JP7396208B2 - Ribosomal protein discrimination method, biological species identification method, mass spectrometer and program - Google Patents

Description

The present invention relates to a method for determining ribosomal proteins, a method for identifying biological species, a mass spectrometer, and a program.

A method using ribosomal proteins as a biomarker is known as a method for identifying bacterial species using mass spectrometry. For example, in Non-Patent Documents 1 and 2, the observed mass of bacterial ribosomal proteins observed on a mass spectrum is compared with the calculated mass obtained from the amino acid sequence registered in a protein database, and bacteria can be quickly identified. Disclosed are methods for selecting suitable biomarkers for and identifying bacterial species that provide information on their amino acid sequences.

Kanae Teramoto et al., “Comparative analysis of ribosomal proteins of lactic acid bacteria genome decoded strains using matrix-assisted laser desorption ionization mass spectrometry,” J Mass Spectrom. Soc. Jpn, Vol. 56, No. 1, 2008. Kanae Teramoto and Hiroaki Sato, "Advances in rapid identification and classification of bacteria by mass spectrometry," J Mass Spectrom. Soc. Jpn, Vol. 56, No. 3, 2008.

However, the calculated mass of a protein obtained from a database often does not match the actually observed mass. One of the reasons for this is that the amino acid sequences registered in databases are mechanically translated DNA sequences of genes, so post-translational modifications of proteins are not taken into account. Because the calculated mass and the observed mass matched, and there were only a limited number of proteins that could be identified as ribosomal proteins, the accuracy of bacterial species identification was not sufficient.

Here, it is thought that ribosomal proteins can be identified if the calculated mass of the protein is calculated taking into account post-translational modifications. However, since the types and patterns of post-translational modifications can be complexly combined, we calculate the calculated masses of many ribosomal proteins that take into account possible post-translational modifications and compare them with the observed masses to determine whether they are ribosomal proteins. It is not reasonable to do so.

An object of the present invention is to provide a method by which it is possible to easily determine whether a protein shown as a peak on a mass spectrum is a ribosomal protein by mass spectrometry.

The present invention compares the observed m/z value of a peak on a mass spectrum detected by mass spectrometry of a sample containing ribosomal proteins with the first calculated m/z value based on a database, and calculates the detected peak. A protein corresponding to a peak with a relative intensity of 50 to 150% with respect to an approximate curve or an approximate straight line obtained from the attribution step of attributing at least a part of to ribosomal proteins and the relative intensity of the peaks assigned to ribosomal proteins. The present invention relates to a method for determining a ribosomal protein, including a step of estimating that the protein is a ribosomal protein.

The present invention includes a mass separator that separates ions based on m/z values, a detector that detects the ions separated by the mass separator, and a mass spectrometer that creates a mass spectrum based on the ions detected by the detector. In the spectrum creation section and the mass spectrum, determine the peaks attributed to ribosomal proteins based on the database, create an approximate curve or approximate straight line of the relative intensity of the peak, and calculate the relative intensity with respect to the approximate curve or approximate straight line. The present invention also relates to a mass spectrometer including a discriminator that selects a peak that is 50% to 150%.

The present invention determines peaks attributed to ribosomal proteins based on a database in a mass spectrum acquired by a mass spectrometer, creates an approximate curve or approximate straight line of the relative intensity of the peak, and On the other hand, the present invention also relates to a program that causes a processing device to select a peak whose relative intensity is 50% to 150%.

According to the present invention, it is possible to easily determine whether a protein shown as a peak on a mass spectrum is a ribosomal protein by mass spectrometry.

FIG. 1 is a diagram showing a schematic configuration of a mass spectrometer according to a ribosomal protein discrimination method according to an embodiment. FIG. 2 is a flowchart showing the flow of a ribosomal protein discrimination method according to one embodiment. FIG. 2 is a flowchart showing the flow of a ribosomal protein discrimination method according to one embodiment. 2 is a scatter diagram showing the observed m/z values of peaks assigned to ribosomal proteins and peaks not assigned to ribosomal proteins, and the relative intensities of the peaks in Experiment 1. FIG. FIG. 2 is a scatter diagram showing the observed m/z values and relative intensities of peaks newly assigned to ribosomal proteins by the ribosomal protein discrimination method in Experiment 1. FIG. FIG. 2 is a scatter diagram showing observed m/z values of peaks assigned to ribosomal proteins and peaks not assigned to ribosomal proteins, and relative intensities of the peaks in Experiment 2. FIG. FIG. 3 is a scatter diagram showing the observed m/z values and relative intensities of peaks newly assigned to ribosomal proteins by the ribosomal protein discrimination method in Experiment 2. FIG. FIG. 3 is a scatter diagram showing observed m/z values of peaks assigned to ribosomal proteins and peaks not assigned to ribosomal proteins, and relative intensities of the peaks in Experiment 3. FIG. FIG. 3 is a scatter diagram showing the observed m/z values and relative intensities of peaks newly assigned to ribosomal proteins by the ribosomal protein discrimination method in Experiment 3. FIG.

The method for determining ribosomal proteins according to the present invention includes:
The observed m/z value shown by the peak on the mass spectrum detected by mass spectrometry of a sample containing ribosomal protein is compared with the first calculated m/z value based on the database, and at least part of the detected peak is an attribution step of attributing the protein to a ribosomal protein;
an estimation step of estimating that a protein corresponding to a peak with a relative intensity of 50% to 150% is a ribosomal protein with respect to an approximate curve or an approximate straight line obtained from the relative intensities of peaks assigned to ribosomal proteins; .

According to the method for determining ribosomal proteins according to the present invention, it is possible to easily determine whether a protein indicated by a peak on a mass spectrum is a ribosomal protein by mass spectrometry. Ribosomes are composed of more than 50 types of proteins, and each protein exists in approximately equal moles. Therefore, when a sample containing ribosomal proteins is subjected to mass spectrometry, ideally the peaks of each ribosomal protein on the mass spectrum are considered to exhibit similar relative intensities. Therefore, it can be determined that a peak showing a relative intensity comparable to that of a peak assigned to a ribosomal protein is likely to be a ribosomal protein.

Each step will be explained in detail below.

[Attribution process]
In the attribution step, the observed m/z value shown by the peak on the mass spectrum detected by mass spectrometry of a sample containing ribosomal proteins is compared with the first calculated m/z value based on the database, and the detected peak is Attribute at least a portion of the protein to ribosomal proteins.

The first calculated m/z value can be calculated by obtaining amino acid sequence information from databases such as GenBank, RefSeq, TPA, SwissProt, PIR, PRF, and PDB. At least one selected from amino acid sequences of proteins and DNA sequences of genes is registered in the database. The database includes information on taxonomic groups (family, genus, species, etc.) to which cells belong. The first calculated m/z value may be calculated taking into account post-translational modifications. This is because it becomes easier to match the observed m/z value and the first calculated m/z value, and it is possible to increase the number of peaks attributed to ribosomal proteins among the peaks detected on the mass spectrum. The post-translational modification is preferably an N-terminal methionine truncation. This is because ribosomal proteins often undergo N-terminal methionine cleavage as a post-translational modification. Particularly in bacteria, many of the first calculated m/z values calculated by considering N-terminal methionine cleavage agree with the observed m/z values, and most of the ribosomal protein peaks can be attributed. N-terminal methionine is defined when the second amino acid residue is glycine (G), alanine (A), serine (S), proline (P), valine (V), threonine (T) and cysteine (C). It is known to be easily cut. Even if these amino acid residues are in the second position, cleavage of the N-terminal methionine may not occur.

In this specification, when the difference between the calculated m/z value and the observed m/z value is within 500 ppm in the case of the external standard method, it is determined that the calculated m/z value and the observed m/z value match. The assigned peak is self-calibrated using the calculated m/z value of the matched protein, and the difference between the calculated m/z value and the observed m/z value of the peak used for self-calibration is within 200 ppm. It is preferable to confirm that.

From the viewpoint of containing equimolar amounts of ribosomal proteins constituting ribosomes, the sample preferably contains cell-derived ribosomal proteins. The sample may contain ribosomes or the cells themselves.

The sample may be of prokaryotic or eukaryotic origin. The sample is preferably of microbial origin and may contain unknown microorganisms. Prokaryotes include bacteria and archaea. Examples of bacteria include bacteria of the genus Escherichia (such as Escherichia coli), bacteria of the genus Bacillus (such as Bacillus subtilis), bacteria of the genus Lactobacillus (such as lactic acid bacteria), bacteria of the genus Synechocystis (such as cyanobacteria), and bacteria of the genus Mycobacteria (such as actinobacteria). It will be done. Archaea include Methanophilus, Methanococcus, Thermococcus, Phyllococcus, and the like. Eukaryotes include animals, plants, fungi and protists. Fungi include filamentous fungi, yeasts, mushrooms, molds, and the like, and include phylum Chytridomycota, Zygomycota, Ascomycota, Basidiomycota, Glomusmycota, Microsporidia, and the like. The phylum Ascomycota includes the genus Aspercillus (Aspergillus mold, etc.), the genus Penicillium (Blue mold, etc.), the genus Saccharomyces (budding yeast, etc.), and the like. In eukaryotes, there are fewer peaks that can be assigned to ribosomal proteins considering only N-terminal methionine cleavage than in bacteria. Ribosomal proteins of eukaryotes, including fungi, often undergo complex post-translational modifications compared to ribosomal proteins of prokaryotes, and the first calculated m/z value calculated from the database and the mass This is because a difference is likely to occur between the m/z value observed by analysis and the observed m/z value. According to the method for determining ribosomal proteins according to the present invention, it is possible to easily determine whether a protein represented by a peak on a mass spectrum is a ribosomal protein even in a sample derived from eukaryotes.

The sample may be appropriately prepared from cells, for example, it may be a fraction obtained by extracting proteins from cells, and preferably it is a fraction obtained by fractionating ribosomal proteins. When a fraction obtained by fractionating ribosomal proteins is used as a sample, the proportion of ribosomal proteins contained in the sample increases, so that ribosomal proteins can be determined with higher accuracy. The method for fractionating ribosomal proteins is not particularly limited as long as ribosomal proteins can be extracted or concentrated, and examples thereof include a method of crushing cells and ultrafiltration, an ultracentrifugation method, and the like.

The mass spectrometry is not particularly limited as long as it is a mass spectrometry method that can quantitatively measure ribosomal proteins, but it is preferably a mass spectrometry method that uses a soft ionization method that does not easily cause decomposition of high molecular weight compounds. Such mass spectrometry methods include analysis methods such as matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS). Mass spectrometry is preferably MALDI-MS. Since most ribosomal proteins are basic proteins with high proton affinity, [M+H] ⁺ ions are easily generated during the MALDI process. Furthermore, the molecular weight of ribosomal proteins is approximately 5,000 to 20,000, and MALDI-MS allows the mass of ribosomal proteins to be determined within an error range of several Da. Therefore, when mass spectrometry is MALDI-MS, ribosomal protein peaks can be easily detected from the sample, and the error between the observed m/z value and the first calculated m/z value can be reduced.

[Estimation process]
In the estimation step, proteins corresponding to peaks with relative intensities of 50 to 150% are estimated to be ribosomal proteins with respect to an approximate curve or an approximate straight line obtained from the relative intensities of peaks assigned to ribosomal proteins.

As described above, since the ribosomal proteins constituting the ribosome exist in approximately equimolar amounts, the relative intensities of the peaks assigned to the ribosomal proteins are ideally at the same level. For example, ribosomal subunit proteins are further prepared from the ribosomal fraction, each subunit protein is separated by liquid chromatography (LC), and each sample is subjected to mass spectrometry along with a standard sample of known concentration to create a mass spectrum. When the assigned peaks are selected and an approximate straight line is created for a scatter diagram showing their relative intensities, the approximate straight line becomes a horizontal straight line (relative intensity is constant) under the process that the ionization efficiency is constant. However, even when proteins present in equal moles are detected, the relative intensities of the peaks on the mass spectrum may not be the same depending on the detection method. For example, when MALDI-MS is performed, due to the mass discrimination effect, when the m/z value is small, the relative intensity is detected to be large, and when the m/z value is large, the relative intensity is detected to be small. Therefore, when a sample is measured by MALDI-MS, ribosomal proteins present in equal moles are approximated by a straight line or curve in which the relative intensity decreases as the m/z value increases. In this specification, the accuracy of an approximated curve or an approximated straight line is determined to be good if the correlation coefficient (R ² ) is 0.70 or more, preferably 0.80 or more. .

Among the unidentified peaks that are not assigned to ribosomal proteins by the first calculated m/z value, peaks whose relative intensity is close to an approximate curve or an approximate straight line are estimated to be ribosomal proteins. The expression that the relative intensity of the peak is close to the approximate curve or approximate straight line means that the relative intensity of the peak is 50 to 150%, preferably 60 to 140%, and more preferably 60 to 140% of the relative intensity of the approximate curve or approximate straight line. is 70 to 130%, more preferably 80 to 120%, and most preferably 90 to 110%.

[Verification process]
The method for identifying ribosomal proteins may further include a verification step of calculating a second calculated m/z value that takes post-translational modification into account for the protein estimated to be a ribosomal protein, and comparing it with the observed m/z value. preferable. For a protein estimated to be a ribosomal protein, when the second calculated m/z value calculated by taking into account post-translational modification and the observed m/z value match, it is more accurate that the peak is a ribosomal protein. Can be distinguished well. Additionally, information on the amino acid sequence and post-translational modification of ribosomal proteins can be obtained.

The second calculated m/z value is calculated taking into account post-translational modifications. Post-translational modifications to be considered here include N-terminal methionine cleavage, acetylation, methylation, phosphorylation, glycosylation, oxidation, ubiquitination, SUMOylation, lipid modification, and combinations thereof. In the verification process, modification patterns that are different from the post-translational modifications in the attribution process are considered. For example, if the first calculated m/z value is calculated in consideration of post-translational modification of N-terminal methionine cleavage in the attribution step, in the verification step, the second calculated m/z value when N-terminal methionine cleavage has not occurred, Alternatively, a second calculated m/z value is calculated when the protein is subjected to other post-translational modifications in addition to N-terminal methionine cleavage. In the estimation process, peaks on the mass spectrum that are estimated to be ribosomal proteins are selected, so there is no need to consider complex post-translational modifications for all peaks on the mass spectrum, making it easier to identify ribosomal proteins. can be determined.

[Method for identifying biological species]
The biological species identification method according to the present invention identifies ribosomal proteins using the above-described method for determining ribosomal proteins, and identifies the species of organisms that provides information on the amino acid sequence of the ribosomal proteins.

Ribosomal proteins are housekeeping genes that are involved in life support and are highly conserved across species. Ribosomal proteins are used as biomarkers to identify biological species because slight differences in amino acid sequences and resulting differences in mass can serve as decisive indicators for evaluating differences in biological species. Furthermore, since ribosomes are structures that exist in large numbers in cells, ribosomal proteins are easily detected by mass spectrometry.

According to the method for determining ribosomal proteins described above, it is possible to increase the number of proteins determined to be ribosomal proteins, so that identification of biological species can be performed more easily and accurately. The organism to be identified is preferably a microorganism whose size is difficult to identify with the naked eye. According to the biological species identification method according to the present invention, useful microorganisms can also be screened.

In particular, when MALDI-MS is used as mass spectrometry, even slight variations in the amino acid sequence of ribosomal proteins can be easily detected as a peak shift. According to the method for identifying biological species according to the present invention, not only species but also subspecies and highly related strains can be identified.

[Mass spectrometer]
As an example of a method for determining ribosomal proteins, a method for determining ribosomal proteins using a mass spectrometer will be described.
A mass spectrometer according to one aspect of the present invention includes:
a mass separation unit that separates ions based on m/z values;
a detection unit that detects ions separated by the mass separation unit;
a mass spectrum creation unit that creates a mass spectrum based on the ions detected by the detection unit;
In the mass spectrum, determine the peaks assigned to ribosomal proteins based on the database, create an approximate curve or approximate straight line of the relative intensity of the peak, and compare the relative intensity with 50 to 150% of the approximate curve or approximate straight line. and a discrimination unit that selects a certain peak.
According to this mass spectrometer, it is possible to easily determine whether a protein shown as a peak on a mass spectrum is a ribosomal protein by mass spectrometry.

FIG. 1 shows a schematic configuration of a mass spectrometer 1 according to an embodiment. The mass spectrometer 1 includes a measurement section 100 and a control section 40. A part or all of the functions of the control section 40 may be placed in a computer, server, etc. that is physically separate from the measurement section 100.

The measurement unit 100 includes an ionization unit 10 that generates protein ions S derived from a sample, an ion acceleration unit 21, a mass separation unit 22, and a detection unit 30. The sample measured by the measurement unit 100 contains ribosomal proteins. The movement of protein ions S generated in the ionization section 10 is schematically shown by arrow A1.

The control section 40 includes an input section 41 , a communication section 42 , a storage section 43 , an output section 44 , and a processing section 50 . The processing section 50 includes an apparatus control section 51, a mass spectrum creation section 52, a discrimination section 53, and preferably a verification section 54. The flow of the detection signal of protein ions S from the detection unit 30 of the measurement unit 100 is schematically shown by arrow A2. Control of the measurement unit 100 by the device control unit 51 is schematically shown by arrow A3.

The ionization unit 10 of the measurement unit 100 includes a sample plate holder (not shown) that supports a sample plate, and an ion source including a laser device (not shown) that irradiates laser light onto the sample plate, and performs matrix-assisted laser desorption ionization of sample components. (MALDI) method. As a method for ionizing the sample, any soft ionization method such as the electrospray (ESI) method can be used in addition to the MALDI method. When performing ionization by the ESI method, it is preferable that the mass spectrometer 1 further includes a liquid chromatograph, and the ionization section 10 ionizes sample components separated by the liquid chromatograph, since high resolution can be obtained. .

After the sample is placed in the sample plate, the matrix is added to the sample and allowed to dry. Thereafter, the sample plate is placed in a sample plate holder within the vacuum container of the ionization section 10. Although the type of matrix is not particularly limited, it is preferable to use sinapinic acid or α-cyano-4-hydroxycinnamic acid (CHCA) from the viewpoint of efficiently ionizing the protein sample.

The ionization unit 10 reduces the pressure of a vacuum container in which a sample plate is installed, and then irradiates each sample on the sample plate with laser light to sequentially ionize the sample. The type of laser device that irradiates laser light is not particularly limited as long as it can emit light that is absorbed by the selected matrix. For example, when the matrix contains sinapinic acid or CHCA, _N2 laser (wavelength 337 nm) is used. etc. can be suitably used. The protein ions S ionized in the ionization section 10 are extracted by an electric field by an extraction electrode (not shown) and introduced into the ion acceleration section 21 .

The ion acceleration unit 21 includes an acceleration electrode 210 and accelerates the introduced protein ions S. The flow of accelerated protein ions S is appropriately focused by an ion lens (not shown) and introduced into the mass separation unit 22.

The mass separation unit 22 includes a flight tube 220, and separates protein ions S based on the difference in flight time when each protein ion S flies inside the flight tube 220. Although a linear type flight tube 220 is shown in FIG. 1, a reflectron type, multiturn type, or the like may be used. The mass spectrometry method is not particularly limited as long as the protein ions S contained in the sample can be separated and detected.

The detection unit 30 includes an ion detector such as a multi-channel plate, detects the protein ions S separated by the mass separation unit 22, and outputs a detection signal with an intensity corresponding to the number of ions incident on the detection unit 30. . The detection signal output from the detection section 30 is input to the processing section 50 of the control section 40.

The input unit 41 of the control unit 40 includes input devices such as a mouse, a keyboard, various buttons, and/or a touch panel. The input unit 41 receives information necessary for controlling the operation of the measuring unit 100, information necessary for processing performed by the processing unit 50, etc. from the user.

The communication unit 42 of the control unit 40 includes a communication device capable of communicating via wireless or wired connection such as the Internet. The communication unit 42 receives data necessary for processing by the determination unit 53 and verification unit 54, transmits data processed by the processing unit 50 such as determination results, and transmits and receives necessary data as appropriate.

The storage unit 43 of the control unit 40 includes a nonvolatile storage medium. The storage unit 43 stores a calculated m/z value based on a database, a mass spectrum created by the mass spectrum creation unit 52, measurement data output from the measurement unit 100, a program for the processing unit 50 to execute processing, etc. .

The output unit 44 of the control unit 40 is configured to include a display device such as a liquid crystal monitor, a printer, etc., and displays information regarding the measurement of the measurement unit 100, the results of the determination unit 53 and the verification unit 54, etc. on the display device. or print it on paper.

The processing unit 50 of the control unit 40 is configured to include a processor such as a CPU, and functions as a main body for controlling the mass spectrometer 1 . The processing unit 50 performs various processes by executing programs stored in the storage unit 43 and the like.

The device control unit 51 of the processing unit 50 controls the operation of the measurement unit 100 based on data related to measurement conditions input from the input unit 41.

The mass spectrum creation unit 52 of the processing unit 50 converts the flight time into an m/z value from the measurement data including the amount of protein detected by the detection unit 30 and the flight time of the protein, and calculates each m/z value. A mass spectrum MS is created that indicates the detected amount corresponding to the value.

The discrimination unit 53 determines peaks attributed to ribosomal proteins in the mass spectrum based on the database, creates an approximate curve or approximate straight line of the relative intensity of the peak, and calculates the relative intensity with respect to the approximate curve or approximate straight line. Select peaks that are between 50 and 150%.

The determining unit 53 calculates the first calculated m/z value of the ribosomal protein based on the database. At least one selected from the amino acid sequence of a protein and the DNA sequence of a gene is registered in the database, and the first calculated m/z value is calculated based on this information. The first calculated m/z value may be calculated taking into account post-translational modifications. The post-translational modification is preferably N-terminal methionine truncation. The observed m/z values of the peaks detected on the mass spectrum are listed and compared with the first calculated m/z value, and the peaks with the matched observed m/z values are attributed to ribosomal proteins.

The discrimination unit 53 creates an approximate curve or an approximate straight line from the relative intensities of the peaks assigned to ribosomal proteins. When a sample is measured by MALDI-MS, ribosomal proteins present in equal moles are approximated by a straight line or curve in which the relative intensity decreases as the m/z value increases.

The discrimination unit 53 selects a peak whose relative intensity is close to an approximate curve or an approximate straight line as a ribosomal protein from among the unidentified peaks that are not assigned to a ribosomal protein by the first calculated m/z value. The expression that the relative intensity of the peak is close to the approximate curve or approximate straight line means that the relative intensity of the peak is 50 to 150%, preferably 60 to 140%, and more preferably 60 to 140% of the relative intensity of the approximate curve or approximate straight line. is 70 to 130%, more preferably 80 to 120%, and most preferably 90 to 110%.

The verification unit 54 attributes the selected peak to a ribosomal protein, further considering post-translational modification.

The verification unit 54 calculates a second calculated m/z value for the peak selected as a ribosomal protein by the discrimination unit 53, preferably taking into account post-translational modification. Post-translational modifications to be considered here include N-terminal methionine cleavage, acetylation, methylation, phosphorylation, glycosylation, oxidation, ubiquitination, SUMOylation, lipid modification, and combinations thereof.

The verification unit 54 compares the second calculated m/z value calculated in consideration of the post-translation modification with the observed m/z value. When the second calculated m/z value calculated in consideration of post-translational modification and the observed m/z value match, the peak can be determined to be a ribosomal protein with higher accuracy. Additionally, information on the amino acid sequence and post-translational modification of ribosomal proteins can be obtained.

The processing unit 50 may include an identification unit. The identification unit identifies the species of the organism that provides the amino acid information on the database for the protein determined to be a ribosomal protein. The more ribosomal proteins whose amino acid sequences are determined, the more accurate the identification will be. The processing performed by the identification unit may be performed by a remotely located server.

FIG. 2 shows a flowchart of an example of a method for determining ribosomal proteins.
First, a sample is set in the measurement section 100 and mass spectrometry is performed. In step S110, the mass spectrum creation unit 52 creates a mass spectrum based on the signal acquired by the detection unit 30.

In step S120, a first calculated m/z value is calculated based on the database. In step S130, the first calculated m/z value is compared with the observed m/z value indicated by the peak on the mass spectrum, and the matched peak is assigned as a ribosomal protein.

In step S140, an approximate curve or an approximate straight line is created from the relative intensities of the peaks assigned to ribosomal proteins. In step S150, among the unidentified peaks that are not assigned to ribosomal proteins by the first calculated m/z value, peaks whose relative intensity is close to an approximate curve or an approximate straight line are estimated to be ribosomal proteins. The expression that the relative intensity of the peak is close to the approximate curve or approximate straight line means that the relative intensity of the peak is 50 to 150%, preferably 60 to 140%, and more preferably 60 to 140% of the relative intensity of the approximate curve or approximate straight line. is 70 to 130%, more preferably 80 to 120%, and most preferably 90 to 110%. In step S160, the results are displayed on the output unit 44.

FIG. 3 shows a flowchart of another example of the method for determining ribosomal proteins. In FIG. 3, after step S150 described above, step S170 and step S180 are further performed. In step S170, a second calculated m/z value is calculated for the protein estimated to be a ribosomal protein, taking post-translational modification into account. In step S180, the second calculated m/z value in consideration of the post-translation modification is compared with the observed m/z value. Matched peaks are determined to be ribosomal proteins. In step S160, the results are displayed on the output unit 44.

[program]
A program for realizing the information processing function of the mass spectrometer 1 is recorded on a computer-readable recording medium, and the above-described ribosomal protein discrimination method and biological species identification method recorded on the recording medium are processed and A computer system may be made to read and execute a program related to control of processing related thereto. Note that the "computer system" herein includes an OS (Operating System) and hardware of peripheral devices. Furthermore, the term "computer-readable recording medium" refers to portable recording media such as flexible disks, magneto-optical disks, optical disks, and memory cards, and storage devices such as hard disks built into computer systems. Furthermore, a "computer-readable recording medium" refers to a storage medium that dynamically stores a program for a short period of time, such as a communication line when transmitting a program via a network such as the Internet or a communication line such as a telephone line. It may also include a device that retains a program for a certain period of time, such as a volatile memory inside a computer system that is a server or client in that case. Further, the above-mentioned program may be for realizing a part of the above-mentioned functions, or may further be for realizing the above-mentioned functions by combining with a program already recorded in the computer system. .

As a program for realizing the above-mentioned information processing function, in the mass spectrum acquired by the mass spectrometer 1, peaks attributed to ribosomal proteins are determined based on the database, and approximate curves or approximate straight lines of the relative intensities of the peaks are determined. For example, there is a program that causes a processing device to create an approximate curve or an approximate straight line and select a peak whose relative intensity is 50 to 150%. According to this program, it is possible to easily determine whether a protein shown as a peak on a mass spectrum by mass spectrometry is a ribosomal protein.

The program may cause the processing device to perform a process of attributing the selected peak to a ribosomal protein, further taking post-translational modification into account. The program calculates a second calculated m/z value for the peak selected as a ribosomal protein, preferably taking into account post-translational modifications. Post-translational modifications to be considered here include N-terminal methionine cleavage, acetylation, methylation, phosphorylation, glycosylation, oxidation, ubiquitination, SUMOylation, lipid modification, and combinations thereof. Compare the second calculated m/z value and the observed m/z value. According to this program, the accuracy of ribosomal protein discrimination can be improved. Additionally, information on the amino acid sequence and post-translational modification of ribosomal proteins can be obtained.

The program may cause the processing device to perform a process of identifying the species of the organism that provides the amino acid sequence information of the ribosomal protein, based on the determined ribosomal protein.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

<Experiment 1>
Escherichia coli (NBRC 3301) was cultured in LB medium at 30 degrees for 8 hours. The cultured bacterial cells were centrifugally washed using ultrapure water and then disrupted for 3 minutes using zirconia silica beads (diameter 0.1 mm). The beads and cell fragments were centrifuged (centrifugation conditions: 15,000 g, 5 minutes), and the supernatant was collected, which was used as a cell disruption solution. The cell disruption solution was ultrafiltered (Amicon Ultra 0.5 mL, NMWL 100 KDa, manufactured by Merck) to obtain a solution in which ribosomes were concentrated.

This solution was subjected to MALDI-MS as a sample containing ribosomal proteins. A matrix solution was prepared by dissolving 10 mg of sinapinic acid in 1 mL of 50% (v/v) acetonitrile solution containing 1% (v/v) trifluoroacetic acid. 7 μL of matrix solution, 1 μL of internal standard reagent, and 1 μL of sample were added to a 0.5 mL plastic tube and stirred, and 1.0 μL was dropped onto a stainless steel sample plate and air-dried. MALDI-MS was observed using Shimadzu AXIMA-Performance in positive ion linear mode (flight length 1.2 m).

Mass spectrum calibration was performed using [M+H] ⁺ (m/z 16952.6) and [M+2H] ²⁺ (m/z 8476.8) for myoglobin, and [M+H] ⁺ (m /z 2465.7) using the internal standard method. Next, among the subunit protein peaks tentatively assigned by the internal standard method, a clear peak with relatively high intensity and a normal distribution is selected, and the final calibration is performed using the self-calibration method. I did it. The m/z value of each peak was determined by three repeated measurements.

(Attribution process)
Regarding the peak on the mass spectrum, if the peak is observed within an error range of 200 ppm with respect to the first calculated m/z value of [M+H] ⁺ ion of each ribosomal protein determined from the amino acid sequence, the protein is considered to have been observed. The first calculated m/z value for each ribosomal protein was determined by obtaining the amino acid sequence from Swiss-Plot/TrEMBL and considering only the N-terminal methionine cleavage. As a result, 27 peaks could be assigned as ribosomal proteins.

(Estimation process)
FIG. 4 shows the observed m/z values of peaks that could be assigned as ribosomal proteins and peaks that could not be assigned (unidentified peaks) and the relative intensities of the peaks as a scatter diagram. Although it is thought that each ribosomal protein exists in equal moles, the relative intensity of the peak assigned as a ribosomal protein was high on the low mass side and low on the high mass side due to the mass discrimination effect. An approximate curve was created from the relative intensities of the peaks assigned to ribosomal proteins, and is shown as a broken line in FIG. Peaks with relative intensities close to the fitted curve were presumed to be ribosomal proteins.

(Verification process)
Here, a protein having an observed peak at m/z 9191, which is presumed to be a ribosomal protein, was further verified. First, a second calculated m/z value was calculated for the ribosomal protein expected to have a peak at m/z 9191, taking into account post-translational modifications other than N-terminal methionine cleavage, but the peak at m/z 9191 Could not be assigned to protein. Here, Table 1 of the literature (V. Ryzhov and C. Fenselau, Anal. Chem. 2001, 73, 746-750) reports that the peak observed at m/z 9191 is ribosomal protein S16. ing. The second amino acid residue of S16 is valine (V), and according to the N-terminal rule, N-terminal methionine cleavage occurs, and the second calculated m/z value is [M+H] ⁺ =9060.4. However, no peak corresponding to m/z 9060.4 on the mass spectrum was observed. Therefore, it was considered that S16 had undergone unexpected post-translational modification, and a peak was observed at m/z 9191 instead of the peak observed at m/z 9060.4. The second calculated m/z value if S16 does not undergo N-terminal methionine cleavage is 9191.6. Therefore, S16 was considered to be exceptionally not subjected to N-terminal methionine cleavage, and the peak at m/z 9191 could be assigned to S16. FIG. 5 shows the relative intensities of the peaks newly assigned to ribosomal proteins.

<Experiment 2>
In Experiment 2, an attempt was made to identify ribosomal proteins using the same method as in Experiment 1, except that yeast (Saccharomyces cerevisiae) was used as the bacterial cell. Yeast was cultured in YM medium (glucose 10 g, peptone 5 g, yeast extract 3 g, malt extract 3 g, distilled water 1 L) at an optimal temperature for 8 hours. A sample in which ribosomes were concentrated from cultured bacterial cells was subjected to MALDI-MS.

(Attribution process)
As a result of obtaining the amino acid sequence from Swiss-Plot/TrEMBL and comparing the first calculated m/z value obtained by considering only the N-terminal methionine cleavage with the observed m/z value, 27 peaks were identified as ribosomal proteins. could be attributed as such.

(Estimation process)
FIG. 6 shows the observed m/z values of peaks that could be assigned as ribosomal proteins and peaks that could not be assigned (unidentified peaks) and the relative intensities of the peaks as a scatter diagram. Although it is thought that each ribosomal protein exists in equal moles, the relative intensity of the peak assigned as a ribosomal protein was high on the low mass side and low on the high mass side due to the mass discrimination effect. An approximate curve was created from the relative intensities of the peaks assigned to ribosomal proteins, and is shown as a broken line in FIG. Peaks with relative intensities close to the fitted curve were presumed to be ribosomal proteins.

(Verification process)
Further verification was performed on peaks with relative intensities close to the approximate curve, which are presumed to be ribosomal proteins. It is known that yeast ribosomal proteins undergo post-translational modification such as acetylation in addition to N-terminal methionine cleavage. Therefore, for the peaks estimated to be ribosomal proteins, a second calculated m/z value was calculated taking into account N-terminal methionine cleavage and acetylation, and when compared with the observed m/z values, nine peaks were found. was newly assigned as a ribosomal protein. FIG. 7 shows the relative intensities of the peaks newly assigned to ribosomal proteins.

<Experiment 3>
In Experiment 3, an attempt was made to identify ribosomal proteins using the same method as in Experiment 1, except that fungal (Aspergillus Kawachi) hyphae were used as bacterial cells. The mold was cultured in potato dextrose medium at optimal temperature for 8 hours. A sample in which ribosomes were concentrated from cultured bacterial cells was subjected to MALDI-MS.

(Attribution process)
As a result of obtaining the amino acid sequence from Swiss-Plot/TrEMBL and comparing the first calculated m/z value obtained by considering only the N-terminal methionine cleavage with the observed m/z value, 16 peaks were identified as ribosomal proteins. could be attributed as such.

(Estimation process)
FIG. 8 shows the observed m/z values of peaks that could be assigned as ribosomal proteins and peaks that could not be assigned (unidentified peaks) and the relative intensities of the peaks as a scatter diagram. Although it is thought that each ribosomal protein exists in equal moles, the relative intensity of the peak assigned as a ribosomal protein was high on the low mass side and low on the high mass side due to the mass discrimination effect. An approximate curve was created from the relative intensities of the peaks assigned to ribosomal proteins, and is shown as a broken line in FIG. Peaks with relative intensities close to the fitted curve were presumed to be ribosomal proteins.

(Verification process)
Further verification was performed on peaks with relative intensities close to the approximate curve, which are presumed to be ribosomal proteins. It is known that fungal ribosomal proteins undergo post-translational modifications such as acetylation and methylation in addition to N-terminal methionine cleavage. Therefore, for the peaks estimated to be ribosomal proteins, a second calculated m/z value was calculated taking into account N-terminal methionine cleavage and acetylation, and when compared with the observed m/z values, eight peaks were found. was newly assigned as a ribosomal protein. FIG. 9 shows the relative intensities of the peaks newly assigned to ribosomal proteins.

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

(Section 1)
A ribosomal protein discrimination method according to one aspect includes an observed m/z value indicated by a peak on a mass spectrum detected by mass spectrometry of a sample containing a ribosomal protein, and a first calculated m/z value based on a database. an attribution step of attributing at least a part of the detected peak to a ribosomal protein, and an approximated curve or an approximated straight line obtained from the relative intensities of the peaks assigned to the ribosomal protein, and the relative intensity is 50 to and an estimating step of estimating that a protein corresponding to a peak of 150% is a ribosomal protein.

According to the method for determining ribosomal proteins described in item 1, it is possible to easily determine whether a protein shown as a peak on a mass spectrum is a ribosomal protein by mass spectrometry.

(Section 2)
In the ribosomal protein discrimination method according to item 1, the first calculated m/z value is calculated in consideration of post-translational modification.

According to the ribosomal protein discrimination method described in Section 2, it becomes easier to match the observed m/z value and the first calculated m/z value, and among the peaks detected on the mass spectrum, it is possible to identify which peaks are attributed to ribosomal proteins. The number of peaks detected can be increased.

(Section 3)
In the method for determining ribosomal proteins according to item 2, the post-translational modification is N-terminal methionine cleavage.

Since many ribosomal proteins undergo post-translational modification such as N-terminal methionine cleavage, according to the ribosomal protein discrimination method described in Section 3, the observed m/z value and the first calculated m/z value are matched. Among the peaks detected on the mass spectrum, the number of peaks attributed to ribosomal proteins can be increased.

(Section 4)
In the method for determining ribosomal proteins according to items 1 to 3, the mass spectrometry is matrix-assisted laser desorption ionization mass spectrometry.

According to the ribosomal protein discrimination method described in Section 4, the ribosomal protein peak can be easily detected from the sample, and the error between the observed m/z value and the first calculated m/z value can be reduced. .

(Section 5)
In the method for determining ribosomal proteins described in items 1 to 4, the sample is derived from eukaryotes.

According to the method for determining ribosomal proteins described in Section 5, it is possible to easily determine whether or not proteins derived from eukaryotes, which often undergo complex post-translational modifications, are ribosomal proteins. be able to.

(Section 6)
In the method for determining ribosomal proteins described in items 1 to 5, the sample is a fraction obtained by fractionating ribosomal proteins.

According to the ribosomal protein discrimination method described in Section 6, the proportion of ribosomal proteins contained in a sample increases, so that ribosomal proteins can be discriminated with higher accuracy.

(Section 7)
In the ribosomal protein discrimination method described in paragraphs 1 to 6, a second calculated m/z value is calculated for the protein estimated to be a ribosomal protein, taking post-translational modification into account, and the observed m/z value is It further includes a verification step of checking against the z-value.

According to the ribosomal protein discrimination method described in Section 7, the accuracy of ribosomal protein discrimination can be improved. Additionally, information on the amino acid sequence and post-translational modification of ribosomal proteins can be obtained.

(Section 8)
A biological species identification method according to one aspect includes determining a ribosomal protein using the ribosomal protein determination method described in items 1 to 7, and identifying the species of the organism that provides amino acid sequence information of the ribosomal protein. .

According to the ribosomal protein discrimination method described in Section 8, biological species can be identified more easily and accurately.

(Section 9)
In the method for identifying biological species according to item 8, the organism is a microorganism.

The biological species identification method described in Section 9 is suitable for identifying microbial species.

(Section 10)
A mass spectrometer according to one embodiment includes a mass separation section that separates ions based on m/z values, a detection section that detects ions separated by the mass separation section, and a detection section that detects ions based on the ions detected by the detection section. a mass spectrum creation unit that creates a mass spectrum based on the mass spectrum; and a mass spectrum creation unit that determines a peak attributed to a ribosomal protein based on a database in the mass spectrum, creates an approximate curve or an approximate straight line of the relative intensity of the peak, and creates an approximate curve or an approximate straight line of the relative intensity of the peak; Alternatively, a discriminator is provided that selects a peak having a relative intensity of 50 to 150% with respect to the approximate straight line.

According to the mass spectrometer described in Item 10, it is possible to easily determine whether a protein shown as a peak on a mass spectrum is a ribosomal protein by mass spectrometry.

(Section 11)
The mass spectrometer according to Item 10 includes a verification unit that attributes the selected peak to a ribosomal protein, further taking post-translational modification into consideration.

According to the mass spectrometer described in Section 11, the accuracy of ribosomal protein discrimination can be improved. Additionally, information on the amino acid sequence and post-translational modification of ribosomal proteins can be obtained.

(Section 12)
The program according to one embodiment determines a peak attributed to a ribosomal protein based on a database in a mass spectrum obtained by a mass spectrometer, creates an approximate curve or an approximate straight line of the relative intensity of the peak, and A processing device is caused to select a peak having a relative intensity of 50 to 150% with respect to a curve or an approximate straight line.

According to the program described in Section 12, it is possible to more easily determine whether a protein indicated by a peak on a mass spectrum is a ribosomal protein by mass spectrometry.

(Section 13)
In the program described in item 12, the processing device causes the processing device to perform a process of attributing the selected peak to a ribosomal protein, further taking post-translational modification into account.

According to the program described in Section 13, the accuracy of ribosomal protein discrimination can be improved. Additionally, information on the amino acid sequence and post-translational modification of ribosomal proteins can be obtained.

The embodiments and examples disclosed this time should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is indicated by the claims rather than the above description, and it is intended that all changes within the meaning and range equivalent to the claims are included.

1 Mass spectrometer, 10 Ionization unit, 21 Ion acceleration unit, 22 Mass separation unit, 30 Detection unit, 40 Control unit, 50 Processing unit, 51 Device control unit, 52 Mass spectrum creation unit, 53 Discrimination unit, 54 Verification unit, 100 Measurement part, S protein ion.

Claims (14)

The observed m/z value of a peak on a mass spectrum detected by mass spectrometry of a sample containing a ribosomal protein and at least one selected from the amino acid sequence of the protein and the DNA sequence of the gene are obtained from a database that is registered. an attribution step of attributing at least a part of the detected peak to the ribosomal protein by comparing the value of the mass of the ribosomal protein calculated from the amino acid sequence or DNA sequence obtained ;
an estimation step of estimating that a protein corresponding to a peak with a relative intensity of 50 to 150% is a ribosomal protein with respect to an approximate curve or an approximate straight line obtained from the relative intensity of the peak attributed to the ribosomal protein;
A method for identifying ribosomal proteins, including 2. The ribosomal protein discrimination method according to claim 1 , wherein the mass value of the ribosomal protein calculated from the amino acid sequence registered in the database is calculated in consideration of post-translational modification. 3. The method for identifying ribosomal proteins according to claim 2, wherein the post-translational modification is N-terminal methionine cleavage. The method for determining ribosomal proteins according to any one of claims 1 to 3, wherein the mass spectrometry is matrix-assisted laser desorption ionization mass spectrometry. The method for determining ribosomal proteins according to any one of claims 1 to 4, wherein the sample is derived from eukaryotes. The method for determining ribosomal proteins according to any one of claims 1 to 5, wherein the sample is a fraction obtained by fractionating ribosomal proteins. Any one of claims 1 to 6, further comprising a verification step of calculating a protein mass value in consideration of post-translational modification for the protein estimated to be a ribosomal protein, and comparing it with the observed m/z value. The method for determining ribosomal proteins according to item 1. 8. The ribosomal protein identification method according to claim 1, wherein the database includes information on the taxonomic group to which the cell belongs . A method for identifying a biological species, which comprises determining a ribosomal protein using the method for determining a ribosomal protein according to any one of claims 1 to 8 , and identifying the species of the organism that provides amino acid sequence information of the ribosomal protein. The method for identifying biological species according to claim 9 , wherein the living organism is a microorganism. a mass separation unit that separates ions derived from a sample containing ribosomal proteins based on m/z values;
a detection unit that detects ions separated by the mass separation unit;
a mass spectrum creation unit that creates a mass spectrum based on the ions detected by the detection unit;
Ribosomal protein calculated from the observed m/z value indicated by the peak on the mass spectrum and the amino acid sequence or DNA sequence obtained from a database in which at least one selected from the amino acid sequence of the protein and the DNA sequence of the gene is registered. The peak attributed to the ribosomal protein is determined by comparing it with the mass value of a discriminator that selects a peak that is ~150%;
A mass spectrometer equipped with. The mass spectrometer according to claim 11 , further comprising a verification unit that attributes the selected peak to a ribosomal protein, taking post-translational modification into account. Based on the observed m/z value indicated by the peak on the mass spectrum obtained by separating ions from a sample containing ribosomal proteins based on the m/z value using a mass spectrometer, and the amino acid sequence of the protein and the DNA sequence of the gene. Determining the peak attributed to the ribosomal protein by comparing it with the value of the mass of the ribosomal protein calculated from the amino acid sequence or DNA sequence obtained from the database in which at least one selected one is registered ,
A program that causes a processing device to perform a process of creating an approximate curve or an approximate straight line of the relative intensity of the peak, and selecting a peak whose relative intensity is 50 to 150% with respect to the approximate curve or the approximate straight line. 14. The program according to claim 13 , which causes a processing device to perform a process of attributing the selected peak to a ribosomal protein, further taking post-translational modification into consideration.

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