JP6775827B2 - Method for measuring hepatitis B virus using mass spectrometry - Google Patents

Description

A technique for detecting hepatitis B virus by mass spectrometry is disclosed.

Hepatitis B virus (HBV) is a virus belonging to the family Hepadnaviridae, which includes envelope, core, DNA polymerase having reverse transcriptase activity, and incomplete circular double-stranded DNA. , A spherical DNA virus with a diameter of about 42 nm. The outer cover of HBV is composed of three types of isoforms: large S (LHBs), meddle S (MHBs), and small S (SHBs). These isoforms are known as HBsAg. Currently, it is estimated that 3.5-4 million people worldwide are infected with HBV, many of whom are at risk of developing cirrhosis and hepatocellular carcinoma in the future.

Currently, the diagnosis of HBV infection is mainly performed by detecting HBs antigens or antibodies thereof present in human serum by an immunological method (Patent Document 1). However, the emergence of antigenic determinant mutants (escape mutants) that can be detected by immunological methods has been reported, which has become a problem.

JP-A-2007-14267

An object of the present invention is to provide a means for more accurately detecting hepatitis B virus.

In order to solve the above objectives, the present inventors have conducted extensive research, came up with the idea of detecting hepatitis B virus by mass spectrometry, and found a peptide fragment of HBs antigen suitable for detection by mass spectrometry. In addition, by using the peptide fragment as a probe, the present inventors can not only detect hepatitis B virus by mass spectrometry, but also identify the genotype of hepatitis B virus and a small amount of sample. It was confirmed that the virus inside could be detected. Furthermore, the present inventors have confirmed that hepatitis B virus antigen in a biological sample can be quantitatively analyzed by isotope dilution mass spectrometry using the peptide and its isotope label. As a result of these findings and further research and examination, inventions represented by the following are provided.

Item 1.
A method for detecting or measuring hepatitis B virus in a biological sample, which comprises detecting or measuring an oligopeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 7 by mass spectrometry.
Item 2.
Item 2. The method according to Item 1, wherein the biological sample is digested with a protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue and the arginine residue.
Item 3.
Item 2. The method according to Item 1 or 2, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, urine, cerebrospinal fluid, bone marrow fluid, lymph, liver tissue, and cultured cells.
Item 4.
Mass spectrometry selected from the group consisting of a quadrupole mass spectrometer, a time-of-flight mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, an ion trap mass spectrometer, and an orbitrap mass spectrometer. Item 6. The method according to any one of Items 1 to 3, wherein an analyzer is used.
Item 5.
An isotope-labeled oligopeptide consisting of at least one amino acid sequence selected from SEQ ID NOs: 1 to 7 is obtained by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue and / or the arginine residue. The resulting precursor peptide.
Item 6.
Item 5. The precursor peptide according to Item 5, which has a length of 70 amino acid residues or less.
Item 7.
One or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1-3 by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue and / or the arginine residue, and Item 5. The precursor peptide according to Item 5 or 6, which yields one or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4 to 7. Item 8.
Item 2. The precursor polypeptide according to any one of Items 5 to 7, which has the amino acid sequence of any of SEQ ID NOs: 8 to 33.
Item 9.
An internal standard substance for detecting or measuring hepatitis B virus in a biological sample by isotope dilution mass spectrometry, which comprises the precursor polypeptide according to any one of Items 5 to 8.
Item 10.
A kit for detecting or measuring hepatitis B virus in a biological sample by an isotope-diluted mass spectrometry method containing the precursor peptide according to any one of Items 5 to 8.
Item 11.
An isotope-labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1, an isotope-labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 2, an isotope-labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: An isotope-labeled oligopeptide consisting of the amino acid sequence of 4, an isotope-labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 5, an isotope-labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 6, and SEQ ID NO: B in a biological sample using at least one isotope-labeled oligopeptide selected from the group consisting of isotope-labeled oligopeptides consisting of an amino acid sequence of 7 as an internal reference material in isotope dilution mass analysis. A method for detecting, measuring, or genotyping a hepatitis virus.
Item 12.
Item 2. The method according to Item 11, wherein the biological sample is digested with a protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue and the arginine residue.
Item 13.
Use of the precursor peptide according to any one of claims 5 to 8 for detecting or measuring hepatitis B virus by isotope dilution mass spectrometry.

According to the present invention, one or more of the following effects can be obtained. Accurate detection of hepatitis B virus is possible without using antibodies. It is possible to detect mutant hepatitis B virus that cannot be detected by immunological methods. Quantitative detection of hepatitis B virus is possible. It is possible to identify the genotype of hepatitis B virus. It is possible to easily detect hepatitis B virus. It is possible to detect hepatitis B virus, which is less invasive.

A typical procedure for detecting a protein of interest in a biological sample by selective reaction monitoring (SRM) using a triple quadrupole mass spectrometer is shown. The position of the probe peptide in the HBsAg (genotype C) sequence is shown. The tandem MS spectrum (left) and SRM chromatograph (right) of the probe peptide (LHBs region; upper, common region; lower) of HBs antigen (genotype C) are shown. A pretreatment scheme for mass spectrometry of protein samples using a soluble polyacrylamide gel is shown. After digesting the protein with protease in the gel, the gel is solubilized by reduction treatment, and the digested peptide is recovered and subjected to mass spectrometry. The probe peptide sequence for discriminating the HBV genotype using mass spectrometry is shown. A stable isotope-labeled peptide for use as an internal standard for isotope dilution mass spectrometry is shown. The tandem MS spectrum of the digested peptide fragment (FLWEWASVX: SEQ ID NO: 2) obtained by trypsin digestion of the internal standard peptide (SEQ ID No: 9) is shown. The tandem MS spectrum of the digested peptide fragment (QPTPISPPLX: SEQ ID NO: 4) obtained by trypsin digestion of the internal standard peptide (SEQ ID No: 6) is shown. An example of SRM analysis of HBs antigens (three types of genotypes A, B, and D) and their internal standard peptides is shown. The HBs antigen contained in the cultured cell extract was digested by the method shown in FIG. 4 and then subjected to SRM analysis. A Western blot image of HBs antigens (wild type and mutant) expressed in cultured cells is shown. The results of quantitative analysis of HBs antigens (wild type and mutants) expressed in cultured cells by the CLIA method are shown. The results of quantitative analysis by SRM of HBs antigens (wild type and mutant) expressed in cultured cells are shown. The amino acid mutation variations known so far in the probe peptide sequence of HBs antigen (genotype C) (LHBs region: QPTPISPPLR (SEQ ID NO: 4) and common region: FLWEWASVR (SEQ ID NO: 2)) are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. The tandem MS spectrum and SRM chromatograph for the amino acid mutation variation are shown. An example of SRM analysis of HBs antigen (genotype C) contained in a human serum sample is shown. Sample A: HBV negative serum, Sample B-D: Hepatitis B patient serum. []: HBsAg value measured by the CLIA method. Serum samples were digested by the method shown in FIG. 4 and then subjected to SRM analysis.

A method for detecting or measuring hepatitis B virus in a biological sample is provided, which comprises detecting or measuring an oligopeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 7 by mass spectrometry.

Selected Reaction Monitoring (SRM), a type of mass spectrometry The principle of detecting hepatitis B virus in biological samples using mass spectrometry is based on the case of using a triple quadrupole mass spectrometer. An example is shown in FIG. (1) A protein component is extracted from a biological sample such as blood by an arbitrary method. (2) Protease digests the extracted protein component. (3) The digested peptide group is separated by means such as liquid chromatography. (4) The separated digested peptide is ionized and subjected to mass spectrometry. (5-1) Only peptide ions having the same mass-to-charge ratio as the mass-to-charge ratio of the digested peptide of HBs antigen (hereinafter, may be referred to as "probe peptide") measured in advance are used in the triple quadrupole mass analyzer. Pass through the Q1 filter. (5-2) Only fragmented peptides having the same mass-to-charge ratio as the mass-to-charge ratio generated when the peptide ions that have passed through the Q1 filter are fragmented by collision-induced dissociation and the probe peptide measured in advance is fragmented. Pass through the Q3 filter. (6) When a fragmented peptide having the same mass-to-charge ratio as that produced when the target peptide is fragmented is obtained, it can be determined that hepatitis B virus is present in the biological sample.

In the above method, the probe peptide is a peptide fragment consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 7. As is clear from the above principle, the probe for detecting HBs antigen by mass spectrometry is an oligopeptide produced by digesting HBs antigen with protease or the like with reproducibility, high ionization efficiency, and translation. In order to avoid a change in mass number due to post-modification, it is preferable to satisfy the conditions such as the absence of methionine residues in the amino acid sequence. Oligopeptides consisting of the amino acid sequences of SEQ ID NOs: 1 to 7 satisfy these conditions. The amino acid sequences of SEQ ID NOs: 1 to 7 and the genotypes and isoforms of the HBsAg in which they are present are shown in the table below.

The type and origin of the biological sample are not particularly limited. For example, the biological sample can be one or more selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, lymph fluid, liver tissue, cultured cells and hepatitis B virus infected cells. In one embodiment, the preferred biological sample is blood, plasma or serum. Further, in one embodiment, the biological sample may be a human-derived biological sample suspected of being infected with hepatitis B virus or a human-derived biological sample confirmed to be infected with hepatitis B virus. preferable.

The biological sample to be subjected to mass spectrometry is preferably digested with a protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue and / or the arginine residue. This is because by digesting the HBs antigen with such a protease, an oligopeptide having the amino acid sequence of any of SEQ ID NOs: 1 to 7 can be obtained. That is, in the amino acid sequence of HBs antigen, an arginine residue or a lysine residue is present on the N-terminal side of the amino acid sequences of SEQ ID NOs: 1 to 7. The protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue and / or the arginine residue is not particularly limited, and may be one type of protease or a combination of two or more types of proteases. Examples of such a protease include trypsin or a combination of the endoproteinase Lys-C (manufactured by Roche) and the endoprotease Arg-C (manufactured by Roche). By treating the biological sample with these proteases under appropriate conditions, a biological sample having an oligopeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 7 can be obtained.

The mass spectrometry method is not particularly limited as long as the HBs antigen in the biological sample can be detected or measured, and a known mass spectrometry method and a mass spectrometry method to be developed in the future can be used. In one embodiment, mass spectrometry consists of a quadrupole mass spectrometer, a time-of-flight mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, an ion trap mass spectrometer, and an orbitrap mass spectrometer. It is preferable to use a more selected mass spectrometer. In one embodiment, the preferred mass spectrometer is a triple quadrupole mass spectrometer.

As shown in Table 1, the genotype of hepatitis B virus can be identified by detecting HBsAg using an oligopeptide consisting of the amino acid sequences of SEQ ID NOs: 1 to 7 as a probe. Specifically, when an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 4 are detected in the biological sample by mass spectrometry, hepatitis B virus contained in the biological sample. The genotype of can be distinguished from A type or G type. Similarly, when an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 5 are detected, the genotype of hepatitis B virus can be distinguished from type B. When an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 2 is detected, the genotype of hepatitis B virus can be identified as type C. When an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 3 is detected, the genotype of hepatitis B virus can be identified as type D. When an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 6 is detected, the genotype of hepatitis B virus can be identified as type E. When an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1 and an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 7 are detected, the genotype of hepatitis B virus can be distinguished from F type or H type.

By using an oligopeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 7 as an internal standard substance, the amount of HBs antigen in a biological sample can be measured (that is, quantitative analysis). When an oligopeptide consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 7 is used as an internal standard, the oligopeptide is preferably labeled. The type of label is not particularly limited as long as the internal standard substance can be measured by mass spectrometry. In one embodiment, the preferred label is an isotope label. Quantitative analysis using isotope-labeled reference materials is also called isotope dilution mass spectrometry. The number and number of amino acid residues to be labeled are arbitrary and are not particularly limited. For example, all carbon atoms of the arginine residue constituting the oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1 to 7 can be labeled with 13C, and all nitrogen atoms can be labeled with 15N. Since the oligopeptide thus isotope-labeled differs from the oligopeptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 7 derived from the biological sample only in mass number, the amount (concentration) of the oligopeptide is measured in advance. By comparing the area of the peak area of the standard substance with the area of the peak area of the oligopeptide derived from the corresponding biological sample, the HBs antigen in the biological sample can be measured.

The oligopeptide used as an internal standard substance may have any structure as long as it functions as an internal standard substance. In one embodiment, the oligopeptide used as the internal standard material is from at least one amino acid sequence selected from SEQ ID NOs: 1 to 7 by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the arginine residue. It preferably has a structure that yields an isotope-labeled oligopeptide. In this document, peptides having such a structure are also referred to as "precursor peptides". That is, the precursor peptide may have an arginine residue or a lysine residue on the N-terminal side of any of the amino acid sequences of SEQ ID NOs: 1 to 7, and an arbitrary amino acid residue on the C-terminal side. Such a precursor peptide is an isotope consisting of at least one amino acid sequence selected from SEQ ID NOs: 1 to 7 by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue or arginine residue. One or more additional amino acid residues may be added to the N-terminal side and / or C-terminal side thereof as long as the body-labeled oligopeptide is produced. The precursor peptide also includes an oligopeptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 7 (that is, an oligopeptide to which one or more other amino acids are not added).

The precursor peptide producing an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 4 preferably has a lysine residue or an arginine residue added to the N-terminal side of the amino acid sequence of SEQ ID NO: 4. Similarly, the precursor peptide producing an oligopeptide consisting of the amino acid sequences of SEQ ID NOs: 5 and 6 preferably has a lysine residue or an arginine residue added to the N-terminal side of the amino acid sequences of SEQ ID NOs: 5 and 6. .. This is because the amino acid sequences of SEQ ID NOs: 4 to 6 have a glutamic acid residue at the N-terminal, and if this is present as an N-terminal residue, it spontaneously becomes Pyro-Glu, which may cause a quantitative bias. A precursor peptide having a lysine residue or an arginine residue on the N-terminal side of an oligopeptide consisting of any of the amino acid sequences of SEQ ID NOs: 4 to 6 further has one or more other amino acid residues on the N-terminal side. It may have one or more other amino acid residues on the C-terminal side.

The structure of the precursor peptide is particularly high as long as digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the arginine residue yields an oligopeptide consisting of at least one amino acid sequence selected from SEQ ID NOs: 1-7. Not limited. In one embodiment, the length of the precursor polypeptide is 70 amino acid residues or less, 65 amino acid residues or less, 60 amino acid residues or less, 55 amino acid residues or less, 50 amino acid residues or less, 45 amino acid residues or less, It is 40 amino acid residues or less, 35 amino acid residues or less, 30 amino acid residues or less, 25 amino acid residues or less, 20 amino acid residues or less, or 15 amino acid residues or less.

In one embodiment, the precursor peptide is one or more oligos selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1-3 by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the arginine residue. It preferably has a structure that yields the peptide and one or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4-7. By using such a precursor peptide, it is possible to estimate the amount of any combination of oligopeptides derived from HBs antigen in the biological sample. Thereby, the isoform ratio of HBs antigen in the biological sample can be determined. Examples of such precursor peptides are shown in Table 2 below.

The amino acid sequences of the precursor peptides illustrated in Table 2 directly (not via the linker sequence) the amino acid sequences of SEQ ID NOs: 1 to 7, but specifically the peptide bond on the carboxyl side of the arginine residue. One or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 3 and one or more selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4 to 7 by digestion with a protease that cleaves. Any linker sequence may be used as long as it produces an oligopeptide. The precursor peptides exemplified in Table 2 are linked from the N-terminal side in ascending order of SEQ ID NO: for convenience, but are sequenced by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the arginine residue. As long as one or more oligopeptides selected from the group consisting of the amino acid sequences of Nos. 1 to 3 and one or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4 to 7 are produced. The arrangement is arbitrary.

As described above, by using an oligopeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 7 as a probe, HBs antigen can be detected without missing a so-called escape mutant by mass spectrometry. However, HBsAg may have mutations in the region corresponding to SEQ ID NOs: 1-7. Specifically, the existence of mutations as shown in FIG. 8 has been reported. In FIG. 8, the amino acid sequence of SEQ ID NO: 4 is shown in the upper row, and the amino acid sequence of SEQ ID NO: 2 is shown in the lower row. The alphabet shown below the alphabet indicating the amino acid residue of each amino acid sequence is the amino acid residue reported as a substitution of the amino acid residue. For example, it has been reported that the N-terminal glutamine (Q) of SEQ ID NO: 4 may be replaced with arginine (R), lysine (K), leucine (L), proline (P), and glutamic acid (E). There is. Therefore, such mutant HBs antigens can be detected by changing the amino acid sequences of SEQ ID NOs: 1 to 7 according to the sequences of the mutants. Therefore, the present invention also includes oligopeptides consisting of amino acid sequences modified according to such variants, and precursor peptides.

The precursor peptide makes it possible to measure the amount of hepatitis B virus protein in a biological sample by isotope dilution mass spectrometry. Therefore, a kit for measuring the amount of hepatitis B virus protein in a biological sample by isotope dilution mass spectrometry containing a precursor peptide is provided. The kit can include other reagents useful for measuring the amount of hepatitis B virus protein in a biological sample by isotope dilution mass spectrometry, instructions for use, and the like.

The oligopeptides and precursor peptides used as the above-mentioned internal standard substances can be produced by any method known in the art. For example, a chemical synthesis method such as the Fmoc method or the Boc method can be used.

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

1. 1. Determination of probe peptides for HBsAg
To construct an SRM assay for HBsAg, peptide mapping analysis using recombinant HBsAg (genotype C) derived from cultured cells as an analysis material was performed in order to obtain probe peptides with high ionization efficiency (also called "Proteotypic peptides"). went. After separating the solubilized HBs antigen by SDS-PAGE, trypsin digestion was performed in the gel, and trypsin digested products derived from each isoform of HBs antigen (LHBs, MHBs, and SHBs) were recovered. As a result of tandem MS analysis using a triple quadrupole mass spectrometer (QTRAP5500, manufactured by AB-SCIEX) using the obtained digested sample, the LHBs-specific sequence was obtained as shown in Table 3 and FIG. 2 below. One type of digestive peptide derived from it and one type of digestive peptide common to all isoforms were detected (Confidence Score 95 or higher).

The peptide FLWEWASVR (SEQ ID NO: 2) was selected as a probe peptide for estimating the total expression level of HBsAg. The peptide QPTPISPPLR (SEQ ID NO: 4), which is derived from the LHBs-specific sequence, has a Gln residue at the N-terminal, which may cause a quantitative bias due to Pyro-Glu formation that occurs after digestion. Therefore, we designed an artificial sequence (FLWEWASVRQPTPISPPLR (SEQ ID NO: 8)) in which FLWEWASVR (SEQ ID NO: 2) and QPTPISPPLR (SEQ ID NO: 4) were combined, and performed isotope-diluted mass spectrometry using the 13C / 15N label as an internal standard. By doing this, the quantitative bias was corrected. FLWEWASVR (SEQ ID NO: 2) and QPTPISPPLR (SEQ ID NO: 4) are both amino acid sequences in the HBs antigen of genotype C. The amino acid sequences in the other genotypes corresponding to these are as shown in Table 1 above.

2. Construction of SRM Assay for HBsAg To construct an SRM assay targeting two selected probe peptides, a triple quadrupole mass connected to a nano flow rate liquid chromatograph (LC) (NanoLC400, manufactured by Ekisigent). An assayer (QTRAP5500, manufactured by AB-SCIEX) was used. Peptide separation was performed by a C18 reverse phase column (75 μm ID x 15 cm). 0.1% formic acid was used for mobile phase A of LC, and 0.1% formic acid / 80% acetonitrile was used for mobile phase B. The flow rate during tandem MS analysis was 300 nL / min, 2-20% mobile phase B / 3 minutes, 20-30% mobile phase B / 32 minutes, 30-90% mobile phase B / 5 minutes, 90-90% migration. Analysis was performed under gradient conditions for phase B / 10 minutes and 2-2% mobile phase B / 10 minutes. Based on the fragmented ion information obtained from tandem MS analysis, the parameters for SRM of HBs antigens (genotypes A, B, C, and D) were determined. The fragmentation information obtained for genotype C is shown in FIG. Based on the information obtained, assay parameters such as SRM transitions and collision energies were set. For transition settings, fragments resulting from the observed divalent precursor ion (eg, for genotype C, m / z 597.3 for FLWEWASVR (SEQ ID NO: 2); m / z 553.3 for QPTPISPPLR (SEQ ID NO: 4)). Among the chemical ions, the y ion group having high ionic strength was used. Using the selected transition, SRM measurement of a tryptic digested sample of recombinant HBs antigen (genotype C) derived from cultured cells was performed, and an SRM chromatograph of the probe peptide was obtained (right side of FIG. 3). Skyline software (MacLean B, et al., Bioinformatics. 2010 Apr 1; 26 (7): 966-8.) Was used to analyze the acquired data.

Internal standard peptides were synthesized labeled with an arginine residue stable isotopes in peptides ^{_{(13 C 6/15 N 4}} ). After determining the absolute amount of the synthetic peptide after purification (95%) by amino acid analysis, the stock solution for internal standard (0.5 pmol / μL) at the time of quantitative analysis was adjusted.

3. 3. Sample pretreatment using soluble polyacrylamide gel In mass spectrometry of HBs antigen, which is a sparingly soluble membrane protein, efficient solubilization and protease digestion in the sample pretreatment step are preferable for achieving reproducible quantitative analysis. .. So far, the present inventors have developed an in-gel protein digestion method for poorly soluble proteins using a soluble polyacrylamide gel (Takemori N. et al., Analyt Technol Biomed Life Sci. 2014 Sep 15; 967: 36-40.), Mass analysis pretreatment of HBs antigen was performed using this method. The pretreatment scheme is shown in FIG. The analysis was performed according to the following procedure, including the pretreatment scheme: (1) HBs antigens were extracted from various biological samples using SDS lysate, and (2) N, N'-bis (acryloyl) cystamine (BAC) -acrylamide solution. To prepare a polyacrylamide gel by mixing with (3) to remove impurities that inhibit ionization by washing the prepared gel, (4) after drying the gel, add an internal standard peptide and trypsin, and add 37 ° C. Trypsin digestion treatment was carried out in (5) Gel was dissolved by reduction treatment, (6) Digested peptide was recovered and then purified by a reverse phase column, (7) SRM analysis.

Specifically, trypsin digestion in the gel was carried out by the following procedure. A protein sample for mass spectrometry (5 μL) obtained from cultured human cells or human serum was reduced with 200 mM dithiothreitol (1 μL) and then alkylated with 1.2 Macrylamide (1 μL). The analytical sample (7 μL) after the reduction alkylation treatment is mixed with a 30% / 0.8% (w / v) acrylamide / BAC solution (5 μL), and then 1.5% ammonium peroxodisulfate (1 μL) and tetramethyl. A polymerization reaction was carried out by adding ethylenediamine (0.5 μL) to prepare a polyacrylamide gel. The prepared acrylamide gel was washed with washing liquid A (50v / v% methanol + 5v / v% acetic acid) and washing liquid B (50 mM ammonium hydrogencarbonate + 50v / v% acetonitrile), then water was removed using acetonitrile, and air-dried at room temperature. did. The gel was collected in a plastic tube, a peptide solution for internal standard (2 μL) was added, and the mixture was allowed to stand at 4 ° C. for 30 minutes. After further adding trypsin (0.1 μg), in-gel digestion (37 ° C., 24 hours) was performed in a 100 mM ammonium hydrogencarbonate solution (40 μL). After completion of the reaction, a 50 mM Tris (2-carboxyethyl) phosphine solution (20 μL) was added to completely dissolve the gel. The obtained gel lysate was mixed with acetonitrile (60 μL) and stirred for 30 seconds. The solution was centrifuged at 15,000 xg for 3 minutes, and the supernatant was collected in a separate tube. The recovered supernatant was mixed with 20 mM ammonium formate (480 μL) and added to a self-made mini-column for solid-phase extraction using 3M Emporedisk SDB-XC. The column was washed with 0.1% trifluoroacetic acid / 10% acetonitrile (40 μL) and then the peptide was recovered with 0.1% trifluoroacetic acid / 30% acetonitrile (30 μL). The recovered peptide solution was mixed with 0.1% trifluoroacetic acid (70 μL) to prepare a sample for mass spectrometry.

4. Construction of SRM assay for genotype identification The probe peptide sequence used in the above assay construction was obtained from gene C type, and other major genotypes (A type, B type, and D type). There is a difference of one amino acid residue in the corresponding amino acid sequence in (Fig. 5A). Therefore, in order to identify each genotype, a conjugate of a probe peptide specific to each genotype was synthesized (FIG. 5B), and an assay was constructed using the conjugate. The acquired MS / MS spectra are shown in FIGS. 5C and 5D. The acquired SRM chromatograph is shown in FIG.

5. Detection of HBV mutants
Mutations within the epitope region of HBsAg (D144A / G145R) are a major cause of false negatives in current immunoassays. Quantitative analysis by various antibody methods (CLIA method and WB method) using recombinant antigens (wild type and mutant D144A / G145R) revealed that the estimated expression ratio of wild type and antigenic determinant mutants was conventional. Similar to the report in (Fig. 7), the CLIA method and the WB method showed significantly different results (Fig. 7). On the other hand, in the quantitative analysis of HBs antigens by SRM, since a sequence other than the epitope region is used as a probe, the problem of false positives in the CLIA method can be avoided. As a result of measuring the absolute amount of recombinant antigen using the SRM assay constructed this time, we succeeded in obtaining a value close to the result of WB.

On the other hand, the appearance of mutant strains in the probe peptide sequence is expected to be the main cause of false negatives in HBV diagnosis using highly specific SRM. Amino acid mutations in the probe sequences of gene C type reported so far are shown in FIG. To solve the problem, it is considered effective to construct an SRM assay for each mutation sequence in advance and perform simultaneous monitoring of mutation variations. In the past, it took time and cost to prepare the standard product required for assay construction, but by utilizing the crude peptide synthesis service, it is possible to synthesize a peptide library covering all mutant sequences quickly and inexpensively. is there. By experimentally synthesizing a crude peptide set covering the major mutants of gene C type and using it for assay construction, we succeeded in rapidly determining the SRM transition corresponding to each mutant (Fig. 9). ). Since the method established this time can respond quickly to the emergence of new mutants, it is also an extremely effective method for constructing an assay for envelope proteins of RNA viruses (for example, HCV) that are more frequently mutated than HBV. Is expected to be.

6. Target analysis of HBs antigens in blood For the diagnosis of HBV infection by mass spectrometry, the serum diagnostic method by absolute quantitative SRM is preferable. In recent years, the assay has been miniaturized toward the realization of less invasive serum diagnosis, and it is hoped that a highly sensitive SRM assay capable of analysis from a trace serum sample (submicroliter level) will be developed. It is rare. Therefore, we performed quantitative analysis of HBs antigens contained in HBV-positive serum samples by utilizing the analytical methods established so far for recombinant antigens. The serum sample to be used was previously mixed with an SDS solution (final concentration 1%) to inactivate the virus activity. Samples for SRM analysis were prepared using a solution equivalent to 0.1 μl of serum. As a result of SRM analysis using three HBV-positive samples, we succeeded in detecting the common sequence FLWEWASVR (SEQ ID NO: 2) in all the samples (Fig. 10).

Claims (6)

An isotope-labeled oligopeptide consisting of at least one amino acid sequence selected from SEQ ID NOs: 1-7 by digestion with a protease that specifically cleaves the carboxyl-side peptide bond of the lysine and / or arginine residues. The resulting precursor peptide, which has an arginine or lysine residue at the N-terminus of at least one amino acid sequence selected from SEQ ID NOs: 1-7 and is 70 amino acid residues or less in length. Peptide . One or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 3 by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the lysine residue and / or the arginine residue, and The precursor peptide according to claim 1, which yields one or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4-7. The precursor polypeptide of claim 1 or 2 , having the amino acid sequence of any of SEQ ID NOs: 8-33. An internal standard substance for detecting or measuring hepatitis B virus in a biological sample by isotope dilution mass spectrometry, which comprises the precursor polypeptide according to any one of claims 1 to 3 . A kit for detecting or measuring hepatitis B virus in a biological sample by isotope dilution mass spectrometry, which comprises the precursor peptide according to claim 1 or 2 . Use of the precursor peptide according to claim 1 or 2 for detecting or measuring hepatitis B virus by isotope dilution mass spectrometry.

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