JPWO2017026241A1 - Method for measuring hepatitis B virus using mass spectrometry - Google Patents

Abstract

Providing means for accurately detecting hepatitis B virus. A method for detecting or measuring hepatitis B virus in a biological sample, comprising detecting or measuring an oligopeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 7 by mass spectrometry.

Description

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

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

  Currently, diagnosis of HBV infection is performed mainly by detecting an HBs antigen or an antibody thereof present in human serum by an immunization method (Patent Document 1). However, the appearance of antigenic determinant mutants (escape mutants) that pass through detection by immunization has been reported and is problematic.

JP2007-14267

    An object of this invention is to provide the means etc. which detect a hepatitis B virus more correctly.

  In order to solve the above-mentioned objects, etc., the present inventors have intensively studied and arrived at 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, the present inventors can not only detect hepatitis B virus by mass spectrometry by using the peptide fragment as a probe, but also identify the genotype of hepatitis B virus and a small amount of sample. It was confirmed that the virus 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 repeating these findings and further studies and studies, the following inventions are provided.

Item 1.
A method for detecting or measuring hepatitis B virus in a biological sample, comprising detecting or measuring an oligopeptide comprising any one of the amino acid sequences 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 peptide bonds on the carboxyl side of lysine residues and arginine residues.
Item 3.
Item 3. The method according to Item 1 or 2, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, lymph fluid, liver tissue, and cultured cells.
Item 4.
A mass selected from the group consisting of a quadrupole analyzer, 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 4. 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 by digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of a lysine residue and / or arginine residue The resulting precursor peptide.
Item 6.
Item 6. 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 a lysine residue and / or arginine residue, and Item 7. The precursor peptide according to Item 5 or 6, which generates one or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4 to 7. Item 8.
Item 8. The precursor polypeptide according to any one of Items 5 to 7, which has an amino acid sequence of any of SEQ ID NOs: 8 to 33.
Item 9.
Item 9. An internal standard for detecting or measuring hepatitis B virus in a biological sample by isotope dilution mass spectrometry, comprising 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 isotope dilution mass spectrometry, comprising the precursor peptide according to any one of Items 5 to 8.
Item 11.
An isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 2, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 3, An isotopically labeled oligopeptide consisting of 4 amino acid sequences, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 5, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 6, and a SEQ ID NO: B in a biological sample using at least one isotopically labeled oligopeptide selected from the group consisting of 7 isotopically labeled oligopeptides consisting of 7 amino acid sequences as an internal standard in isotope dilution mass spectrometry Detection, measurement, or genotype identification method for hepatitis B virus.
Item 12.
Item 12. The method according to Item 11, comprising digesting the biological sample with a protease that specifically cleaves peptide bonds on the carboxyl side of lysine residues and arginine residues.
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. Hepatitis B virus can be detected with high accuracy without using an antibody. Mutant hepatitis B virus that cannot be detected by immunization can be detected. Quantitative detection of hepatitis B virus is possible. The genotype of hepatitis B virus can be identified. A simple detection of hepatitis B virus is possible. Detection of hepatitis B virus with low invasiveness is possible.

A typical procedure for detecting a target protein 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 HBs antigen (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. 1 shows a pretreatment scheme for mass spectrometry of protein samples using a soluble polyacrylamide gel. 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 an HBV genotype using mass spectrometry is shown. 1 shows a stable isotope labeled peptide for use as an internal standard for isotope dilution mass spectrometry. 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. Fig. 2 shows a tandem MS spectrum of a digested peptide fragment (QPTPISPPLX: SEQ ID NO: 4) obtained by trypsin digestion of an internal standard peptide (SEQ ID No: 6). Examples of SRM analysis of HBs antigens (genotypes A, B, and D) and their internal standard peptides are shown. The HBs antigen contained in the cultured cell extract was digested by the technique shown in FIG. 4 and then subjected to SRM analysis. The western blot image of HBs antigen (wild type and mutant) expressed in cultured cells is shown. The quantitative analysis result by CLIA method of HBs antigen (wild type and mutant) expressed in cultured cells is shown. The quantitative analysis result by SRM of HBs antigen (wild type and mutant) expressed in cultured cells is shown. The amino acid variation variation known so far in the probe peptide sequence (LHBs region: QPTPISPPLR (SEQ ID NO: 4) and common region: FLWEWASVR (SEQ ID NO: 2)) of HBs antigen (genotype C) is shown. The tandem MS spectrum and SRM chromatograph concerning amino acid variation are shown. The tandem MS spectrum and SRM chromatograph concerning amino acid variation are shown. The tandem MS spectrum and SRM chromatograph concerning amino acid variation are shown. The tandem MS spectrum and SRM chromatograph concerning amino acid variation are shown. The tandem MS spectrum and SRM chromatograph concerning amino acid variation are shown. The tandem MS spectrum and SRM chromatograph concerning amino acid variation are shown. The tandem MS spectrum and SRM chromatograph concerning amino acid variation are shown. The tandem MS spectrum and SRM chromatograph concerning amino acid 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. []: HBs antigen value measured by CLIA method. Serum samples were subjected to SRM analysis after digestion by the method of FIG.

  There is provided a method for detecting or measuring hepatitis B virus in a biological sample, comprising detecting or measuring an oligopeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 7 by mass spectrometry.

  When using a triple quadrupole mass spectrometer, the principle of detecting hepatitis B virus in a biological sample using the selected reaction monitoring (SRM) mass spectrometry, which is a type of mass spectrometry. An example is shown in FIG. (1) A protein component is extracted from a biological sample such as blood by an arbitrary method. (2) The extracted protein component is digested with protease. (3) The digested peptide group is separated using means such as liquid chromatography. (4) The separated digested peptide is ionized and subjected to mass spectrometry. (5-1) Only a peptide ion having the same mass-to-charge ratio as the mass-to-charge ratio of a digested peptide of an HBs antigen measured in advance (hereinafter sometimes referred to as “probe peptide”) is measured with a triple quadrupole mass spectrometer. Pass through Q1 filter. (5-2) Fragmentation (fragmentation) of peptide ions that have passed through the Q1 filter by collision-induced dissociation, and only fragmented peptides having the same mass-to-charge ratio as the mass-to-charge ratio generated when the probe peptide measured in advance is fragmented Is passed through the Q3 filter. (6) When a fragmented peptide having the same mass-to-charge ratio as that generated 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, a peptide fragment consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 7 becomes a probe peptide. As is clear from the above principle, the probe for detecting HBs antigen by mass spectrometry is an oligopeptide that is reproducibly produced by digesting HBs antigen with a protease, etc., high ionization efficiency, and translation. In order to avoid mass number changes due to post-modification, it is preferable to satisfy the conditions such as the absence of a methionine residue in the amino acid sequence. The oligopeptide consisting of the amino acid sequences of SEQ ID NOs: 1 to 7 satisfies these conditions. The amino acid sequences of SEQ ID NOs: 1 to 7 and the genotypes and isoforms of HBs antigens in which they exist are shown in the table below.

  The type and origin of the biological sample are not particularly limited. For example, the biological sample may 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. In one embodiment, the biological sample may be a biological sample derived from a human suspected of being infected with hepatitis B virus or a human being 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 peptide bonds on the carboxyl side of lysine residues and / or arginine residues. This is because an oligopeptide having any one of the amino acid sequences of SEQ ID NOs: 1 to 7 can be obtained by digesting the HBs antigen with such a protease. That is, in the amino acid sequence of the 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 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 proteases include trypsin or a combination of endoprotease Lys-C (Roche) and endoprotease Arg-C (Roche). By treating a biological sample under appropriate conditions using these proteases, a biological sample having an oligopeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7 can be obtained.

  Mass spectrometry is not particularly limited as long as HBs antigen in a biological sample can be detected or measured, and known mass spectrometry and mass spectrometry developed in the future can be used. In one embodiment, the mass spectrometry is a group consisting of a quadrupole analyzer, 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 preferred 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, hepatitis B virus genotype can be identified by detecting HBs antigen using an oligopeptide consisting of the amino acid sequences of SEQ ID NOs: 1 to 7 as a probe. Specifically, when mass spectrometry detects 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 from a biological sample, hepatitis B virus contained in the biological sample 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 the B type. 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 distinguished from D type. When an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 6 is detected, the genotype of hepatitis B virus can be distinguished from E type. 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 any one of the amino acid sequences of SEQ ID NOs: 1 to 7 as an internal standard substance, the amount of HBs antigen in a biological sample can be measured (ie, quantitative analysis). When an oligopeptide consisting of any one of the amino acid sequences 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 an isotope-labeled standard substance is also called isotope dilution mass spectrometry. The amino acid residue and number to be labeled are arbitrary and are not particularly limited. For example, all carbon atoms of an arginine residue constituting an oligopeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7 can be labeled with 13C, and all nitrogen atoms can be labeled with 15N. The oligopeptide thus labeled with an isotope is different from the oligopeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7 derived only from the biological sample. 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.

  An oligopeptide used as an internal standard substance can have any structure as long as it functions as an internal standard substance. In one embodiment, the oligopeptide used as the internal standard substance is derived 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 results in an isotopically labeled oligopeptide. In this document, a peptide having such a structure is also referred to as a “precursor peptide”. That is, the precursor peptide may have an arginine residue or a lysine residue on the N-terminal side of any one of the amino acid sequences of SEQ ID NOS: 1 to 7, and may have any 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 a lysine residue or arginine residue. One or more additional amino acid residues may be added to the N-terminal side and / or the C-terminal side as long as the body-labeled oligopeptide is generated. The precursor peptide also includes an oligopeptide consisting of any one 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 that generates an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 4 preferably has a lysine residue or arginine residue added to the N-terminal side of the amino acid sequence of SEQ ID NO: 4. Similarly, the precursor peptide that generates an oligopeptide consisting of the amino acid sequences of SEQ ID NOs: 5 and 6 preferably has a lysine residue or arginine residue added to the N-terminal side of the amino acid sequences of SEQ ID NOs: 5 and 6. . This is because in the amino acid sequences of SEQ ID NOs: 4 to 6, the N-terminus is a glutamic acid residue, and if it exists as an N-terminal residue, it spontaneously becomes pyro-Glu, which may cause a quantitative bias. The 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 not limited as long as digestion with a protease that specifically cleaves the peptide bond on the carboxyl side of the arginine residue results in 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, 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 is preferable to have a structure that gives rise to one or more oligopeptides selected from the group consisting of peptides and amino acid sequences of SEQ ID NOs: 4-7. By using such precursor peptides, the amount of any oligopeptide combination derived from the HBs antigen in the biological sample can be assumed. Thereby, the isoform ratio of the HBs antigen in the biological sample can also be determined. Examples of such precursor peptides are shown in Table 2 below.

  In the amino acid sequence of the precursor peptide exemplified in Table 2, the amino acid sequences of SEQ ID NOs: 1 to 7 are directly linked (without the linker sequence), but the peptide bond on the carboxyl side of the arginine residue is specifically bound. 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 cleaves, and one or more types selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4 to 7 As long as the oligopeptide is generated, it may be via any linker sequence. In addition, the precursor peptides exemplified in Table 2 are linked from the N-terminal side in order of increasing sequence number for convenience, but the sequence is obtained 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 of amino acid sequences of SEQ ID Nos. 4 to 7 are generated, Arrangement is arbitrary.

  As described above, by using an oligopeptide consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 7 as a probe, the HBs antigen can be detected without missing a so-called escape mutant by mass spectrometry. However, the HBs antigen may have a mutation in the region corresponding to SEQ ID NOs: 1-7. Specifically, the presence 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 part, and the amino acid sequence of SEQ ID NO: 2 is shown in the lower part. The alphabet shown below the alphabet showing the amino acid residue of each amino acid sequence is the amino acid residue reported as a substitution for that amino acid residue. For example, it has been reported that N-terminal glutamine (Q) of SEQ ID NO: 4 may be substituted with arginine (R), lysine (K), leucine (L), proline (P), and glutamic acid (E). Yes. Therefore, such a mutant HBs antigen can be detected by changing the amino acid sequence of SEQ ID NOs: 1 to 7 in accordance with the mutant sequence. Accordingly, 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 the precursor peptide is provided. The kit can contain 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 oligopeptide and precursor peptide used as the above-mentioned internal standard substance can be produced by any technique known in the art. For example, a chemical synthesis method such as the Fmoc method or the Boc method can be used.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these.

1. Determination of probe peptides for HBs antigen
For the construction of SRM assay for HBs antigen, peptide mapping analysis using recombinant HBs antigen (genotype C) derived from cultured cells as analysis material is required to obtain probe peptides with high ionization efficiency (also called “Proteotypic peptides”). went. After solubilized HBs antigen was separated by SDS-PAGE, trypsin digestion was performed in the gel, and trypsin digestion products derived from HBs antigen isoforms (LHBs, MHBs, and SHBs) were recovered. As a result of performing tandem MS analysis using a triple quadrupole mass spectrometer (QTRAP5500, manufactured by AB-SCIEX) using the obtained digested sample, as shown in Table 3 and FIG. One derived digested peptide and one digested peptide common to all isoforms were detected (Confidence Score 95 or higher).

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

2. Construction of SRM assay for HBs antigen For construction of SRM assay targeting two types of selected probe peptides, triple quadrupole mass connected to a nano flow liquid chromatograph (LC) (NanoLC400, manufactured by Ekisigent) An analyzer (QTRAP5500, manufactured by AB-SCIEX) was used. Peptide separation was performed with a C18 reverse phase column (75 μm ID × 15 cm). LC mobile phase A was 0.1% formic acid, and mobile phase B was 0.1% formic acid / 80% acetonitrile. The flow rate during tandem MS analysis is 300 nL / min, 2-20% mobile phase B / 3 min, 20-30% mobile phase B / 32 min, 30-90% mobile phase B / 5 min, 90-90% mobile Analysis was carried out under gradient conditions of phase B / 10 min, 2-2% mobile phase B / 10 min. Based on fragmented ion information obtained from tandem MS analysis, SRM parameters for HBs antigens (genotypes A, B, C, and D) were determined. The fragmentation information obtained for genotype C is shown in FIG. Based on the obtained information, assay parameters such as SRM transition and collision energy were set. For the transition setting, a fragment generated from the observed divalent precursor ion (for example, in the case of genotype C, m / z 597.3 for FLWEWASVR (SEQ ID NO: 2); m / z 553.3 for QPTPISPPLR (SEQ ID NO: 4)) Among ionized ions, a group of y ions having high ionic strength was used. Using the selected transition, SRM measurement of a trypsin 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). For analysis of the acquired data, Skyline software (MacLean B, et al., Bioinformatics. 2010 Apr 1; 26 (7): 966-8.) Was used.

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, an internal standard stock solution (0.5 pmol / μL) at the time of quantitative analysis was prepared.

3. Sample pretreatment using soluble polyacrylamide gel In mass spectrometry of HBs antigen, a poorly soluble membrane protein, efficient solubilization and protease digestion in the sample pretreatment process are preferable for realizing reproducible quantitative analysis . So far, the present inventors have developed a method for in-gel protein digestion of sparingly soluble proteins using soluble polyacrylamide gels (Takemori N. et al., Analyt Technol Biomed Life Sci. 2014 Sep 15; 967: 36-40.), Mass spectrometry pretreatment of HBs antigen was performed using this technique. The pretreatment scheme is shown in FIG. The analysis was carried out according to the following procedure including the pretreatment scheme: (1) Extraction of HBs antigen from various biological samples by SDS lysis solution, (2) N, N'-bis (acryloyl) cystamine (BAC) -acrylamide solution (3) Remove the impurities that inhibit ionization by washing the prepared gel, (4) Add the internal standard peptide and trypsin after drying the gel, (5) Dissolve the gel by reduction treatment, (6) After recovering the digested peptide, purify by reverse phase column, (7) SRM analysis.

  Specifically, in-gel trypsin digestion was performed according to the following procedure. A protein sample for mass spectrometry (5 μL) obtained from human cultured cells or human serum was subjected to reduction treatment with 200 mM dithiothreitol (1 μL), and then subjected to alkylation treatment with 1.2 M acrylamide (1 μL). An analytical sample (7 μL) after the reductive alkylation treatment was mixed with a 30% / 0.8% (w / v) acrylamide / BAC solution (5 μL), and then 1.5% ammonium peroxodisulfate (1 μL) and tetramethyl were mixed. Ethylenediamine (0.5 μL) was added to carry out a polymerization reaction to prepare a polyacrylamide gel. The prepared acrylamide gel was washed with washing solution A (50 v / v% methanol + 5 v / v% acetic acid) and washing solution B (50 mM ammonium bicarbonate + 50 v / v% acetonitrile), then water was removed using acetonitrile and air-dried at room temperature. did. The gel was collected in a plastic tube, an internal standard peptide solution (2 μL) was added, and the mixture was allowed to stand at 4 ° C. for 30 minutes. Further, trypsin (0.1 μg) was added, followed by in-gel digestion (37 ° C., 24 hours) in a 100 mM ammonium hydrogen carbonate solution (40 μL). After completion of the reaction, 50 mM tris (2-carboxyethyl) phosphine solution (20 μL) was added to completely dissolve the gel. The obtained gel solution was mixed with acetonitrile (60 μL) and stirred for 30 seconds. The solution was centrifuged at 15,000 × g for 3 minutes, and the supernatant was collected in another tube. The collected supernatant was mixed with 20 mM ammonium formate (480 μL) and added to a minicolumn for solid phase extraction, which was self-made using 3M Empordisk SDB-XC. The column was washed with 0.1% trifluoroacetic acid / 10% acetonitrile (40 μL), and then the peptide was recovered using 0.1% trifluoroacetic acid / 30% acetonitrile (30 μL). The collected peptide solution was mixed with 0.1% trifluoroacetic acid (70 μL) to obtain a sample for mass spectrometry.

4). Construction of SRM assay for genotype discrimination 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) In FIG. 5A, there is a difference of one amino acid residue in the corresponding amino acid sequence (FIG. 5A). Therefore, in order to identify each genotype, a ligated probe peptide specific to each genotype was synthesized (FIG. 5B), and an assay was constructed using it. The acquired MS / MS spectra are shown in FIGS. 5C and 5D. The acquired SRM chromatograph is shown in FIG.

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

  On the other hand, the appearance of a mutant strain in the probe peptide sequence is expected to be a major cause of false negatives in HBV diagnosis using highly specific SRM. The amino acid mutations reported in the gene C-type probe sequence reported so far are shown in FIG. To solve the problem, it is considered effective to construct an SRM assay for each mutant sequence in advance and perform simultaneous monitoring of mutation variations. Conventionally, it took time and cost to prepare standard products required for assay construction, but using a crude peptide synthesis service, it is possible to quickly and inexpensively synthesize peptide libraries covering all mutated sequences. is there. By experimentally synthesizing a crudely purified peptide set that covers the major variants of gene C type and using them for assay construction, we succeeded in rapidly determining the SRM transitions corresponding to each variant (FIG. 9). ). The newly established method can respond quickly to the emergence of new mutants, so it is extremely effective in constructing assays for envelope proteins of RNA viruses (such as HCV) that have a higher frequency of mutation than HBV. It is expected to be

6). Target analysis of blood HBs antigen For diagnosis of HBV infection by mass spectrometry, a serum diagnostic method by absolute quantitative SRM is preferred. In recent years, the development of a highly sensitive SRM assay that can analyze small amounts of serum samples (sub-microliter level) is expected because the scale of the assay has been reduced to achieve a less invasive serum diagnosis. It is rare. Thus, quantitative analysis of HBs antigens contained in HBV-positive serum samples was performed by utilizing analytical techniques established so far with recombinant antigens. Serum samples to be used were inactivated by premixing with SDS solution (final concentration 1%). Samples for SRM analysis were prepared using a solution corresponding to 0.1 μl of serum. As a result of SRM analysis using three HBV positive samples, the common sequence FLWEWASVR (SEQ ID NO: 2) was successfully detected in all samples (FIG. 10).

Claims (13)

A method for detecting or measuring hepatitis B virus in a biological sample, comprising detecting or measuring an oligopeptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 7 by mass spectrometry. The method according to claim 1, wherein the biological sample is digested with a protease that specifically cleaves the peptide bond on the carboxyl side of lysine residues and arginine residues. The method of claim 1 or 2, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, urine, spinal fluid, bone marrow fluid, lymph fluid, and liver tissue. A mass selected from the group consisting of a quadrupole analyzer, a time-of-flight mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, an ion trap mass spectrometer, and an orbitrap mass spectrometer. The method according to claim 1, wherein an analyzer is used. An isotope-labeled oligopeptide 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 a lysine residue and / or arginine residue The resulting precursor peptide. The precursor peptide according to claim 5, which has a length of 70 amino acid residues or less. 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 a lysine residue and / or arginine residue, and Precursor peptide according to claim 5 or 6, which produces one or more oligopeptides selected from the group consisting of the amino acid sequences of SEQ ID NOs: 4-7. The precursor polypeptide according to any one of claims 5 to 7, which has an amino acid sequence of any of SEQ ID NOs: 8 to 33. An internal standard substance for detecting or measuring hepatitis B virus in a biological sample by isotope dilution mass spectrometry, comprising the precursor polypeptide according to claim 5. A kit for detecting or measuring hepatitis B virus in a biological sample by isotope dilution mass spectrometry, comprising the precursor peptide according to claim 5. An isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 2, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 3, An isotopically labeled oligopeptide consisting of 4 amino acid sequences, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 5, an isotopically labeled oligopeptide consisting of the amino acid sequence of SEQ ID NO: 6, and a SEQ ID NO: B in a biological sample using at least one isotopically labeled oligopeptide selected from the group consisting of 7 isotopically labeled oligopeptides consisting of 7 amino acid sequences as an internal standard in isotope dilution mass spectrometry Detection, measurement, or genotype identification method for hepatitis B virus. The method according to claim 11, comprising digesting the biological sample with a protease that specifically cleaves peptide bonds on the carboxyl side of lysine residues and arginine residues. 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.

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