JP2021189111A - Ribosomal protein determination method, species identification method, mass analysis device, and program - Google Patents

Abstract

To provide a method capable of easily determining whether protein showing a peak on a mass spectrum is ribosomal protein or not with mass analysis.SOLUTION: A ribosomal protein determination method comprises: an attribution step of collating an observed m/z value indicated by a peak on a mass spectrum detected by mass analysis of a sample containing ribosomal protein with a first calculated m/z value based on a database, to attribute at least a portion of the detected peak to ribosomal protein; and an estimation step of estimating protein corresponding to a peak having a relative intensity of 50 to 150% to be 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.SELECTED DRAWING: Figure 2

Description

The present invention relates to a method for discriminating ribosomal proteins, a method for identifying an organism, a mass spectrometer and a program.

As a method for identifying bacterial species using mass spectrometry, a method using a ribosomal protein as a biomarker is known. For example, in Non-Patent Documents 1 and 2, the observed mass of the ribosomal protein of the bacterium observed on the mass spectrum is compared with the calculated mass obtained from the amino acid sequence registered in the protein database, and the bacterium is rapidly identified. A method for selecting a suitable biomarker for a protein, and a method for identifying a bacterial species that gives information on the amino acid sequence thereof are disclosed.

Kanae Teramoto et al., "Comparative Analysis of Ribosomal Proteins of Lactobacillus Genome Decoding Strains by Matrix-Assisted Laser Desorption / Ionization Mass Spectrometry", J Mass Spectrum. 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 Spectrum. Soc. Jpn, Vol. 56, No. 3,2008.

However, the calculated mass of the protein obtained from the database often does not match the actual observed mass. One of the reasons for this is that the amino acid sequences registered in the database are mechanically translated DNA sequences of genes, and therefore post-translational modification of proteins is not considered. Since the calculated mass and the observed mass were in agreement and the proteins that could be identified as ribosomal proteins were limited, the identification accuracy of the bacterial species was not sufficient.

Here, it is considered that ribosomal proteins can be discriminated by obtaining the calculated mass of the protein in consideration of post-translational modification. However, since the types and patterns of post-translational modifications can be combined in a complex manner, for many ribosomal proteins, the calculated mass considering possible post-translational modifications is obtained, and whether it is a ribosomal protein or not is determined by comparing it with the observed mass. It is not reasonable to do.

It is an object of the present invention to provide a method capable of easily determining whether or not a protein peaked on a mass spectrum is a ribosomal protein by mass spectrometry.

The present invention collates the observed m / z value indicated by the peak on the mass spectrum detected by mass analysis of a sample containing ribosomal protein with the first calculated m / z value based on the database, and the detected peak. The protein corresponding to the peak whose relative intensity is 50 to 150% with respect to the approximate curve or straight line obtained from the attribution step of assigning at least a part of the protein to the ribosomal protein and the relative intensity of the peak assigned to the ribosomal protein. The present invention relates to a method for discriminating a ribosomal protein, which comprises an estimation step of presuming that the protein is a ribosomal protein.

In the present invention, a mass separator that separates ions based on the m / z value, a detection unit that detects the ions separated by the mass separation unit, and a mass that creates a mass spectrum based on the ions detected by the detection unit. In the spectrum creator and mass spectrum, the peaks attributed to the ribosome protein are determined based on the database, an approximate curve or straight line of the relative intensity of the peak is created, and the relative intensity is relative to the approximate curve or straight line. It also relates to a mass spectrometer comprising a discriminator for selecting peaks of 50-150%.

In the present invention, in the mass spectrum acquired by the mass spectrometer, the peaks attributed to the ribosome protein are determined based on the database, an approximate curve or a straight line of the relative intensity of the peak is created, and the approximate curve or the approximate straight line is obtained. On the other hand, the present invention also relates to a program in which a processing apparatus is allowed to perform a process of selecting a peak having a relative intensity of 50 to 150%.

According to the present invention, it is possible to easily determine whether or not the protein indicated by the peak on the mass spectrum is a ribosomal protein by mass spectrometry.

It is a figure which shows the schematic structure of the mass spectrometer which concerns on the method of discriminating the ribosomal protein of one Embodiment. It is a flowchart which shows the flow of the discriminating method of the ribosomal protein of one Embodiment. It is a flowchart which shows the flow of the discriminating method of the ribosomal protein of one Embodiment. It is a scatter diagram which shows the observed m / z value of the peak which was attributed to a ribosomal protein and the peak which was not attributed to a ribosomal protein in Experiment 1, and the relative intensity of a peak. It is a scatter diagram which shows the observed m / z value and the relative intensity of the peak newly assigned to the ribosomal protein by the method of discriminating the ribosomal protein in Experiment 1. It is a scatter diagram which shows the observed m / z value of the peak which was attributed to a ribosomal protein and the peak which was not attributed to a ribosomal protein in Experiment 2, and the relative intensity of a peak. It is a scatter diagram which shows the observed m / z value and the relative intensity of the peak newly assigned to the ribosomal protein by the method of discriminating the ribosomal protein in Experiment 2. FIG. 3 is a scatter plot showing the observed m / z values of peaks attributed to ribosomal proteins and peaks not attributed to ribosomal proteins in Experiment 3 and the relative intensities of the peaks. It is a scatter diagram which shows the observed m / z value and the relative intensity of the peak newly assigned to the ribosomal protein by the method of discriminating the ribosomal protein in Experiment 3.

The method for discriminating ribosomal proteins according to the present invention is as follows.
At least a part of the detected peaks are collated with the observed m / z value indicated by the peak on the mass spectrum detected by mass spectrometry of the sample containing ribosomal protein and the first calculated m / z value based on the database. And the attribution process to attribute to ribosomal proteins,
Includes an estimation step of estimating that a protein corresponding to a peak having a relative intensity of 50-150% is a ribosomal protein with respect to an approximate curve or straight line obtained from the relative intensity of the peaks assigned to the ribosomal protein. ..

According to the method for discriminating ribosomal proteins according to the present invention, it is possible to easily discriminate whether or not the protein peaked on the mass spectrum is a ribosomal protein by mass spectrometry. Ribosomes are composed of more than 50 kinds of proteins, and each protein is present in approximately equimolar amounts. 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 show the same relative intensity. Therefore, it can be determined that a peak showing a relative intensity similar to the relative intensity of the peak attributed to the ribosomal protein is likely to be a ribosomal protein.

Hereinafter, each step will be described in detail.

[Attribution process]
In the attribution step, the observed m / z value indicated by the peak on the mass spectrum detected by mass spectrometry of the sample containing ribosomal protein is compared with the first calculated m / z value based on the database, and the detected peak is collated. At least part of is attributed 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 the amino acid sequence of a protein and the DNA sequence of a gene is registered in the database. The database contains information on the taxa (family, genus, species, etc.) to which the cell belongs. The first calculated m / z value may be calculated in consideration of post-translational modification. This is because the observed m / z value and the first calculated m / z value can be easily matched, and the number of peaks attributed to the ribosomal protein among the peaks detected on the mass spectrum can be increased. Post-translational modification is preferably N-terminal methionine cleavage. This is because ribosomal proteins often undergo N-terminal methionine cleavage as a post-translational modification. Especially in bacteria, most of the first calculated m / z values calculated in consideration of N-terminal methionine cleavage coincide with the observed m / z values, and most of the peaks of ribosomal proteins can be assigned. N-terminal methionine is 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 at the second position, cleavage of the N-terminal methionine may not occur.

In the present 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 peaks are self-calibrated using the calculated m / z values of the matched proteins, and the difference between the calculated m / z values and the observed m / z values of the peaks used for self-calibration is within 200 ppm. It is preferable to confirm that.

From the viewpoint of containing equimolar ribosomal proteins constituting ribosomes, it is preferable that the sample contains cell-derived ribosomal proteins. The sample may contain ribosomes or the cells themselves.

The sample may be of prokaryotic origin or eukaryotic origin. The sample is preferably derived from a microorganism and may contain an unknown microorganism. Prokaryotes include bacteria and archaea. Bacteria include Esqueriquia bacteria (Escherichia coli, etc.), Bacillus bacteria (Bacteria, etc.), Lactobacillus bacteria (Lactobacillus, etc.), Synechocystis spp. Is done. Archaea include the genus Methanococcus, the genus Methanococcus, the genus Thermococcus, the genus Philococcus, and the like. Eukaryotes include animals, plants, fungi and protists. Fungi include filamentous fungi, yeasts, mushrooms, molds and the like, including chytrids, zygomycota, ascomycetes, basidiomycetes, glomus and microsporidia. Ascomycetes include Aspergillus (Aspergillus, etc.), Penicillium (Penicillium, etc.), Saccharomyces (Saccharomyces, etc.) and the like. In eukaryotes, the number of peaks that can be assigned to ribosomal proteins considering only N-terminal methionine cleavage is considered to be smaller than that in bacteria. Eukaryotic ribosomal proteins, including fungi, are often more complex post-translational modifications than prokaryotic ribosomal proteins, with the first calculated m / z value calculated from the database and mass. This is because a difference is likely to occur between the m / z value observed by analysis. According to the method for discriminating ribosomal proteins according to the present invention, it is possible to easily discriminate whether or not the protein indicated by the peak on the mass spectrum is a ribosomal protein even in a sample derived from eukaryote.

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

The mass spectrometry is not particularly limited as long as it is a mass spectrometry method capable of quantitatively measuring ribosomal proteins, but a mass spectrometry method using a soft ionization method in which decomposition of a high molecular weight compound is unlikely to occur is preferable. Examples of such mass spectrometry 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, they tend ^{to generate [M + H] +} ions in the MALDI process. The molecular weight of the ribosomal protein is about 5,000 to 20,000, and according to MALDI-MS, the mass of the ribosomal protein can be determined within an error range of several Da. Therefore, when the mass spectrometry is MALDI-MS, the peak of ribosomal protein 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, the protein corresponding to the peak having a relative intensity of 50 to 150% with respect to the approximate curve or the approximate straight line obtained from the relative intensity of the peak attributed to the ribosomal protein is estimated to be a ribosomal protein.

As described above, since the ribosomal proteins constituting the ribosome are present in approximately equimolar amounts, the relative intensities of the peaks assigned to the ribosomal proteins are ideally about the same. For example, a ribosomal subunit protein is further prepared from the ribosomal fraction, each subunit protein is separated by liquid chromatography (LC), and each sample is mass spectrometrically analyzed together with a standard sample having a known concentration to prepare a mass spectrum, which is used as a ribosomal protein. When the assigned peak is selected and an approximate straight line is created for the scatter diagram showing the relative intensity, 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 existing in equal moles are detected, the relative intensities of peaks on the mass spectrum may not be about the same depending on the detection method. For example, when MALDI-MS is performed, the relative intensity is detected when the m / z value is small, and the relative intensity is detected when the m / z value is large due to the mass discrimination effect. Therefore, when the sample is measured by MALDI-MS, the ribosomal proteins existing in equal moles are approximated to a straight line or a curve whose relative intensity decreases as the m / z value increases. In the present specification, if the approximation curve or the approximate straight line has a correlation coefficient (R ² ) of 0.70 or more, preferably 0.80 or more, it is judged that the accuracy of the approximate curve or the approximate straight line is good. ..

Among the unidentified peaks not attributed to the ribosomal protein by the first calculated m / z value, the peak whose relative intensity is close to the approximate curve or the approximate straight line is estimated to be the ribosomal protein. When the relative intensity of the peak is close to the approximate curve or the approximate straight line, the relative intensity of the peak is 50 to 150%, preferably 60 to 140%, more preferably with respect to the relative intensity of the approximate curve or the approximate straight line. Is 70 to 130%, more preferably 80 to 120%, and most preferably 90 to 110%.

[Verification process]
The method for discriminating a ribosomal protein may further include a verification step of calculating a second calculated m / z value in consideration of post-translational modification for a protein presumed to be a ribosomal protein and collating it with the observed m / z value. preferable. For a protein presumed to be a ribosomal protein, when the second calculated m / z value calculated in consideration of post-translational modification and the observed m / z value match, the peak is more accurate as a ribosomal protein. It can be discriminated well. In addition, information on the amino acid sequence of ribosomal protein and post-translational modification can be obtained.

The second calculation m / z value is calculated in consideration of 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. In the verification process, a modification pattern different from the post-translational modification in the attribution process is considered. For example, when the first calculated m / z value is calculated in consideration of the post-translational modification of N-terminal methionine cleavage in the attribution step, the second calculated m / z value when the N-terminal methionine cleavage is not received in the verification step, Alternatively, the second calculated m / z value when undergoing other post-translational modifications in addition to N-terminal methionine cleavage is calculated. In the estimation process, peaks on the mass spectrum presumed to be ribosomal proteins are selected, so that it is not necessary to consider complicated post-translational modifications for all peaks on the mass spectrum, and it is easier to use ribosomal proteins. Can be determined.

[Method of identifying organisms]
In the method for identifying an organism according to the present invention, the ribosomal protein is identified by using the above-mentioned method for determining ribosomal protein, and the species of the organism that gives the amino acid sequence information of the ribosomal protein is identified.

Ribosomal proteins are housekeeping genes that are involved in life support and are very well conserved across species. Ribosomal proteins are used as biomarkers to identify species because they can be a definitive indicator for assessing species differences due to slight amino acid sequence differences and resulting mass differences. In addition, since ribosomes are structures that are abundantly present in cells, ribosomal proteins are easily detected during mass spectrometry.

According to the above-mentioned method for discriminating ribosomal proteins, the number of proteins discriminated as ribosomal proteins can be increased, so that species can be identified more easily and accurately. The organism to be identified is preferably a microorganism having a size that is difficult to identify with the naked eye. According to the method for identifying an organism according to the present invention, screening of useful microorganisms can also be performed.

In particular, when MALDI-MS is used for mass spectrometry, even slight mutations in the amino acid sequence of ribosomal proteins can be easily detected as peak shifts. According to the method for identifying an organism 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 the method for discriminating ribosomal proteins, a method for discriminating ribosomal proteins by a mass spectrometer will be described.
The mass spectrometer according to one aspect of the present invention is
A mass separator that separates ions based on the m / z value,
A detector that detects ions separated by the mass separator, and a detector
A mass spectrum creation unit that creates a mass spectrum based on the ions detected by the detection unit,
In the mass spectrum, the peaks attributed to ribosomal proteins are determined based on the database, and an approximate curve or straight line of the relative intensity of the peak is created, and the relative intensity is 50 to 150% with respect to the approximate curve or straight line. A discriminating unit for selecting a certain peak is provided.
According to this mass spectrometer, it is possible to easily determine whether or not the protein indicated by the peak on the 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 measuring unit 100 and a control unit 40. A part or all of the functions of the control unit 40 may be arranged in a computer, a server, or the like physically separated from the measurement unit 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 measuring unit 100 contains a ribosomal protein. The movement of the protein ion S generated in the ionization unit 10 is schematically shown by an arrow A1.

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

The ionization unit 10 of the measurement unit 100 includes a sample plate holder (not shown) that supports the sample plate and an ion source equipped with a laser device (not shown) that irradiates the sample plate with a laser beam, and matrix-assisted laser desorption / ionization of the sample components. It is ionized by the (MALDI) method. As a method for ionizing the sample, any soft ionization method such as an electrospray (ESI) method can be used in addition to the MALDI method. When ionization is performed by the ESI method, it is preferable that the mass spectrometer 1 further includes a liquid chromatograph and the sample component separated by the liquid chromatograph is ionized by the ionization unit 10 because high separation ability can be obtained. ..

After the sample is placed on the sample plate, the matrix is added to the sample and dried. After that, the sample plate is installed in the sample plate holder in the vacuum container of the ionization unit 10. The type of matrix is not particularly limited, but it is preferable to use cinapinic acid, α-cyano-4-hydroxycylic acid (CHCA), or the like from the viewpoint of efficiently ionizing a protein sample.

The ionization unit 10 decompresses the vacuum container in which the 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 the laser light is not particularly limited as long as it can oscillate the light absorbed by the selected matrix, for example, when the matrix contains sinapinic acid or CHCA, an N ₂ laser (wavelength 337 nm). Etc. can be preferably used. The protein ion S ionized by the ionization unit 10 is extracted by an electric field using an extraction electrode (not shown) or the like, and is introduced into the ion acceleration unit 21.

The ion acceleration unit 21 includes an acceleration electrode 210 and accelerates the introduced protein ion S. The accelerated flow of the protein ion S is appropriately converged 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 the protein ions S due to the difference in flight time when each protein ion S flies inside the flight tube 220. Although the linear type flight tube 220 is shown in FIG. 1, it may be a reflector type, a multi-turn type, or the like. The method of mass spectrometry is not particularly limited as long as the protein ion 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 ion S separated by the mass separation unit 22, and outputs a detection signal having an intensity corresponding to the number of ions incident on the detection unit 30. .. The detection signal output from the detection unit 30 is input to the processing unit 50 of the control unit 40.

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

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

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

The output unit 44 of the control unit 40 includes a display device such as a liquid crystal monitor, a printer, and the like, and displays information on the measurement of the measurement unit 100, the results of the discrimination unit 53 and the verification unit 54, and the like on the display device. Or print on paper media.

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 of an operation for controlling the mass spectrometer 1. The processing unit 50 performs various processes by executing a program stored in the storage unit 43 or the like.

The device control unit 51 of the processing unit 50 controls the operation of the measurement unit 100 based on the data regarding the 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 detection amount of the protein detected by the detection unit 30 and the flight time of the protein, and each m / z. Create a mass spectrum MS showing the amount of detection corresponding to the value.

In the mass spectrum, the discriminator 53 determines the peak attributed to the ribosome protein based on the database, creates an approximate curve or an approximate straight line of the relative intensity of the peak, and determines the relative intensity with respect to the approximate curve or the approximate straight line. Select peaks that are 50-150%.

The discrimination 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 the protein and the DNA sequence of the 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 in consideration of post-translational modification. Post-translational modification is preferably N-terminal methionine cleavage. The observed m / z values of the peaks detected on the mass spectrum are listed, collated with the first calculated m / z value, and the peaks of the matched observed m / z values are assigned to the ribosomal protein.

The discriminant unit 53 creates an approximate curve or an approximate straight line from the relative intensities of the peaks attributed to the ribosomal protein. When the sample is measured by MALDI-MS, the ribosomal proteins present in equimolar amounts are approximated to a straight line or a curve whose relative intensity decreases as the m / z value increases.

The discriminant unit 53 selects, as the ribosomal protein, a peak whose relative intensity is close to an approximate curve or an approximate straight line among unidentified peaks not attributed to the ribosomal protein by the first calculated m / z value. When the relative intensity of the peak is close to the approximate curve or the approximate straight line, the relative intensity of the peak is 50 to 150%, preferably 60 to 140%, more preferably with respect to the relative intensity of the approximate curve or the approximate straight line. Is 70 to 130%, more preferably 80 to 120%, and most preferably 90 to 110%.

The verification unit 54 assigns the selected peak to the ribosomal protein in consideration of further post-translational modification.

The verification unit 54 calculates a second calculated m / z value for the peak selected as the ribosomal protein by the discrimination unit 53, preferably considering 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 collates the second calculated m / z value calculated in consideration of post-translational 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 more accurately determined to be a ribosomal protein. In addition, information on the amino acid sequence of ribosomal protein and post-translational modification can be obtained.

The processing unit 50 may have an identification unit. The discriminator identifies the species of the organism that gives the amino acid information on the database for the protein identified as a ribosomal protein. The more ribosomal proteins for which the amino acid sequence has been determined, the better the accuracy of identification. The processing performed by the identification unit may be performed by a server located remotely.

The flow chart of an example of the method for discriminating ribosomal proteins is shown in FIG.
First, the sample is set in the measuring unit 100 and mass spectrometry is executed. 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, the first calculated m / z value is calculated based on the database. In step S130, the calculated first calculated m / z value is collated with the observed m / z value indicated by the peak on the mass spectrum, and the matching peak is assigned as a ribosomal protein.

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

The flow chart of another example of the method for discriminating ribosomal proteins is shown in FIG. In FIG. 3, after the above-mentioned step S150, further steps S170 and S180 are performed. In step S170, the second calculated m / z value considering post-translational modification is calculated for the protein presumed to be a ribosomal protein. In step S180, the second calculated m / z value in consideration of post-translational modification is collated with the observed m / z value. Matched peaks are determined to be ribosomal proteins. In step S160, the result is displayed on the output unit 44.

[program]
A program for realizing the information processing function of the mass analyzer 1 is recorded on a computer-readable recording medium, and the processing and processing of the above-mentioned ribosomal protein discrimination method and biological species identification method recorded on the recording medium are performed. A computer system may be loaded with a program related to the control of related processing and executed. The term "computer system" as used herein includes hardware of an OS (Operating System) and peripheral devices. Further, the "computer-readable recording medium" refers to a portable recording medium such as a flexible disk, a magneto-optical disk, an optical disk, or a memory card, and a storage device such as a hard disk built in a computer system. Further, a "computer-readable recording medium" is a communication line for transmitting a program via a network such as the Internet or a communication line such as a telephone line, and dynamically holds the program for a short period of time. It may include a program that holds a program for a certain period of time, such as a volatile memory inside a computer system that is a server or a client in that case. Further, the above-mentioned program may be for realizing a part of the above-mentioned functions, and may be further realized by combining the above-mentioned functions 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, the peak attributed to the ribosome protein is determined based on the database, and an approximate curve or an approximate straight line of the relative intensity of the peak is determined. There is a program that causes a processing device to perform a process of selecting a peak having a relative intensity of 50 to 150% with respect to an approximate curve or an approximate straight line. According to this program, it is possible to easily determine whether or not the protein peaked on the mass spectrum is a ribosomal protein by mass spectrometry.

The program may allow the processing device to perform a process of attributing the selected peak to the ribosomal protein in consideration of further post-translational modification. The program calculates a second calculated m / z value for peaks selected as ribosomal proteins, 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. The second calculation m / z value and the observed m / z value are collated. According to this program, the accuracy of discrimination of ribosomal proteins can be improved. In addition, information on the amino acid sequence of ribosomal protein and post-translational modification can be obtained.

The program may have the processing apparatus perform a process of identifying the species of the organism that provides the amino acid sequence information of the ribosomal protein based on the identified ribosomal protein.

Hereinafter, the present invention will be described 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 ° C. for 8 hours. The cultured cells were washed centrifugally with ultrapure water and then crushed with zirconia silica beads (diameter 0.1 mm) for 3 minutes. The beads and cell fragments were centrifuged (centrifugal conditions 15000 g, 5 minutes), and the supernatant was collected and used as a cell disruption solution. The cell disruption solution was ultrafiltered (Amicon Ultra 0.5 mL, NMWL 100KDa, manufactured by Merck) to obtain a ribosome-concentrated solution.

This solution was subjected to MALDI-MS as a sample containing ribosomal protein. A matrix solution was prepared by dissolving 10 mg of sinapic acid in 1 mL of a 50% (v / v) acetonitrile solution containing 1% (v / v) trifluoroacetic acid. 7 μL of the matrix solution, 1 μL of the internal standard reagent and 1 μL of the sample were added to a plastic tube having a capacity of 0.5 mL, and the mixture was stirred. 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).

The mass spectrum was calibrated with myoglobin [M + H] ⁺ (m / z 16952.6) and [M + 2H] ²⁺ (m / z 8476.8), and the adrenocorticotropic hormone clip18-39 [M + H] ⁺ (m). Each peak of / z 2465.7) was used by the internal standard method. Next, among the peaks of subunit proteins tentatively assigned by the internal standard method, relatively high-intensity and well-distributed peaks are selected, and the final calibration is performed by the self-calibration method. Was done. The m / z value of each peak was determined by repeated measurement three times.

(Attribution process)
Regarding the peak on the mass spectrum, when a 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 obtained from the amino acid sequence, the protein is found.} Considered to be observed. The first calculated m / z value of each ribosomal protein was determined by obtaining the amino acid sequence from Swiss-Plot / TREMBL and considering only 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 the peaks that could be assigned as ribosomal proteins and the peaks that could not be assigned (unidentified peaks), and the relative intensities of the peaks as a scatter plot. It is considered that each ribosomal protein exists in equimolar amounts, but the relative intensity of the peaks assigned as ribosomal proteins was high on the low mass side and low on the high mass side due to the mass discrimination effect. An approximate curve is created from the relative intensities of the peaks attributed to ribosomal proteins and is shown by the dashed line in FIG. Peaks with relative intensities close to the approximation 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, the second calculated m / z value was calculated for the ribosomal protein expected to have a peak at m / z 9191 in consideration of post-translational modifications other than N-terminal methionine cleavage. It could not be attributed to the protein. Here, in Table 1 of the literature (V. Ryzhov and C. Fensellau, Anal. Chem. 2001, 73, 746-750), it is reported 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 was observed at m / z 9060.4 on the mass spectrum. Therefore, it is possible that S16 received an unexpected post-translational modification, and a peak was observed at m / z 9191 instead of a peak at m / z 9060.4. The second calculated m / z value when S16 does not undergo N-terminal methionine cleavage is 9191.6. Therefore, it was considered that S16 was exceptionally not subjected to N-terminal methionine cleavage, and the peak of m / z 9191 could be assigned to S16. The relative intensities of the peaks newly assigned to ribosomal proteins are shown in FIG.

<Experiment 2>
In Experiment 2, an attempt was made to discriminate ribosomal proteins by the same method as in Experiment 1 except that yeast (Saccharomyces cerevisiae) was used as a 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 optimum temperature for 8 hours. A sample enriched with ribosomes from the cultured cells was subjected to MALDI-MS.

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

(Estimation process)
FIG. 6 shows the observed m / z values of the peaks that could be assigned as ribosomal proteins and the peaks that could not be assigned (unidentified peaks), and the relative intensities of the peaks as a scatter plot. It is considered that each ribosomal protein exists in equimolar amounts, but the relative intensity of the peaks assigned as ribosomal proteins was high on the low mass side and low on the high mass side due to the mass discrimination effect. An approximate curve is created from the relative intensities of the peaks attributed to ribosomal proteins and is shown by the dashed line in FIG. Peaks with relative intensities close to the approximation curve were presumed to be ribosomal proteins.

(Verification process)
Further verification was performed on peaks having relative intensities close to the approximate curve, which are presumed to be ribosomal proteins. Yeast ribosomal proteins are known to undergo post-translational modifications of acetylation in addition to N-terminal methionine cleavage. Therefore, for the peaks presumed to be ribosomal proteins, the second calculated m / z value was calculated in consideration of N-terminal methionine cleavage and acetylation, and the peaks were compared with the observed m / z values. Was newly assigned as a ribosomal protein. The relative intensities of the peaks newly assigned to ribosomal proteins are shown in FIG.

<Experiment 3>
In Experiment 3, the ribosomal protein was discriminated by the same method as in Experiment 1 except that the mycelium of Aspergillus Kawachi was used as the bacterial cell. The mold was cultured in potato dextrose medium at an optimum temperature for 8 hours. A sample enriched with ribosomes from the cultured cells was subjected to MALDI-MS.

(Attribution process)
The amino acid sequence was obtained from Swiss-Plot / TREMBL, and the first calculated m / z value obtained by considering only N-terminal methionine cleavage was compared with the observed m / z value. As a result, 16 peaks were found in ribosomal proteins. Could be attributed as.

(Estimation process)
FIG. 8 shows the observed m / z values of the peaks that could be assigned as ribosomal proteins and the peaks that could not be assigned (unidentified peaks), and the relative intensities of the peaks as a scatter plot. It is considered that each ribosomal protein exists in equimolar amounts, but the relative intensity of the peaks assigned as ribosomal proteins was high on the low mass side and low on the high mass side due to the mass discrimination effect. An approximate curve is created from the relative intensities of the peaks attributed to ribosomal proteins and is shown by the dashed line in FIG. Peaks with relative intensities close to the approximation curve were presumed to be ribosomal proteins.

(Verification process)
Further verification was performed on peaks having relative intensities close to the approximate curve, which are presumed to be ribosomal proteins. Ribosomal proteins in molds are known to undergo post-translational modifications of acetylation and methylation in addition to N-terminal methionine cleavage. Therefore, for the peaks presumed to be ribosomal proteins, the second calculated m / z value was calculated in consideration of N-terminal methionine cleavage and acetylation, and compared with the observed m / z value, eight peaks were obtained. Was newly assigned as a ribosomal protein. The relative intensities of the peaks newly assigned to ribosomal proteins are shown in FIG.

[Mode]
It will be understood by those skilled in the art that the plurality of exemplary embodiments and examples described above are specific examples of the following embodiments.

(Section 1)
In the method for discriminating a ribosomal protein according to one embodiment, an observed m / z value indicated by a peak on a mass spectrum detected by mass analysis of a sample containing a ribosomal protein and a first calculated m / z value based on a database are used. The relative intensity is 50 to 50 to the approximation step obtained by collating and assigning at least a part of the detected peak to the ribosomal protein and the approximate curve or straight line obtained from the relative intensity of the peak assigned to the ribosomal protein. It comprises an estimation step of estimating the protein corresponding to the peak of 150% as a ribosomal protein.

According to the method for discriminating ribosomal proteins according to the first item, it is possible to easily determine whether or not the protein indicated by the peak on the mass spectrum is a ribosomal protein by mass spectrometry.

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

According to the method for discriminating ribosomal proteins according to the second item, it becomes easy to match the observed m / z value with the first calculated m / z value, and among the peaks detected on the mass spectrum, they belong to the ribosomal protein. The number of peaks to be made can be increased.

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

Since most ribosomal proteins undergo post-translational modification of N-terminal methionine cleavage, the observed m / z value and the first calculated m / z value are matched according to the ribosomal protein discrimination method described in Section 3. This facilitates the increase in the number of peaks attributed to ribosomal proteins among the peaks detected on the mass spectrum.

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

According to the method for discriminating ribosomal proteins according to the fourth item, the peak of ribosomal proteins 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 discriminating ribosomal proteins according to paragraphs 1 to 4, the sample is derived from eukaryote.

According to the method for discriminating ribosomal proteins according to Section 5, it is easy to discriminate whether or not a protein derived from a eukaryotic organism, which is often subjected to complicated post-translational modification, is a ribosomal protein. be able to.

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

According to the method for discriminating ribosomal proteins according to the sixth item, since the proportion of ribosomal proteins contained in the sample increases, the ribosomal proteins can be discriminated more accurately.

(Section 7)
In the method for discriminating a ribosomal protein according to the first to sixth paragraphs, a second calculated m / z value considering post-translational modification was calculated for the protein presumed to be the ribosomal protein, and the observation m /. It further includes a verification step of collating with the z value.

According to the method for discriminating ribosomal proteins according to the seventh item, the accuracy of discriminating ribosomal proteins can be improved. In addition, information on the amino acid sequence of ribosomal protein and post-translational modification can be obtained.

(Section 8)
In the method for identifying a biological species according to one embodiment, the ribosomal protein is identified by using the ribosomal protein discrimination method according to the first to seventh paragraphs, and the species of the organism that gives the amino acid sequence information of the ribosomal protein is identified. ..

According to the method for discriminating ribosomal proteins according to Item 8, the species can be identified more easily and accurately.

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

The method for identifying an organism species described in Section 9 is suitable for identifying a species of a microorganism.

(Section 10)
The mass spectrometer according to one embodiment is based on a mass separator that separates ions based on an m / z value, a detection unit that detects ions separated by the mass separator, and an ion detected by the detection unit. In the mass spectrum creating unit that creates the mass spectrum, the peaks attributed to the ribosome protein are determined based on the database, and an approximate curve or straight line of the relative intensity of the peak is created, and the approximate curve is created. Alternatively, the discriminator for selecting a peak having a relative intensity of 50 to 150% with respect to the approximate straight line is provided.

According to the mass spectrometer according to the tenth item, it is possible to easily determine whether or not the protein peaked on the mass spectrum is a ribosomal protein by mass spectrometry.

(Section 11)
The mass spectrometer according to Item 10 includes a verification unit for assigning the selected peak to a ribosomal protein in consideration of post-translational modification.

According to the mass spectrometer according to the eleventh item, the accuracy of discrimination of ribosomal proteins can be improved. In addition, information on the amino acid sequence of ribosomal protein and post-translational modification can be obtained.

(Section 12)
In the program according to one aspect, in the mass spectrum acquired by the mass spectrometer, the peak attributed to the ribosome protein is determined based on the database, an approximate curve or a straight line of the relative intensity of the peak is created, and the approximation is performed. Have the processing device perform a process of selecting 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 or not the protein peaked on the mass spectrum is a ribosomal protein by mass spectrometry.

(Section 13)
In the program according to Section 12, the processing apparatus is allowed to perform a process of assigning the selected peak to a ribosomal protein in consideration of post-translational modification.

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

The embodiments and examples disclosed this time should be considered to be exemplary and not restrictive in all respects. The scope of the present invention is shown by the scope of claims rather than the above description, and is intended to include all modifications within the meaning and scope equivalent to the scope of claims.

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 Measuring unit, S protein ion.

Claims (13)

The observed m / z value indicated by the peak on the mass spectrum detected by mass spectrometry of the sample containing ribosomal protein is compared with the first calculated m / z value based on the database, and at least one of the detected peaks is compared. The attribution step of assigning the part to the ribosomal protein,
An estimation step of estimating that a protein corresponding to a peak having 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 discriminating ribosomal proteins, including. The method for discriminating a ribosomal protein according to claim 1, wherein the first calculated m / z value is calculated in consideration of post-translational modification. The method for discriminating a ribosomal protein according to claim 2, wherein the post-translational modification is N-terminal methionine cleavage. The method for discriminating a ribosomal protein according to any one of claims 1 to 3, wherein the mass spectrometry is matrix-assisted laser desorption / ionization mass spectrometry. The method for discriminating a ribosomal protein according to any one of claims 1 to 4, wherein the sample is derived from eukaryote. The method for discriminating a ribosomal protein according to any one of claims 1 to 5, wherein the sample is a fraction obtained by fractionating a ribosomal protein. Claims 1 to 6 further include a verification step of calculating a second calculated m / z value in consideration of post-translational modification of the protein presumed to be a ribosomal protein and collating it with the observed m / z value. The method for discriminating a ribosomal protein according to any one item. A method for identifying an organism, wherein the ribosomal protein is discriminated using the method for discriminating a ribosomal protein according to any one of claims 1 to 7, and the species of the organism that gives the amino acid sequence information of the ribosomal protein is identified. The method for identifying an organism according to claim 8, wherein the organism is a microorganism. A mass separator that separates ions based on the m / z value,
A detection unit that detects the ions separated by the mass separation unit, and a detection unit.
A mass spectrum creating unit that creates a mass spectrum based on the ions detected by the detection unit,
In the mass spectrum, the peak attributed to the ribosomal protein is determined based on the database, an approximate curve or a straight line of the relative intensity of the peak is created, and the relative intensity is 50 to 50 to the approximate curve or the approximate straight line. A discriminator that selects a peak that is 150%,
A mass spectrometer equipped with. The mass spectrometer according to claim 10, further comprising a verification unit for assigning the selected peak to a ribosomal protein in consideration of post-translational modification. In the mass spectrum obtained by the mass spectrometer, the peak attributed to the ribosomal protein was determined based on the database, and the peak was determined.
A program that creates an approximate curve or an approximate straight line of the relative intensity of the peak, and causes a processing device to perform a process of selecting a peak having a relative intensity of 50 to 150% with respect to the approximate curve or the approximate straight line. 12. The program of claim 12, wherein the processing apparatus is allowed to perform a process of attributing the selected peak to a ribosomal protein in consideration of post-translational modification.

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