stirring is performed so that the matrix solution 23 and the eluate 22 become uniform in the concave portion 21a. After that, a drying air is applied to the sample slide 21 to evaporate a solvent or the like and blow it off, leaving a mixture of the matrix and the substance to be analyzed in the recess 21a. Note that such operations may be performed manually, or may be performed using the above-described automation device. The sample slide 21 created in this way is set in the MALDI-TOFMS, and the analysis is executed according to a predetermined procedure.
[0023]
In the above embodiment, the control signal for fractionation is generated corresponding to one peak appearing in the mass chromatogram. However, for example, the control signal appears in the mass chromatogram having a different mass number or mass number range. A logical operation such as a logical sum or a logical product of peaks may be executed, and a control signal for sorting may be generated based on the result.
[0024]
Further, in the above embodiment, the mass spectrometer is used as the detector of the liquid chromatograph, but another detector such as an ultraviolet-visible spectrophotometer may be used. However, if a detector other than a mass spectrometer is used, it is necessary to determine the fractionation timing only by the peak appearing in the chromatogram (that is, information on the mass number cannot be used). Preliminary liquid chromatographic analysis is preferably performed to confirm the appearance. In this way, a chromatogram is created, and by specifying the peak appearing on the chromatogram by the analysis operator, it is possible to instruct an operation at the time of subsequent fractionation. Such a procedure is similar to a normal preparative liquid chromatograph.
[0025]
The above embodiment is merely an example, and it is apparent that changes and modifications can be made as appropriate within the scope of the present invention.
[Brief description of the drawings]
FIG. 1 is an overall configuration diagram of a sample preparation device for MALDI-TOFMS according to one embodiment of the present invention.
FIG. 2 is a diagram illustrating a sample preparation operation using the present embodiment.
FIG. 3 is a diagram for explaining the operation of the sample preparation device of the present embodiment.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Eluent tank 2 ... Liquid sending pump 3 ... Sample introduction part 4 ... Column 5 ... Channel branch part 6 ... Mass spectrometer 7 ... Fraction collector 8 ... Signal processing part 9 ... Fractionation control part 10 ... Input setting part 20 ... Needle 21 ... Sample slide 21a ... Recess 22 ... Eluate 23 ... Matrix solution

Claims (2)

A sample preparation device used to prepare a sample for MALDI-TOFMS in which a mixture of a substance to be analyzed and a matrix is accommodated in a recess formed in a sample slide,
a) a column of a liquid chromatograph for separating a sample component contained in a sample solution in a time direction;
b) a detector for sequentially detecting an eluate eluted from the column;
c) a fraction collector for collecting a part of the eluate and dropping it into the concave portion of the sample slide;
d) control means for controlling the fraction collector so that the eluate is collected at a timing corresponding to a part of the target peak appearing in the chromatogram created based on the detection signal from the detector.
A sample preparation device for MALDI-TOFMS, comprising: The detector is a mass spectrometer, and further includes an input setting unit for specifying a sample component or a sample component group that is a set thereof to be collected in a concave portion of the sample slide by a mass number or a mass number range, The control means is configured to collect the eluate at a timing corresponding to a part of a peak appearing in a mass chromatogram created for each mass number or mass number range designated by the input setting means, so that the fraction collector is collected. The sample preparation apparatus for MALDI-TOFMS according to claim 1, wherein

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