CN114524863B - Polypeptide sequence for quantitatively detecting content of G protein in rabies vaccine based on mass spectrometry and application of polypeptide sequence - Google Patents
Polypeptide sequence for quantitatively detecting content of G protein in rabies vaccine based on mass spectrometry and application of polypeptide sequence Download PDFInfo
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- CN114524863B CN114524863B CN202210161790.6A CN202210161790A CN114524863B CN 114524863 B CN114524863 B CN 114524863B CN 202210161790 A CN202210161790 A CN 202210161790A CN 114524863 B CN114524863 B CN 114524863B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a polypeptide sequence for quantitatively detecting the content of G protein in rabies vaccine based on mass spectrometry, which comprises 10 specific polypeptide sequence groups capable of repeatedly detecting rabies virus G protein. The invention also discloses a method for quantitatively detecting the G protein content in the rabies vaccine by using the polypeptide sequence, which comprises the steps of screening the polypeptide sequence specific to the G protein in a sample, adopting corresponding heavy standard quantitative peptide, rabies virus G protein or inactivated rabies virus sample containing corresponding peptide segment sequences to perform serial dilution as a standard substance, drawing a standard curve, and analyzing detection signals of a preset target peptide segment by using a parallel reaction monitoring technology of a mass spectrometer, thereby realizing quantitative detection of the rabies virus G protein. The method has expandability, high sensitivity and strong specificity, does not depend on antibodies, can quantitatively detect, and can be applied to content measurement and quality assessment of rabies vaccine stock solution and finished products.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a polypeptide sequence for quantitatively detecting the content of G protein in rabies vaccine based on mass spectrometry and application thereof.
Background
The rabies virus antigen content is one of the main quality indexes of rabies vaccine, wherein glycoprotein (G protein) is the only protein of rabies virus which can induce a host to produce and combine with neutralizing antibodies. The protection titer of the rabies vaccine is mainly acted by the G protein, and the protection titer of the vaccine can be determined by the content of the G protein in the vaccine to a certain extent. As the potency measurement by the mouse method reflects the in vivo biological activity of the vaccine, the EIA method can detect the relative content of glycoprotein antigen in the product, and the results of the two detection methods are positively correlated under the condition of good biological activity of virus antigen. Therefore, the mouse method and the EIA method are commonly adopted in the development and production of the rabies vaccine in China at present, and the quality control of the effective components of the vaccine is carried out.
The mouse method comprises virus titer measurement, NIH method efficacy measurement and the like, the test period of the method needs 14-28 days, the use of experimental animals has strict ethical constraint, and the test result is easily influenced by uncontrollable factors such as individual differences of the animals, so the method has the obvious limitations of poor stability of the measurement result.
The EIA method is a detection method established by utilizing the characteristic of specific binding of an anti-rabies virus G protein monoclonal antibody and a G protein antigen, and comprises a unidirectional radial immunodiffusion test (SRD), an enzyme-linked immunosorbent assay (ELISA), a time-resolved immunofluorescence assay (TRFIA) and the like. Compared with the mouse method, the EIA method is relatively simple, convenient and rapid, has strong specificity, and has the advantages of relatively quantitative detection and the like; however, this type of method also has a number of disadvantages:
(1) Antibody specificity cannot cover rabies strains, and usually only specific strains can be detected;
(2) The antigen-antibody binding reaction has strict requirements on test conditions, and the reaction temperature and time are often required to be accurately controlled;
(3) The measurement result is influenced by the buffer solution and the sample composition, the variation of the non-antigen components of the sample often leads to the deviation of the test, and the antigen content in the process of technological development cannot be accurately estimated.
Therefore, combining the above situations, the protein Parallel Reaction Monitoring (PRM) quantification technology based on mass spectrometry has the characteristics of high sensitivity, high specificity, no dependence on antibodies, quantification and the like, is hopeful to overcome the defects of the EIA method, has important significance in developing a quantitative detection method for measuring the G protein content of rabies virus, and is hopeful to become a new generation quality control technology different from the EIA principle.
Disclosure of Invention
Aiming at the defects of the prior art, the technical problem to be solved by the invention is to provide a polypeptide sequence for quantitatively detecting the content of G protein in the rabies vaccine based on a mass spectrometry, and the polypeptide sequence is applied to quantitatively detecting the content of G protein in the rabies vaccine by the mass spectrometry, so that the quality control level of the rabies vaccine can be improved.
In order to solve the technical problems, the invention provides a polypeptide sequence for quantitatively detecting the content of G protein in rabies vaccine based on mass spectrometry, which comprises 10 specific polypeptide sequence groups capable of repeatedly detecting rabies virus G protein, wherein the amino acid sequence is as follows:
(1)VGYISAIK；
(2)YEESLHNPYPDYH；
(3)TTKESLIIISPSVTDLDPYDK；
(4)ESLIIISPSVTDLDPYDK；
(5)LMDGTWVAMQTSDETK；
(6)SDEIEHLVVEELVK；
(7)LVPGFGK；
(8)TWNEIIPSK；
(9)MHPLADPSTVFK；
(10)EGDEAEDFVEVHLPDVYK。
the invention provides a screening method of polypeptide sequences, which comprises the following steps:
(1) Enzymolysis of the inactivated rabies virus sample FASP;
(2) LC-MS/MS (DDA experiment), adopting software to perform data analysis, screening repeated peptide segments with good mass spectrum signal response, and selecting 17 polypeptide sequences;
(4) LC-MS/MS (PRM experiment); screening a polypeptide sequence of the rabies virus G protein with good specificity and repeated occurrence, introducing the screened 17 peptide fragment sequence information into software for PRM method setting, and taking 0.5 mug peptide fragment from a rabies virus G protein standard for chromatographic separation; the separated peptide segment directly enters a mass spectrometer Thermo SCIENTIFIC Q Exactive for PRM mass spectrum detection; and (3) carrying out RPM experimental data analysis by adopting software, deducting background, screening out peptide fragments with good mass spectrum signal response, and finally obtaining 10 polypeptide sequences as described above.
Wherein, the enzymolysis of the inactivated rabies virus sample FASP in the step (1) comprises the following specific steps:
(1) 3 different batches of samples containing inactivated rabies virus were taken: adding TCEP, reacting for 1 hour at 60 ℃, adding MMTS, and carrying out reductive alkylation at room temperature for 45 minutes;
(2) Adding the protein solution subjected to reductive alkylation into a 10K ultrafiltration tube, and centrifuging at 4 ℃ for 20min with 12000 g;
(3) Adding 8M urea (pH 8.5), centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 2 times;
(4) Adding 0.25M TEAB, centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 3 times;
(5) The new collection tube was replaced, 0.5M TEAB, trypsin (trypsin to protein mass ratio 1:50) were added, and 37℃overnight;
(6) Trypsin (trypsin to protein mass ratio 1:100) was added the next day and reacted at 37℃for 4 hours; centrifuging for 20min at 12000 g;
(7) Centrifuging the peptide solution subjected to enzymolysis and digestion at the bottom of a collecting pipe; adding 0.5MTEAB into an ultrafiltration tube, centrifuging for 20min at 4 ℃ and 12000g, and combining the solution at the bottom of a collecting tube with the above step to obtain an enzymolysis sample;
(8) And (5) vacuum-pumping the sample after enzymolysis to carry out the next experiment.
In addition, in order to improve the quality control level of the rabies vaccine, the invention also provides a method for quantitatively detecting the content of G protein in the rabies vaccine based on a mass spectrometry, the method selects the polypeptide sequence as a target sequence, serial dilution is carried out by using rabies virus G protein or inactivated rabies virus samples as a standard, mass spectrum signals of the target sequence are collected, a coordinate system is established by using the mass spectrum signals of the concentration/content of the rabies virus G protein and the target sequence, and a linear regression equation is fitted to realize the content determination of the rabies virus G protein.
Preferably, in the method, polypeptide sequences as described above are adopted, and a re-standard quantitative peptide is prepared and used as an internal standard substance to be added into a sample to be detected, so that accurate quantification of the rabies virus G protein content is realized.
Preferably, the method comprises the following specific steps:
(1) Performing FASP enzymolysis on a sample and a standard substance, wherein the standard substance is a rabies virus G protein or a standard substance obtained by serial dilution of an inactivated rabies virus sample;
(2) LC-MS/MS (PRM experiment): adding the sample and the standard substance diluted according to the gradient into a sample introduction cup for LC-MS/MS;
(3) After the experiment is completed, 3 secondary sub-ion signals with highest ion intensity in the target polypeptide sequence are summed to be used as mass spectrum ion intensity signal values of the target polypeptide sequence, mass spectrum data are exported to be subjected to data analysis, the concentration of the rabies virus G protein standard substance solution is used as an abscissa, the mass spectrum ion intensity signal values are used as an ordinate to draw a standard curve, a linear fitting equation is obtained, the mass spectrum ion intensity signal values of the target polypeptide sequence of the sample to be detected are substituted into the equation, the concentration of the sample on-machine test solution is combined, and the rabies virus G protein content of the sample to be detected is calculated according to the sample pretreatment process.
Wherein, FASP enzymolysis of the sample and the standard in the step (1) is specifically as follows:
Respectively taking 200 mug standard substance and sample to be tested (protein content 157 mug/ml, taking 1274 mug for enzymolysis), adding 4 mug TCEP, placing at 60 ℃ for reaction for 1 hour, adding 2 mug MMTS, and reacting at room temperature for 45 minutes for reductive alkylation; adding into a 10K ultrafiltration tube, and centrifuging at 4deg.C for 20min at 12000 g; discarding the filtrate, adding 100 μl of 8M urea (pH 8.5), centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 2 times; adding 0.25M TEAB 100 μl, centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 3 times; the new collection tube was replaced, 50. Mu.l of 0.5M TEAB was added to the ultrafiltration tube, trypsin (trypsin to protein mass ratio 1:50) was added, and the reaction was carried out overnight at 37 ℃; adding trypsin (trypsin to protein mass ratio is 1:100) the next day, reacting for 4 hours at 37 ℃, centrifuging for 20 minutes at 12000g, and centrifuging the peptide fragment solution after enzymolysis and digestion at the bottom of a collecting pipe; 50 μl of 0.5M TEAB was added to the ultrafiltration tube, and the mixture was centrifuged at 12000g for 20min at 4deg.C, and the bottom solution of the collection tube was combined with the above step to obtain 100 μl of the sample after enzymatic hydrolysis, and vacuum-dried.
Wherein, in the step (2), the LC-MS/MS (PRM experiment) is specifically as follows:
(1) PRM method setting is carried out in mass spectrometer (Thermo SCIENTIFIC Q Exactive) software Xcalibur, and target polypeptide sequence information including mass-to-charge ratio, charge number, peptide fragment sequence, protein name and time is input;
(2) 200 mu l of a peptide segment of the standard substance which is pumped out is dissolved by using a sample dissolving solution (0.1% formic acid and 5% acetonitrile) to obtain a rabies virus G protein standard substance solution stock solution with the concentration of 1 mu G/mu l; respectively sucking 5 mu l, 7.5 mu l, 10 mu l, 12.5 mu l, 15 mu l and 20 mu l of standard solution stock solution, adding the sample solution to 40 mu l, and preparing standard solution with the concentration of 125 ng/mu l, 187.5 ng/mu l, 250 ng/mu l, 312.5 ng/mu l, 375 ng/mu l and 500 ng/mu l, adding the standard solution into a sample injection cup for LC-MS/MS, and setting the sample injection amount of the upper machine to 8 mu l/times;
(3) 200 μl of sample-pumped polypeptide is dissolved by using a sample dissolving solution (0.1% formic acid and 5% acetonitrile) to obtain a sample polypeptide solution stock solution to be tested with a concentration of 1 μg/μl; 12.5. Mu.l of the stock solution was aspirated, 27.5. Mu.l of the sample solution was added to make the total volume 40. Mu.l, and LC-MS/MS was performed in a sample introduction cup, and the on-press sample introduction amount was set to 8. Mu.l/time.
Compared with the prior art, the technical scheme of the invention at least comprises the following beneficial effects:
According to the scheme, a mass spectrometry technology is utilized to screen out a specific polypeptide sequence of rabies virus glycoprotein (G protein), the method is applied to quantitative detection of quality control of rabies vaccine, specifically, the method is used for screening out the specific polypeptide sequence of the G protein in a sample, serial dilution is carried out on a corresponding heavy standard quantitative peptide, the rabies virus G protein or an inactivated rabies virus sample containing a corresponding peptide fragment sequence to serve as a standard substance, a standard curve is drawn, and a Parallel Reaction Monitoring (PRM) technology of a mass spectrometer is utilized to analyze detection signals of a preset target peptide fragment, so that quantitative detection of the rabies virus G protein is realized. The method has expandability, high sensitivity and strong specificity, does not depend on antibodies, can quantitatively detect, and is expected to be applied to content measurement and quality assessment of rabies vaccine stock solution and finished products.
Drawings
In order to more clearly illustrate the technical solution of the embodiments of the present invention, the drawings of the embodiments will be briefly described below.
Fig. 1: recombinant rabies virus G protein purification profile. Wherein LaneM: SDS-PAGE Protein Marker, lane1: supernatant after inclusion body dissolution and centrifugation, lane2: supernatant was incubated with Ni-IDA followed by effluent, lane3:50mM Imidazole elution component Lane4-9:300mM Imidazole elution component.
Fig. 2: recombinant rabies virus G protein identification map. Wherein LaneM: wester Blot Marker, lane1: recombinant rabies virus G protein.
Fig. 3: and (3) a standard curve for calculation of rabies virus G protein quantitative detection.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: screening of rabies virus G protein stabilizing peptide
1 Enzymatic hydrolysis of inactivated rabies virus sample FASP
(1) 3 Different batches of samples containing inactivated rabies virus were taken: adding TCEP, reacting for 1 hour at 60 ℃, adding MMTS, and carrying out reductive alkylation at room temperature for 45 minutes;
(2) Adding the protein solution subjected to reductive alkylation into a 10K ultrafiltration tube, and centrifuging at 4 ℃ for 20min with 12000 g;
(3) Adding 8M urea (pH 8.5), centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 2 times;
(4) Adding 0.25M TEAB, centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 3 times; (5) The new collection tube was replaced, 0.5M TEAB, trypsin (trypsin to protein mass ratio 1:50) was added,
Overnight at 37 ℃;
(6) Trypsin (trypsin to protein mass ratio 1:100) was added the next day and reacted at 37℃for 4 hours; centrifuging for 20min at 12000 g;
(7) Centrifuging the peptide solution subjected to enzymolysis and digestion at the bottom of a collecting pipe; adding 0.5MTEAB into an ultrafiltration tube, centrifuging for 20min at 4 ℃ and 12000g, and combining the solution at the bottom of a collecting tube with the above step to obtain an enzymolysis sample;
(8) And (5) vacuum pumping.
2LC-MS/MS (DDA experiment)
2.1 Experimental details
(1) Mobile phase information:
Mobile phase a:0.1% of formic acid, and the mixture is mixed with water,
Mobile phase B:0.1% formic acid, 80% ACN
(2) Chromatographic operation:
The peptide fragment was dissolved in a sample solution (0.1% formic acid, 5% acetonitrile), vortexed with sufficient shaking, centrifuged at 13500rpm at 4℃for 20min, the supernatant was transferred to a coupon, 3 replicates were performed for each sample, and 7. Mu.l each sample was subjected to two-dimensional liquid chromatography. The elution conditions were as follows, the analysis time of each sample was 90min, and the flow rate was 300nL/min.
(3) Mass spectrometry parameters:
The separated peptide fragment directly enters a mass spectrometer for on-line detection, and the specific parameters are as follows:
Primary mass spectrometry parameters:
Resolution:70,000
Maximum ionic strength AGC TARGET:3e6
Maximum injection time Maximum IT:60ms
Scan range SCAN RANGE:350to 1800m/z
Secondary mass spectrometry parameters:
Resolution:17,500
Maximum ionic strength AGC TARGET:5e4
Maximum injection time Maximum IT:100ms of
Parent ion selection 20: topN:20
Impact energy NCE/STEPPEDNCE:27
2.2 Data analysis
Adopting software (Skyline) to perform data analysis, and screening peptide fragments which repeatedly appear and have good mass spectrum signal response; finally, 17 polypeptide sequences are selected, and the sequence information is shown in Table 1.
TABLE 1 repeatable, good signal response polypeptide sequence information for DDA screening
3LC-MS/MS (PRM experiment)
The above-mentioned 17 peptide fragment sequence information was imported into software (Xcalibur) for PRM method setting. Taking 0.5 mug peptide fragment from rabies virus G protein standard substance for chromatographic separation; the separated peptide segment directly enters a mass spectrometer Thermo SCIENTIFIC Q Exactive for PRM mass spectrum detection; and adopting software to perform RPM experimental data analysis, deducting background, screening out peptide fragments with good mass spectrum signal response, wherein the peptide fragments are respectively as follows:
(1)VGYISAIK(SEQ ID NO：3)；
(2)YEESLHNPYPDYH(SEQ ID NO：4)；
(3)TTKESLIIISPSVTDLDPYDK(SEQ ID NO：5)；
(4)ESLIIISPSVTDLDPYDK(SEQ ID NO：6)；
(5)LMDGTWVAMQTSDETK(SEQ ID NO：7)；
(6)SDEIEHLVVEELVK(SEQ ID NO：8)；
(7)LVPGFGK(SEQ ID NO：9)；
(8)TWNEIIPSK(SEQ ID NO：10)；
(9)MHPLADPSTVFK(SEQ ID NO：11)；
(10)EGDEAEDFVEVHLPDVYK(SEQ ID NO：12)。
Example 2: preparation of recombinant rabies virus G protein
1 Gene synthesis and vector construction
The gene sequence of rabies virus G protein (sequence table: SEQ ID No. 2) is optimized by adopting codon optimization software MaxCodonTM Optimization Program (V13), then the sequence is synthesized by a total gene synthesis method, the G protein gene is inserted into an expression vector pET30a (Kan+) through restriction enzyme cleavage sites NdeI and HindIII, and the correctness of the final expression vector is confirmed by an enzyme cleavage method and sequencing.
2 Rabies virus G protein expression purification and identification
2.1 Transformation of expression vectors and inducible expression
The constructed plasmid containing rabies virus G protein gene was transformed into BL21 (DE 3) competent cells, then uniformly spread on LB plates (containing 50. Mu.g/mL kanamycin sulfate), and inverted in an incubator at 37℃overnight. From the transformed plates, single clones were selected, inoculated into 4mL of LB medium (containing 50. Mu.g/mL kanamycin sulfate), cultured until OD600 was 0.5-0.8, and 0.2mM IPTG was added to the test tube culture medium at a final concentration, and the culture medium was placed at 15℃and 37℃for induction of expression, respectively, and identification was performed.
2.2 Engineering bacteria culture
And (3) confirming engineering bacteria and induction expression conditions thereof through analysis and identification. Culturing 28L engineering bacteria, after the engineering bacteria grow to OD600 of 0.8, adding IPTG with final concentration of 0.2mM, and collecting thalli after induction for 16h at 15 ℃.
2.3 Purification of target proteins
The rabies virus G protein is expressed in inclusion bodies by Ni-IDA affinity chromatography purification analysis, the inclusion bodies are washed by 20mM PB (pH 7.8) and 300mM NaCl containing 1% Triton X-100,2mM EDTA,5mM DTT, then the inclusion bodies are dissolved by 20mMPB (pH 7.8) buffer solution and balanced with Ni-IDA column, finally target proteins are eluted by balanced buffer solutions with different concentrations of imidazole, and each eluted component is collected for SDS-PAGE analysis detection, as a result, see figure 1, lane 4-8 is collected, added into a dialysis bag after treatment, and the treated dialysis bag is subjected to renaturation in buffer solution [1 XPBS (pH 7.8), 4mM GSH,0.4mM GSSG,0.4ML-Arginine,1M Urea,5%Glycerol ] at 4 ℃, and G protein after renaturation is finally dialyzed in storage solution 1 XPBS (pH 7.8) and 5% glycerol solution for about 6-8 hours. After dialysis renaturation, the supernatant was filtered through a 0.22 μm filter and then sub-packaged, and frozen to-80 ℃.
2.5 Identification of target proteins
Taking 1.2 mug purified rabies virus G protein (amino acid sequence: SEQ ID NO: 1), carrying out electrophoresis, membrane transfer and sealing, respectively incubating by a primary antibody and a secondary antibody, and developing the results, wherein the developing results are shown in the accompanying figure 2: the target protein identification result meets the expectations, and can be used as a standard for detecting the rabies virus G protein content by a PRM method.
Example 3: detection of rabies virus G protein content in sample
1 Sample FASP enzymatic hydrolysis
Respectively taking 200 mug standard substance and sample to be tested (protein content 157 mug/ml, taking 1274 mug for enzymolysis), adding 4 mug TCEP, placing at 60 ℃ for reaction for 1 hour, adding 2 mug MMTS, and reacting at room temperature for 45 minutes for reductive alkylation; adding into a 10K ultrafiltration tube, and centrifuging at 4deg.C for 20min at 12000 g; discarding the filtrate, adding 100 μl of 8M urea (pH 8.5), centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 2 times; adding 0.25M TEAB 100 μl, centrifuging at 4deg.C for 20min at 12000g, discarding the waste liquid, and repeating for 3 times; the new collection tube was replaced, 50. Mu.l of 0.5M TEAB was added to the ultrafiltration tube, trypsin (trypsin to protein mass ratio 1:50) was added, and the reaction was carried out overnight at 37 ℃; adding trypsin (trypsin to protein mass ratio is 1:100) the next day, reacting for 4 hours at 37 ℃, centrifuging for 20 minutes at 12000g, and centrifuging the peptide fragment solution after enzymolysis and digestion at the bottom of a collecting pipe; 50 μl of 0.5M TEAB was added to the ultrafiltration tube, and the mixture was centrifuged at 12000g for 20min at 4deg.C, and the bottom solution of the collection tube was combined with the above step to obtain 100 μl of the sample after enzymatic hydrolysis, and vacuum-dried.
2PRM procedure
(1) PRM method setup was performed in mass spectrometer (Thermo SCIENTIFIC Q Exactive) software Xcalibur, and selected target polypeptide sequence information (exemplified by "SDEIEHLVVEELVK") was entered, specifically including: mass to charge ratio, number of charges, peptide fragment sequence, protein name, time, etc.
(2) 200 Mu l of a peptide segment of the standard substance which is pumped out is dissolved by using a sample dissolving solution (0.1% formic acid and 5% acetonitrile) to obtain a rabies virus G protein standard substance solution stock solution with the concentration of 1 mu G/mu l; stock solutions of 5. Mu.l, 7.5. Mu.l, 10. Mu.l, 12.5. Mu.l, 15. Mu.l and 20. Mu.l were aspirated, and the sample solutions were fed to 40. Mu.l to prepare standard solutions of concentrations 125 ng/. Mu.l, 187.5 ng/. Mu.l, 250 ng/. Mu.l, 312.5 ng/. Mu.l, 375 ng/. Mu.l and 500 ng/. Mu.l, and added to a sample cup for LC-MS/MS, and the amount of the upper sample was set to 8. Mu.l/time.
(3) 200 Μl of sample-pumped polypeptide is dissolved by using a sample dissolving solution (0.1% formic acid and 5% acetonitrile) to obtain a sample polypeptide solution stock solution to be tested with a concentration of 1 μg/μl; 12.5. Mu.l of the stock solution was aspirated, 27.5. Mu.l of the sample solution was added to make the total volume 40. Mu.l, and LC-MS/MS was performed in a sample introduction cup, and the on-press sample introduction amount was set to 8. Mu.l/time.
3PRM results calculation
After the experiment is completed, the 3 secondary sub-ion signals with highest ion intensity in the target polypeptide sequence are summed to be used as mass spectrum ion intensity signal values of the target polypeptide sequence, and mass spectrum data are exported for data analysis.
And drawing a standard curve (figure 3) by taking the concentration of the rabies virus G protein standard substance solution as an abscissa and the mass spectrum ion intensity signal value as an ordinate, wherein a linear fitting equation is as follows:
Y＝(4.31E+07)X－(4.20E+09)，R2＝0.9933
Substituting the mass spectrum ion intensity signal value of the target polypeptide sequence of the sample to be detected into the equation, and calculating the concentration of the on-machine test solution of the sample to be detected to be 145 ng/. Mu.l.
According to the sample pretreatment process, the rabies virus G protein content of the sample to be detected can be known as follows:
I.e. 72.8 mug/ml.
According to the method, the polypeptide sequence of the specificity of the G protein in the sample is firstly screened, the corresponding re-standard quantitative peptide, rabies virus G protein or an inactivated rabies virus sample containing the corresponding peptide fragment sequence is adopted for serial dilution to be used as a standard substance, a standard curve is drawn, and a detection signal of a preset target peptide fragment is analyzed by utilizing a Parallel Reaction Monitoring (PRM) technology of a mass spectrometer, so that the quantitative detection of the rabies virus G protein is realized. The method has expandability, high sensitivity and strong specificity, does not depend on antibodies, can quantitatively detect, and is expected to be applied to content measurement and quality assessment of rabies vaccine stock solution and finished products.
The foregoing disclosure is merely illustrative of the preferred embodiments of the present invention and is not intended to limit the scope of the claims herein, as equivalent changes may be made in the claims herein without departing from the scope of the invention.
Sequence listing
<110> Guangzhou Nocheng biological products, inc
<120> Polypeptide sequence for quantitatively detecting G protein content in rabies vaccine based on mass spectrometry and application thereof
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Arg Thr Thr Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp
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His Leu Val Val Glu Glu Leu Val Lys Lys Arg Glu Glu Cys Leu Asp
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Ser His Leu Arg Lys Leu Val Pro Gly Phe Gly Lys Ala Tyr Thr Ile
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<210> 2
<211> 1575
<212> DNA
<213> Rabies virus (Rabies virus)
<400> 2
atggttcctc aggttctttt gtttgtaccc cttctgggtt tttcgttgtg tttcgggaag 60
ttccccattt acacgatacc agacgaactt ggtccctgga gccctattga catacaccat 120
ctcagctgtc caaataacct ggttgtggag gatgaaggat gtaccaacct gtccgagttc 180
tcctacatgg aactcaaagt gggatacatc tcagccatca aagtgaacgg gttcacttgc 240
acaggtgttg tgacagaggc agagacctac accaactttg ttggttatgt cacaaccaca 300
ttcaagagaa agcatttccg ccccacccca gacgcatgta gagccgcgta taactggaag 360
atggccggtg accccagata tgaagagtcc ctacacaatc cataccccga ctaccactgg 420
cttcgaactg taagaaccac caaagagtcc ctcattatca tatccccaag tgtgacagat 480
ttggacccat atgacaaatc ccttcactca agggtcttcc ctggcggaaa gtgctcagga 540
ataacggtgt cctctaccta ctgctcaact aaccatgatt acaccatttg gatgcccgag 600
aatccgagac caagtacacc ttgtgacatt tttaccaata gcagagggaa gagagcatcc 660
aaagggaaca agacttgcgg ctttgtggat gaaagaggcc tgtataagtc tctaaaagga 720
gcatgcaggc tcaagttatg tggagttctt ggacttagac ttatggatgg aacatgggtc 780
gcgatgcaaa catcagatga gaccaaatgg tgccctccag ataagttggt gaatttgcac 840
gactttcgct cagacgagat tgagcatctc gttgtggagg agttagtcaa gaaaagagag 900
gaatgtctgg atgcattaga gtccatcatg accaccaagt cagtaagttt cagacgtctc 960
agtcacctga gaaaacttgt cccagggttt ggaaaagcat ataccatatt caacaaaacc 1020
ttgatggagg ctgatgctca ctacaagtca gtccggacct ggaatgagat catcccctca 1080
aaagggtgtt tgaaagttgg aggaaggtgc catcctcatg tgaacggggt gtttttcaat 1140
ggtataatat tagggcctga cggccatgtc ctaatcccag agatgcaatc atccctcctc 1200
cagcaacata tggagttgtt gaaatcttca gttatccccc tgatgcaccc cctggcagac 1260
ccttctacag ttttcaaaga aggtgatgag gctgaggatt ttgttgaagt tcacctcccc 1320
gatgtgtaca aacagatctc aggggttgac ctgggtctcc cgaactgggg aaagtatgta 1380
ttgatgactg caggggccat gattggcctg gtgttgatat tttccctaat gacatggtgc 1440
agaagagcca atcgaccaga atcgaaacaa cgcagttttg gagggacagg gaggaatgtg 1500
tcagtcactt cccaaagcgg aaaagtcata ccttcatggg aatcatataa gagtggaggt 1560
gagatcagac tgtga 1575
<210> 3
<211> 8
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 3
Val Gly Tyr Ile Ser Ala Ile Lys
1 5
<210> 4
<211> 13
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 4
Tyr Glu Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His
1 5 10
<210> 5
<211> 21
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 5
Thr Thr Lys Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp Leu
1 5 10 15
Asp Pro Tyr Asp Lys
20
<210> 6
<211> 18
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 6
Glu Ser Leu Ile Ile Ile Ser Pro Ser Val Thr Asp Leu Asp Pro Tyr
1 5 10 15
Asp Lys
<210> 7
<211> 16
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 7
Leu Met Asp Gly Thr Trp Val Ala Met Gln Thr Ser Asp Glu Thr Lys
1 5 10 15
<210> 8
<211> 14
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 8
Ser Asp Glu Ile Glu His Leu Val Val Glu Glu Leu Val Lys
1 5 10
<210> 9
<211> 7
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 9
Leu Val Pro Gly Phe Gly Lys
1 5
<210> 10
<211> 9
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 10
Thr Trp Asn Glu Ile Ile Pro Ser Lys
1 5
<210> 11
<211> 12
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 11
Met His Pro Leu Ala Asp Pro Ser Thr Val Phe Lys
1 5 10
<210> 12
<211> 18
<212> PRT
<213> Rabies virus (Rabies virus)
<400> 12
Glu Gly Asp Glu Ala Glu Asp Phe Val Glu Val His Leu Pro Asp Val
1 5 10 15
Tyr Lys
Claims (7)
1. A polypeptide sequence for quantitatively detecting the content of G protein in rabies vaccine based on mass spectrometry comprises 10 specific polypeptide sequence groups of repeatedly-detected rabies virus G protein, and the amino acid sequence is as follows:
(1)VGYISAIK；
(2)YEESLHNPYPDYH；
(3)TTKESLIIISPSVTDLDPYDK；
(4)ESLIIISPSVTDLDPYDK；
(5)LMDGTWVAMQTSDETK；
(6)SDEIEHLVVEELVK；
(7)LVPGFGK；
(8)TWNEIIPSK；
(9)MHPLADPSTVFK；
(10)EGDEAEDFVEVHLPDVYK。
2. use of the polypeptide sequence as claimed in claim 1 in a reagent for quantitatively detecting the G protein content in rabies vaccine.
3. A method for quantitatively detecting the content of G protein in rabies vaccine based on mass spectrometry is characterized by comprising the following steps: according to the method, the polypeptide sequence as claimed in claim 1 is selected as a target sequence, rabies virus G protein or inactivated rabies virus samples are adopted for serial dilution to serve as a standard, mass spectrum signals of the target sequence are collected, a coordinate system is established by using the 'concentration/content of rabies virus G protein-mass spectrum signals of the target sequence', and a linear regression equation is fitted to realize the content measurement of rabies virus G protein.
4. A method for quantitatively detecting G protein content in rabies vaccine based on mass spectrometry as claimed in claim 3, characterized in that: in the method, the polypeptide sequence of claim 1 is adopted, and a prepared re-standard quantitative peptide is used as an internal standard and added into a sample to be detected, so that the accurate quantification of the rabies virus G protein content is realized.
5. A method for quantitative detection of G protein content in rabies vaccine based on mass spectrometry as claimed in claim 3, comprising the specific steps of:
(1) Performing FASP enzymolysis on a sample and a standard substance, wherein the standard substance is a rabies virus G protein or a standard substance obtained by serial dilution of an inactivated rabies virus sample;
(2) LC-MS/MS, PRM experiment: adding the sample and the standard substance diluted according to the gradient into a sample introduction cup for LC-MS/MS;
(3) After the experiment is completed, 3 secondary sub-ion signals with highest ion intensity in the target polypeptide sequence are summed to be used as mass spectrum ion intensity signal values of the target polypeptide sequence, mass spectrum data are exported to be subjected to data analysis, the concentration of the rabies virus G protein standard substance solution is used as an abscissa, the mass spectrum ion intensity signal values are used as an ordinate to draw a standard curve, a linear fitting equation is obtained, the mass spectrum ion intensity signal values of the target polypeptide sequence of the sample to be detected are substituted into the equation, the concentration of the sample on-machine test solution is combined, and the rabies virus G protein content of the sample to be detected is calculated according to the sample pretreatment process.
6. The method for quantitatively detecting G protein content in rabies vaccine based on mass spectrometry according to claim 5, wherein FASP enzymatic hydrolysis of the sample and standard in step (1) is specifically:
Respectively taking 200 mug of standard substance and sample to be tested, wherein the protein content of the sample to be tested is 157 mug/ml, taking 1274 mug of the sample to be tested for enzymolysis, adding 4 mug of TCEP, placing at 60 ℃ for reaction for 1 hour, adding 2 mug of MMTS, and reacting at room temperature for 45 minutes for reductive alkylation; adding into a 10K ultrafiltration tube, and centrifuging at 4deg.C for 20min at 12000 g; discarding the filtrate, adding 100 μl of 8M urea, centrifuging at 8.5,4 deg.C with 12000g of urea for 20min, discarding the waste liquid, and repeating for 2 times; 100 μl of 0.25MTEAB was added, and the mixture was centrifuged at 12000g for 20min at 4deg.C, and the waste liquid was discarded and repeated 3 times; the new collection tube was replaced, 50. Mu.l of 0.5M TEAB was added to the ultrafiltration tube, trypsin was added, the trypsin was reacted with protein in a 1:50 mass ratio, 37℃overnight; adding trypsin in the next day, reacting at 37 ℃ for 4 hours with the trypsin and protein in a mass ratio of 1:100, centrifuging 12000g for 20min, and centrifuging the peptide fragment solution after enzymolysis and digestion at the bottom of a collecting pipe; 50 μl of 0.5M TEAB was added to the ultrafiltration tube, and the mixture was centrifuged at 12000g for 20min at 4deg.C, and the bottom solution of the collection tube was combined with the above step to obtain 100 μl of the sample after enzymatic hydrolysis, and vacuum-dried.
7. The method for quantitative detection of G protein content in rabies vaccine based on mass spectrometry according to claim 5, wherein LC-MS/MS, PRM assay in step (2) is specifically:
(1) PRM method setting is carried out in mass spectrometer (Thermo SCIENTIFIC Q Exactive) software Xcalibur, and target polypeptide sequence information including mass-to-charge ratio, charge number, peptide fragment sequence, protein name and time is input;
(2) Dissolving the peptide fragments of the standard substance by 200 mu l of sample dissolving liquid, wherein the sample dissolving liquid comprises 0.1% formic acid and 5% acetonitrile, so as to obtain rabies virus G protein standard substance solution stock solution with the concentration of 1 mu G/mu l; respectively sucking 5 mu l, 7.5 mu l, 10 mu l, 12.5 mu l, 15 mu l and 20 mu l of standard solution stock solution, adding the sample solution to 40 mu l, and preparing standard solution with the concentration of 125 ng/mu l, 187.5 ng/mu l, 250 ng/mu l, 312.5 ng/mu l, 375 ng/mu l and 500 ng/mu l, adding the standard solution into a sample injection cup for LC-MS/MS, and setting the sample injection amount of the upper machine to 8 mu l/times;
(3) Dissolving the polypeptide pumped by the sample with 200 mu l of sample dissolving solution, wherein the sample dissolving solution comprises 0.1% formic acid and 5% acetonitrile, so as to obtain a polypeptide solution stock solution of the sample to be tested with the concentration of 1 mu g/mu l; 12.5. Mu.l of the stock solution was aspirated, 27.5. Mu.l of the sample solution was added to make the total volume 40. Mu.l, and LC-MS/MS was performed in a sample introduction cup, and the on-press sample introduction amount was set to 8. Mu.l/time.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924571A (en) * | 2012-10-29 | 2013-02-13 | 复旦大学 | Rabies virus glycoprotein and nucleoprotein antigen epitope polypeptides, and screening and identification method and application thereof |
| RU2020142861A3 (en) * | 2021-02-08 | 2021-04-16 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102924571A (en) * | 2012-10-29 | 2013-02-13 | 复旦大学 | Rabies virus glycoprotein and nucleoprotein antigen epitope polypeptides, and screening and identification method and application thereof |
| RU2020142861A3 (en) * | 2021-02-08 | 2021-04-16 |
Non-Patent Citations (2)
| Title |
|---|
| Novel mass spectrometry based detection and identification of variants of rabies virus nucleoprotein in infected brain tissues;Matthew Reed等;PLOS Neglected Tropical Diseases;第12卷(第12期);第1-18页 * |
| Proteomic Profiling of Purified Rabies Virus Particles;Yan Zhang等;Virologica Sinica;第35卷;第143–155页 * |
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