WO2008065704A1

WO2008065704A1 - Apparatus for analyzing mass spectrometric data

Info

Publication number: WO2008065704A1
Application number: PCT/JP2006/323611
Authority: WO
Inventors: Norio Mukai
Original assignee: Shimadzu Corporation
Priority date: 2006-11-27
Filing date: 2006-11-27
Publication date: 2008-06-05
Also published as: JP4788773B2; JPWO2008065704A1

Abstract

An apparatus for analyzing mass spectrometric data by which samples before and after a chemical change are individually separated by chromatography and then subjected to MS measurement and MSMS measurement by using a mass spectrometer and then the product formed by the chemical change is searched for based on the data thus obtained, wherein the chromatograms of the samples before and after the chemical change at the mass/charge ratio of a precursor ion in the MSMS measurement are compared and a peak appearing exclusively in the sample after the change is judged as the peak assignable to the product. Thus, the product can be searched for with the use of the chromatogram containing the sample component alone as the subject to be analyzed and, as a result, it can be avoided to judge a peak originating in noises as being assignable a product by error, which contributes to the increase in the searching accuracy. By narrowing the search to the subject while eliminating chromatograms free from the sample component, moreover, the analysis speed can be remarkably elevated.

Description

Specification

Mass spectrometry data analyzer

Technical field

TECHNICAL FIELD [0001] The present invention relates to a mass spectrometry data analysis apparatus, and more particularly to searching for substances generated by chemical changes such as metabolites using data obtained by a mass spectrometer capable of collecting MSMS spectra. The present invention relates to a data analysis apparatus.

Background art

[0002] Metabolites that are the products of chemical changes in vivo in various fields such as diagnosis of various diseases and diseases, evaluation of efficacy and safety of pharmaceuticals and functional foods, research on lifestyle and health In recent years, a method for comprehensive analysis of metabolites called metabolomics has attracted attention.

[0003] To analyze such metabolites, a technique that combines chromatography (typically high-performance liquid chromatography) and a high-accuracy mass spectrometer is useful (see Non-Patent Document 1, etc.). ). In this case, it is necessary to search for metabolites by analyzing the mass spectrum obtained by the mass spectrometer. In general, in the conventional metabolite search, a database in which the mass (strictly speaking, the mass-to-charge ratio m / z) of a metabolite with a known composition and structure is registered in advance. The metabolite is identified by comparing the mass of the peak appearing in the mass spectrum obtained by the analysis with a database. However, in such a search method, even if an unknown metabolite is registered in the database, it cannot be found even if it exists in the sample, and a certain peak is attributed to the unknown metabolite. Even if I can guess, I cannot know the composition of the metabolite.

[0004] However, since metabolites are metabolized through a predetermined metabolic path of the original substance (original substance), if the original substance is metabolized through a known metabolic path, the structural change is caused. Can be predicted. Therefore, in such a case, identification can be performed relatively easily even if the metabolite is not registered in the database. However, when the raw material is metabolized via an unexpected metabolic path or multiple metabolic paths, it is difficult to estimate the structure of the metabolic product in advance. [0005] Therefore, in order to search for such metabolites, chromatograph mass spectrometry is performed on samples before and after metabolism, and components that exist only after metabolism are listed as metabolite candidates. Yes. In other words, in the mass spectrometry data of post-metabolism samples, a chromatogram (this is called “extracted ion chromatogram”) is created by continuously cutting out the measured mass range with a certain mass width (strictly, mass-to-charge ratio width) Compared to a chromatogram cut out from the pre-metabolism sample with the same mass width, it is compared whether a chromatographic peak exists in the same time zone. Here, if the peak detected in the post-metabolism sample is also present in the pre-metabolism sample, it is determined that it is a substance that originally existed rather than the metabolite. On the other hand, if the same peak does not exist in the pre-metabolism sample, it is determined that the substance (ie, metabolite) generated during the metabolic process is highly likely!

[0006] Non-Patent Document 1: “Shimadzu Metabolomitas Solution”, [Searched on November 8, 2006], Shimadzu Corporation, Internet <URL: http://www.an.shimadzu.co.jp/ topics / 2006 1200b 丄 0 / metabo / metabolome.htm

Disclosure of the invention

Problems to be solved by the invention

[0007] However, in the conventional metabolite search methods as described above, fluctuations on the chromatogram that are originally noise may be determined as metabolite candidates, and therefore, the chromatograms listed as search results are included. The number of peaks tends to be enormous. In addition, the resolution of mass spectrometers in recent years has made it possible to set the width of the chromatogram cut out more precisely, resulting in a dramatic increase in the number of chromatograms to be compared. The number of noise peaks determined as metabolite candidates tends to increase. In addition, the processing time becomes longer as the number of chromatograms to be processed increases.

[0008] For example, FIG. 7 shows chromatograms cut out for each O.lm / z of data obtained by performing chromatographic mass spectrometry for each of the pre-metabolized sample and the post-metabolized sample. Here, only in the sample after metabolism, a peak (location indicated by an arrow) appears in the chromatogram of m / z = 455.3, and this is determined as a metabolite candidate. In the figure, only the chromatogram of m / z = 455.1 to 455.5 is illustrated. In this case, chromatogram generation and comparison processing are performed over the entire measurement mass range. Therefore, for example, when measurement is performed in the range of m / z = 100 to 800, it is necessary to process about 7000 chromatograms if processing is performed for each O.lm / ζ. With a high resolution and mass spectrometer, the chromatogram can be drawn with a finer mass width, which increases the number of treatments.

[0009] It should be noted that the above-mentioned problem is not limited to the search for metabolites, but the situation is the same for the search for products generated as a result of some chemical change of a certain substance.

[0010] The present invention has been made to solve such problems, and the purpose thereof is caused by a chemical change based on mass spectral data obtained by mass spectrometry. An object of the present invention is to provide a mass spectrometry data analysis apparatus capable of increasing the accuracy of the search and reducing the time required for the analysis process in the mass spectrometry data analysis apparatus for searching for a product.

Means for solving the problem

[0011] A mass spectrometry data analysis apparatus according to the present invention, which has been made to solve the above-mentioned problems, separates a sample before and after a chemical change with a chromatograph, and then performs MS measurement and MSMS using a mass spectrometer. A mass spectrometry data analysis device for searching for a product by the chemical change based on data obtained by measurement,

Product search means for comparing the chromatograms of the mass-to-charge ratios of the precursor ions in the MSMS measurement with respect to the sample before and after the chemical change, and determining the peak existing only in the sample after the change as the peak derived from the product It is characterized by having.

[0012] Here, the above-mentioned "i-change" is typically metabolism, and in this case, "product" means a metabolite (metabolite).

[0013] As shown in Fig. 6, during MSMS measurement, the precursor ion (precursor ion) present in the mass spectrum (hereinafter referred to as "MS spectrum") obtained by MS measurement without ion cleavage is cleaved. As a result, a mass spectrum (hereinafter, “MSMS spectrum”) of the resulting fragment ions is obtained. At this time, in general, a peak satisfying a predetermined condition in the MS spectrum, for example, a spectrum peak having an intensity equal to or higher than a predetermined threshold value. Or the peak having the maximum intensity at the time when the intensity of the chromatogram or the chromatogram intensity exceeds a predetermined threshold is selected as the precursor ion. Therefore, the acquisition of MSMS spectra clearly indicates that the precursor ions are derived from some material and not electrical or chemical noise on the chromatogram.

[0014] Therefore, in the mass spectrometry data analysis apparatus according to the present invention described above, by comparing the chromatogram generated for the mass-to-charge ratio of the precursor ion in such MSMS measurement with respect to the sample before and after the chemical change, It is possible to search for a product due to the chemical change by analyzing only a chromatogram including a sample component.

The invention's effect

[0015] According to the mass spectrometry data analysis apparatus of the present invention having the above-described configuration, it is possible to prevent a peak due to noise from being determined as a metabolite and to improve the accuracy of metabolite search. it can. In addition, the analysis speed can be significantly reduced by narrowing down the processing targets by eliminating chromatograms that do not contain sample components.

[0016] Note that the mass spectrometry data analysis apparatus according to the present invention further includes a chromatogram including a peak determined as a product-derived peak by the product search means, and an MS spectrum of the precursor ion and It is desirable to have display means to display with MSMS spectrum.

[0017] From this, it becomes possible to easily refer to the MS spectrum and fragment pattern of the MSMS spectrum related to the product searched as described above together with the chromatogram, thereby saving the user's identification operation. be able to.

[0018] When using a high resolution mass spectrometer, the number of mass digits obtained ranges from 4 to 5 digits after the decimal point. Therefore, by using this, it is possible to estimate to some extent the elemental composition of the target substance from the mathematical combination of elemental masses. Therefore, it is more desirable that the mass spectrometry data analysis apparatus according to the present invention further includes a composition estimation unit that estimates the composition of the product based on the mass-to-charge ratio of the precursor ions.

[0019] This makes it possible to estimate the yarn formation of the product mentioned by the product search means, and display the result together with the retention time of the chromatogram, for example. Can help. Metabolite is the structure of the substance before metabolism If metabolite composition candidates are displayed as described above, the composition before metabolism, which is often maintained to some extent, is very useful information for identification. Brief Description of Drawings

FIG. 1 is a schematic configuration diagram of an embodiment of a mass spectrometry system including a data analysis apparatus according to the present invention.

FIG. 2 is a flowchart showing an example of the procedure of an unknown metabolite analysis processing operation by the mass spectrometry system of the present embodiment.

FIG. 3 is a diagram for explaining an unknown metabolite search method in the data analysis apparatus of the present embodiment.

FIG. 4 is a diagram showing a state in which a chromatogram, an MS spectrum, and an MSMS spectrum of an unknown metabolite are displayed in the data analysis apparatus of this example.

FIG. 5 is a diagram showing a state in which the composition estimation result of an unknown metabolite is displayed in the data analysis apparatus of this example.

FIG. 6 is a diagram for explaining an MSMS spectrum acquisition method.

FIG. 7 is a diagram for explaining an unknown metabolite search method in a conventional data analysis apparatus.

Explanation of symbols

20 · Mass spectrometer

30 ... Data processing section

31 ··· Data acquisition part

32 '· Chromatogram creation part

33 · Metabolite search unit

34 ··· Composition estimation section

35 ··· Display control unit

40 ··· Display

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, an embodiment of a mass spectrometry data analysis apparatus according to the present invention will be described in detail with reference to the drawings.

FIG. 1 shows an embodiment of a mass spectrometry system including a mass spectrometry data analysis apparatus according to the present invention. It is a schematic block diagram of an example. The mass spectrometer 20 is, for example, an ion trap time-of-flight mass spectrometer (IT—TOFMS), which sequentially ionizes samples eluted from the high performance liquid chromatograph (HPLC) 10 to perform MS measurement and MSMS measurement. The acquired data is sent to the data processing unit 30. The data processing unit 30 includes a data acquisition unit 31, a chromatogram creation unit 32, a metabolite search unit 33, a composition estimation unit 34, and a display control unit 35 as functional blocks. The data acquisition unit 31 receives data (for example, MS spectrum and MSMS spectrum) transmitted from the mass spectrometer 20, and the chromatogram creation unit 32 is based on the MS spectrum data acquired by the data acquisition unit 31. Thus, an extracted ion chromatogram (Extracted Ion Chromatogram: XIC) in which the ionic strength at a predetermined mass charge ratio is plotted on the vertical axis and the holding time on the horizontal axis is created (details will be described later). The metabolite search unit 33 compares the extracted ion chromatogram between the pre-metabolism sample and the post-metabolism sample, and extracts a peak that exists only in the post-metabolism sample as a metabolite-derived peak. The composition estimation unit 34 estimates the composition of the metabolite based on the mass-to-charge ratio of the precursor ions. In addition, the display control unit 35 performs processing for causing the display unit 40 connected to the data processing unit 30 to display the data output from the above-described units. Note that the data processing unit 30 is usually embodied by a personal computer, and the analysis processing function as described above is achieved by executing a predetermined program installed in the computer.

Next, an example of metabolite search and identification processing by the mass spectrometry system will be described with reference to the flowchart of FIG. First, the pre-metabolism sample and the post-metabolism sample are analyzed by HPLC 10 and mass spectrometer 20, respectively, to obtain mass spectrum data (Step Sl l). At this time, the mass spectrometer 20 acquires MS spectrum data by mass analysis (MS measurement) without ion cleavage operation, and automatically selects an ion having an intensity equal to or higher than a predetermined threshold as a precursor ion. MSMS spectral data is obtained by mass analysis (MSMS measurement) of fragment ions generated by cleavage of the precursor ions.

The data acquired as described above is sent from the mass spectrometer 20 to the data processing unit 30. In the chromatogram creation unit 32, data on the mass-to-charge ratio of the precursor ion in the MSMS measurement is obtained from the data set of the MS spectrum acquired over the entire measurement time. Extraction is performed to produce an extracted ion chromatogram at the mass-to-charge ratio (step 12). Subsequently, the extraction chromatograms of the pre-metabolized sample and the post-metabolized sample are compared in the metabolite search unit 33, and a peak that exists only in the post-metabolized sample is extracted (step S13).

[0026] Specifically, first, a peak existing on the extracted ion chromatogram of the sample after metabolism is detected, and then a peak is detected at the same time zone in the extracted ion chromatogram at the same mass-to-charge ratio of the sample before metabolism. It is determined whether or not there is an existing force. At this time, if a similar peak is also present in the pre-metabolized sample, it is determined that the peak is derived from the original substance. If a similar peak does not exist in the pre-metabolic sample, the peak is It is determined that the substance originates from a substance (metabolite) newly generated by the process of metabolism.

FIG. 3 shows an example of the extracted ion chromatogram generated in step S12. Fig 3

(b) is an extracted ion chromatogram of the precursor ion mass-to-charge ratio prepared for the post-metabolized sample, and Fig. 3 (a) is an extracted ion chromatogram of the pre-metabolized sample generated for the same mass-to-charge ratio. Gram. In the post-metabolized sample, the extracted ion chromatogram of the mass-to-charge ratio of the precursor ions, m / z = 442.28, m / z = 367.72, m / z = 455.30, m / z = 842.97, m / z = 247.16, Each has a peak derived from the sample component. When these are compared with the extracted ion chromatograms of the premetabolized samples prepared for the same mass-to-charge ratio, the extracted ion chromatograms of m / z = 442.28, m / z = 455.30, m / z = 842.97 are obtained in the post-metabolized samples. Since the peak present in the gram does not exist in the pre-metabolism sample, it is determined that these peaks are derived from metabolites. According to the present invention, only the extracted ion chromatogram created for the mass-to-charge ratio of the precursor ions as described above is used as a target for comparison processing, so that it is created for each predetermined mass-to-charge ratio width as in the past. Compared with the case of processing all extracted ion chromatograms, metabolite searches can be performed more efficiently and reliably.

[0028] When the metabolite search is completed, the result is displayed on the display unit 40 (step S14). Fig. 4 shows an example of the display screen at this time. For the peak determined as a metabolite, together with the extracted ion chromatogram ("precursor ion mass chromatogram" in the figure), the above precursor ion Is the MS spectrum and MSMS spectrum of . When finally identifying the metabolite searched by the above, the fragment ion information of the MSMS spectrum as well as the MS spectrum is an effective hand. Therefore, by performing such display, the user can easily refer to necessary information, and the efficiency of identification work by the user can be improved. In the case of using a mass spectrometer having a function to obtain an MS ⁿ Supegairu further cleaves MSMS spectrum peak, the MS ⁿ spectra, the MS spectrum, be displayed with MSMS spectra desirable.

[0029] Subsequently, the composition estimation unit 34 estimates the elemental composition of the metabolite based on the mathematical combination of the element masses from the mass-to-charge ratio of the precursor ions (step S15). The composition candidate and the retention time of the chromatogram peak are displayed on the display unit 40 in a table format (step S16). Fig. 5 shows an example of a screen display showing such a composition estimation result. Here, the display screen shows the retention time (RT), precursor ion mass (Ion m / z), area percentage (Area%) of the chromatogram peak, and estimated elemental composition for the searched metabolite. (Formula (M)), the degree of agreement of the estimated elemental composition (Score), the difference between the mass of the estimated elemental composition and the mass of the precursor ion (Diff (mDa)) t, and other information are displayed. By performing such display, it is possible to provide information useful for metabolite identification by the user.

[0030] It should be noted that the above-described embodiment is an example of the present invention, and it is obvious that modifications, changes, additions, and the like as appropriate within the scope of the present invention are included in the scope of the claims of the present application. For example, the present invention can be used not only for changes due to metabolism but also for searching for unknown substances that are generated with general chemical changes such as chemical changes due to the synthesis of polymer compounds.

Claims

The scope of the claims

[1] Mass spectrometry in which samples before and after a chemical change are chromatographed, and then products are searched for by the chemical change based on the data obtained by performing MS measurement and MSMS measurement using a mass spectrometer. A data analysis device,

Product search means for comparing the chromatograms of the mass-to-charge ratios of the precursor ions in the MSMS measurement with respect to the sample before and after the chemical change, and determining the peak existing only in the sample after the change as the peak derived from the product A mass spectrometry data analysis apparatus comprising:

[2] In addition,

The mass spectrometric data according to claim 1, further comprising display means for displaying a chromatogram including a product-derived peak determined by the product search means together with an MS spectrum and an MSMS spectrum of the precursor ion. Analysis device.

[3] In addition,

3. The mass spectrometry data analysis apparatus according to claim 1, further comprising a composition estimation unit that estimates a composition of the product based on a mass-to-charge ratio of the precursor ions.

[4] A program for causing a computer to function as the mass spectrometry data analysis apparatus according to any one of claims 1 to 3.