CN114166966B - Method for determining pro-SFTPB value in serum - Google Patents

Method for determining pro-SFTPB value in serum Download PDF

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CN114166966B
CN114166966B CN202111421732.4A CN202111421732A CN114166966B CN 114166966 B CN114166966 B CN 114166966B CN 202111421732 A CN202111421732 A CN 202111421732A CN 114166966 B CN114166966 B CN 114166966B
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sftpb
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magnetic beads
serum
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CN114166966A (en
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高俊顺
高俊莉
江南
王洪
宋林珍
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Hangzhou Guangke Ander Biotechnology Co ltd
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Abstract

The invention discloses a method for determining pro-SFTPB in serum, which comprises the steps of preparing antibody coated magnetic beads, performing immune enrichment and purification on pro-SFTPB in serum, performing denaturation, reduction, alkylation and enzymolysis on protein in a serum sample, desalting, drying, re-dissolving and nano liter liquid chromatography-mass spectrometry analysis on the sample after enzymolysis; the beneficial effects of the invention are as follows: the method fills the blank of the prior art, solves the problem of large result deviation caused by nonspecific interference of antigen-antibody immune reaction in the traditional immunoassay, ensures the accuracy, reliability and traceability of the fixed value result, and provides reference for the serum standard substance development of pro-SFTPB and the measurement method research of reference values; the quantitative analysis method provided by the invention has guiding and reference significance for quantitative and traceable analysis and research of other protein markers in serum.

Description

Method for determining pro-SFTPB value in serum
Technical Field
The invention belongs to a method for determining a value of a standard substance, and particularly relates to a method for determining a value of pro-SFTPB in serum.
Background
The precursor protein (Pro-surfactant protein B, pro-SFTPB) of the lung surfactant protein can promote the modification and maturation of the lung surfactant protein B, and has the functions of regulating and maintaining the internal environment stability of the lung surfactant and promoting the reuse of the lung surfactant lipid and protein. The research shows that pro-SFTPB in blood is an independent predictive factor of lung cancer and can provide important supplement for the existing lung cancer risk prediction model. The conventional detection method of pro-SFTPB is an immunological method, but the accuracy and reliability thereof depend on the performance of specific affinity reactions based on antigen and antibody, and the quality difference between batches of antibody is a major factor causing a large deviation of measurement results.
At present, no quantitative traceability analysis and research on pro-SFTPB in serum has been reported, and therefore, development and establishment of a measurement method of a pro-SFTPB reference value in serum are urgently needed. The isotope dilution mass spectrometry has the characteristics of high sensitivity, high accuracy and good repeatability, and the measurement result can be traced to an international SI unit, thus being a currently internationally recognized protein fixed value method. For example, song Dewei et al, national academy of sciences of metering, established a method for determining the isotopic dilution mass spectrum of C-reactive proteins in serum.
Disclosure of Invention
The main purpose of the application is to provide a method for determining the value of pro-SFTPB in serum with good accuracy, reliability and traceability.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for the determination of pro-SFTPB in serum, comprising the steps of:
(1) Preparing antibody coated magnetic beads: activating magnetic beads, adding antibodies into the activated magnetic beads for coupling reaction, separating and cleaning the magnetic beads after the reaction is finished, adding a sealing agent to seal non-specific binding sites on the magnetic beads, separating and cleaning the magnetic beads, and re-suspending the magnetic beads by using PBS solution to obtain antibody-coated magnetic beads;
(2) Adding antibody-coated magnetic beads into a serum sample, diluting with PBS, shaking and incubating to enable pro-SFTPB in the serum to be bound to the magnetic bead-coupled antibodies, and cleaning the magnetic beads to remove non-specifically bound proteins on the magnetic beads;
(3) Sequentially adding a denaturing reagent, a reducing reagent and an alkylating reagent into the magnetic bead sample combined with the pro-SFTPB obtained in the step (2) to react with the pro-SFTPB in the sample; then adding trypsin solution for enzymolysis to obtain a sample containing characteristic peptide segment SFTPBb;
(4) Adding an isotope labeled internal standard peptide segment SFTPBb_IS into the sample of the characteristic peptide segment SFTPBb obtained in the step (3), purifying by a desalting column, and collecting eluent;
(5) And (3) re-dissolving the eluent obtained in the step (4) after freeze drying, carrying out nano liter liquid chromatography-mass spectrometry analysis, obtaining the concentration of the characteristic peptide fragment in the on-line sample through the peak area ratio of the characteristic peptide fragment to the internal standard peptide fragment, and calculating the concentration of pro-SFTPB in the serum sample according to the concentration of the characteristic peptide fragment.
The above-mentioned method for determining pro-SFTPB in serum, as a preferred embodiment,
in the step (1), the activated magnetic beads are: sequentially adding EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and NHS reagent (N-hydroxy thiosuccinimide) into magnetic beads, and performing rotary incubation for 30min at room temperature for activation; after the magnetic beads are activated, separating the magnetic beads by a magnetic frame to remove EDC and NHS reagents, adding a pro-SFTPB monoclonal antibody into the activated magnetic beads, mixing uniformly, and incubating for 2 hours at room temperature in a rotating way to perform a coupling reaction, wherein the weight ratio of the added pro-SFTPB monoclonal antibody to the magnetic beads is as follows: 30ug to 1mg.
The magnetic beads are carboxyl magnetic beads, the concentration is 20-100mg/mL, and the particle size is 1-3um.
The final concentration of the added EDC and NHS reagent is 0.2-1.0 mg/mL and 0.1-0.5 mg/mL respectively.
In the method for determining pro-SFTPB in serum, as a preferred embodiment, in the step (1), the blocking agent is PBS buffer solution containing 1% BSA, the blocking agent is added to rotationally incubate for 2 hours at room temperature, and then the mixture is kept stand at 4 ℃ for 15-24 hours to block the non-specific binding sites on the magnetic beads.
In the method for determining pro-SFTPB in serum, as a preferred embodiment, in the step (1), the step of re-suspending is to add PBS buffer containing 0.1% BSA to the magnetic beads to a concentration of 1-10mg/mL, prepare antibody-coated magnetic beads, and store the antibody-coated magnetic beads at 4 ℃ for later use.
In the method for determining the value of pro-SFTPB in serum, as a preferred embodiment, in the step (3), the denaturing agent is 50mM ammonium bicarbonate solution containing 5M urea, and after the denaturing agent is added to the sample, the sample is incubated for 10min at 2000rpm at room temperature with shaking; adding a reducing reagent of 500mM TCEP solution, uniformly mixing, and carrying out shaking incubation for 60min at 37 ℃ and 2000 rpm; adding 500mM iodoacetamide solution as alkylating agent, mixing, incubating at 2000rpm for 60min at room temperature in dark condition, adding trypsin solution to reduce urea concentration in denaturing agent to below 1M, and incubating at 37deg.C and 1500rpm for 24-48 hr to obtain sample containing characteristic peptide SFTPBb.
Preferably, the concentration of the trypsin solution is 10ng/uL, and the trypsin solution is prepared by dissolving trypsin in 50mM ammonium bicarbonate solution.
In the method for determining pro-SFTPB in serum, as a preferred embodiment, the sequence of the characteristic peptide segment SFTPBb is as follows: LVLPVLPGALQAR.
In the method for determining pro-SFTPB in serum, as a preferred embodiment, in the step (4), 10% formic acid aqueous solution and isotope labeled internal standard peptide segment SFTPBb_IS are sequentially added into a sample containing characteristic peptide segment SFTPBb, and after uniform mixing, the mixture IS added into a pre-balanced desalting column, firstly, the mixture IS washed for 2 times by 15% acetonitrile aqueous solution, then eluted for 3 times by 90% acetonitrile aqueous solution containing 0.1% formic acid, and the eluent IS collected and combined.
Preferably, the sequence of the internal standard peptide segment sftpbb_is IS: LVLPVLPGALQAR x 13 C 6 ， 15 N 4 。
In the above method for determining pro-SFTPB in serum, as a preferred embodiment, in the step (5), the reconstitution is: the reconstitution was performed with 15% acetonitrile in water containing 0.1% formic acid.
In the above method for determining pro-SFTPB in serum, as a preferred embodiment, in the step (5), the characteristic peptide fragment solutions of pro-SFTPB with different concentrations and the internal standard peptide fragment solution with fixed concentrations are mixed in equal volumes to prepare a standard series of solutions, and then nano liter liquid chromatography-mass spectrometry analysis is performed, wherein the concentration of the characteristic peptide fragment is taken as the abscissa, and the characteristic peptide fragmentAnd the peak area ratio of the internal standard peptide fragment is taken as an ordinate, a standard curve is established, and then the peak area ratio of the characteristic peptide fragment and the internal standard peptide fragment in the sample after the re-dissolution is substituted into the standard curve, so as to obtain the concentration C of the characteristic peptide fragment in the sample peptide 。
As a preferred embodiment, the method for determining the pro-SFTPB concentration in serum as described above, in step (5), the concentration C of pro-SFTPB in the serum sample protein The calculation formula of (2) is as follows:
C protein ＝C peptide ×V Dilute ×P UV ×M protein /V sample ×M peptide
wherein:
C protein representing the protein concentration in the original blood sample;
C peptide representing the concentration of the characteristic peptide fragments measured in the sample of the machine;
V Dilute representing the reconstituted volume of the sample after desalting and drying;
P UV indicating the determined UV purity of the characteristic peptide fragment;
M protein indicating the molecular weight of the protein measured;
V sample representing the volume of the original serum sample;
M peptide representing the molecular weight of the characteristic peptide fragment measured.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for determining the value of pro-SFTPB in serum, fills the blank of the prior art, solves the problem of large result deviation caused by nonspecific interference of antigen-antibody immune reaction in traditional immunoassay, ensures the accuracy, reliability and traceability of the result of the determination, and provides reference for developing serum standard substances of pro-SFTPB and researching a reference value measuring method.
Drawings
FIG. 1 is a chromatogram of a characteristic peptide fragment SFTPBb;
FIG. 2 IS a chromatogram of an internal standard peptide fragment SFTPBb_IS;
FIG. 3 is a standard curve of the characteristic peptide fragment SFTPBb.
Detailed Description
In order that those skilled in the art will better understand the present application, a technical solution in the embodiments of the present application will be clearly and completely described in the following in connection with examples, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.
The application discloses a method for determining pro-SFTPB in serum, which fills the blank of the prior art and solves the problem of large result deviation caused by nonspecific interference of antigen-antibody immune reaction in traditional immunoassay.
The method specifically comprises the following steps:
(1) Preparing antibody coated magnetic beads: activating magnetic beads, adding antibodies into the activated magnetic beads for coupling reaction, separating and cleaning the magnetic beads after the reaction is finished, adding a sealing agent to seal non-specific binding sites on the magnetic beads, separating and cleaning the magnetic beads, and re-suspending the magnetic beads by using PBS solution to obtain antibody-coated magnetic beads;
(2) Adding antibody-coated magnetic beads into a serum sample, diluting with PBS, shaking and incubating to enable pro-SFTPB in the serum to be bound to the magnetic bead-coupled antibodies, and cleaning the magnetic beads to remove non-specifically bound proteins on the magnetic beads;
(3) Sequentially adding a denaturing reagent, a reducing reagent and an alkylating reagent into the magnetic bead sample combined with the pro-SFTPB obtained in the step (2) to react with the pro-SFTPB in the sample; then adding trypsin solution for enzymolysis to obtain a sample containing characteristic peptide segment SFTPBb;
(4) Adding isotope labeled internal standard peptide segment SFTPBb_IS into the sample containing characteristic peptide segment SFTPBb obtained in the step (3), purifying by a desalting column, and collecting and combining eluents;
(5) And (3) re-dissolving the eluent obtained in the step (4) after freeze drying, carrying out nano liter liquid chromatography-mass spectrometry analysis, obtaining the concentration of the characteristic peptide fragment in the on-line sample through the peak area ratio of the characteristic peptide fragment to the internal standard peptide fragment, and calculating the concentration of pro-SFTPB in the serum sample according to the concentration of the characteristic peptide fragment.
Specific examples:
a method for the determination of pro-SFTPB in serum, comprising the steps of:
(1) Preparing antibody coated magnetic beads: sequentially adding EDC and NHS reagents into magnetic beads, and performing rotary incubation for 30min at room temperature for activation; after the magnetic beads are activated, separating the magnetic beads by a magnetic frame, removing EDC and NHS reagents, adding a pro-SFTPB monoclonal antibody into the activated magnetic beads, and carrying out coupling reaction by rotating and incubating for 2 hours at room temperature after uniformly mixing, wherein the weight ratio of the added pro-SFTPB monoclonal antibody to the magnetic beads is as follows: 30ug to 1mg;
after the reaction is finished, separating the magnetic beads by using a magnetic frame, removing unreacted antibodies, washing the magnetic beads by using a PBS solution, adding a PBS buffer solution containing 1% BSA as a blocking agent, rotating and incubating for 2 hours at room temperature, and standing for 15-24 hours at 4 ℃ to block non-specific binding sites on the magnetic beads; separating the magnetic beads by a magnetic frame, removing the blocking agent, washing the magnetic beads by a PBS solution, adding a PBS buffer solution containing 0.1% BSA for resuspension to prepare antibody coated magnetic beads with the concentration of 1mg/mL, and preserving the antibody coated magnetic beads at the temperature of 4 ℃ for later use;
(2) Adding 200uL of antibody-coated magnetic beads into a serum sample, diluting to 1mL with PBS, and incubating for 2h at room temperature under 2000rpm with shaking, so that pro-SFTPB in the serum is bound to the magnetic bead-coupled antibodies; after incubation is finished, adsorbing magnetic beads by using a magnetic rack, absorbing supernatant to perform chemiluminescence analysis, determining the magnetic bead enrichment efficiency of pro-SFTPB, and then washing the magnetic beads by using PBS to remove non-specifically bound proteins on the magnetic beads;
because of the complex matrix of the serum sample, interference is caused to immunoaffinity enrichment of pro-SFTPB in serum, so that the pro-SFTPB captured by the magnetic beads cannot truly reflect the content in the actual serum. The method is used for examining the enrichment efficiency of the magnetic beads, enriching human negative serum labeled samples containing pro-SFTPB with different concentrations by using the magnetic beads, wherein each concentration level is parallel for 3 times, and then detecting the concentration of the residual pro-SFTPB in supernatant after incubating the magnetic beads by using a chemiluminescent instrument, and the results are shown in Table 1.
As can be seen from Table 1, the magnetic bead enrichment efficiency of pro-SFTPB in serum samples with different concentrations is greater than 99.5%, which indicates that the method can efficiently enrich and extract pro-SFTPB in serum.
TABLE 1 evaluation results of magnetic bead enrichment efficiency
(3) Adding 50uL of a 50mM ammonium bicarbonate solution containing 5M urea into the magnetic bead sample combined with pro-SFTPB obtained in the step (2), and carrying out shaking incubation for 10min at the room temperature of 2000rpm to denature proteins in the sample; adding 2.5uL of a reducing reagent of 500mM TCEP solution, uniformly mixing, and then carrying out shaking incubation at 37 ℃ and 2000rpm for 60min to reduce disulfide bonds of the protein; adding 2.5uL of 500mM iodoacetamide solution serving as an alkylating reagent, uniformly mixing, and carrying out oscillation incubation at 2000rpm for 60min under the condition of being protected from light at room temperature, so as to carry out alkylation on sulfhydryl generated after reduction and prevent the sulfhydryl from being closed again to generate disulfide bonds; then adding trypsin solution to reduce the concentration of urea in the denaturing reagent to below 1M, and placing the mixture at 37 ℃ and 1500rpm for shaking incubation for 24-48 hours for enzymolysis to obtain a sample containing characteristic peptide SFTPBb; the sequence of the characteristic peptide segment SFTPBb is as follows: LVLPVLPGALQAR.
(4) Sequentially adding 10uL of 10% formic acid aqueous solution and 50uL of 5ng/mL isotope labeled internal standard peptide segment SFTPBb_IS into the sample containing the characteristic peptide segment SFTPBb obtained in the step (3), uniformly mixing, adding into a pre-balanced desalting column, washing for 2 times with 15% acetonitrile aqueous solution, eluting for 3 times with 90% acetonitrile aqueous solution containing 0.1% formic acid, and collecting and combining eluates; the sequence of the internal standard peptide segment SFTPBb_IS IS: LVLPVLPGALQAR x 13 C 6 ， 15 N 4 ；
(5) Freeze-drying the eluent obtained in the step (4), re-dissolving the eluent to 100uL by using 15% acetonitrile water solution containing 0.1% formic acid, and then carrying out nano liter liquid chromatography-mass spectrometry analysis:
the conditions for the nanoliter liquid chromatography were:
the analytical column adopts a C18 capillary chromatographic column (3 um,75um multiplied by 15 cm), the column temperature is 55 ℃, and the mobile phase A is 0.1% formic acid water solution; mobile phase B was 80% acetonitrile in water (0.1% formic acid); the loading solution is 0.1% formic acid water solution, and the volume is 10uL; gradient elution is adopted for 16min, the elution program is shown in table 2, the sample injection amount is 2uL, and the detection system is quadrupole-orbitrap tandem mass spectrum.
TABLE 2 gradient elution procedure for nanoliter liquid chromatography
Time (min) Flow rate (nL/min) Comparative example B (%)
0 500 20
10 500 54
11 1000 100
16 1000 100
The mass spectrometry conditions were: capillary voltage 2.1kV, collision energy NCE 27, resolution 30000, isolation window 1.6m/z, data were collected in PRM scan mode according to the parameters of Table 3, and chromatograms were recorded, as shown in FIGS. 1 and 2.
TABLE 3 PRM Scan information of pro-SFTPB
Concentration determination of characteristic peptide fragments in the sample: mixing characteristic peptide fragment solutions of pro-SFTPB with different concentrations and internal standard peptide fragment solutions of 5ng/mL in equal volume to prepare a standard series solution, then carrying out nano liter liquid chromatography-mass spectrometry analysis according to the conditions, taking the concentration of the characteristic peptide fragment as an abscissa, taking the peak area ratio of the characteristic peptide fragment and the internal standard peptide fragment as an ordinate, establishing a standard curve with the linear range of 0.25-50ng/mL and the linear correlation coefficient R 2 0.9983, weight 1/X, as shown in FIG. 3. Then substituting the peak area ratio of the characteristic peptide fragment and the internal standard peptide fragment in the re-dissolved on-line sample into a standard curve to obtain the concentration C of the characteristic peptide fragment in the on-line sample peptide And calculating the concentration of pro-SFTPB in the serum sample according to the concentration of the characteristic peptide fragment.
Concentration degree C of pro-SFTPB in serum sample protein The calculation formula of (2) is as follows:
C protein ＝C peptide ×V Dilute ×P UV ×M protein /V sample ×M peptide
wherein:
C protein representing the protein concentration in the original blood sample;
C peptide representing the concentration of the characteristic peptide fragments measured in the sample of the machine;
V Dilute representing the reconstituted volume of the sample after desalting and drying;
P UV indicating the determined UV purity of the characteristic peptide fragment;
M protein indicating the molecular weight of the protein measured;
V sample representing the volume of the original serum sample;
M peptide representing the molecular weight of the characteristic peptide fragment measured.
And (3) verifying a fixed value method: preparing pro-SFTPB (pro-SFTPB) labeled serum with 5 concentration levels as a quality control sample, preparing the sample by adopting the method, processing the quality control sample with each concentration for 3 times in parallel, respectively carrying out nano liter liquid chromatography-mass spectrometry analysis, determining the concentration of peptide fragments in the sample, and then calculating the content of pro-SFTPB in the quality control sample according to the formula so as to obtain the recovery rate and the variation Coefficient (CV) of the quality control samples with different concentrations, wherein the result is shown in the table 4, and the accuracy and the repeatability of the quantitative method are evaluated.
TABLE 4 constant value results for quality control samples
As can be seen from Table 4, the measured concentration of pro-SFTPB in the quality control samples with different concentrations is very close to the theoretical concentration, and the recovery rate is 92.1% -95.2%. And CV of quality control samples with different concentrations is 1.7% -4.4%, which shows that the precision of the sample treatment process is better. The result meets the requirements of clinical mass spectrometry that the recovery rate of the verification index is 80% -120% and the CV is less than 15%, and the fixed value method is proved to be high in accuracy and good in repeatability, and is a reliable detection method.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.

Claims (10)

1. A method for the determination of pro-SFTPB in serum, comprising the steps of:
(1) Preparing antibody coated magnetic beads: activating magnetic beads, adding antibodies into the activated magnetic beads for coupling reaction, separating and cleaning the magnetic beads after the reaction is finished, adding a sealing agent to seal non-specific binding sites on the magnetic beads, separating and cleaning the magnetic beads, and re-suspending the magnetic beads by using PBS solution to obtain antibody-coated magnetic beads;
(2) Adding antibody-coated magnetic beads into a serum sample, diluting with PBS, shaking and incubating to enable pro-SFTPB in the serum to be bound to the magnetic bead-coupled antibodies, and cleaning the magnetic beads to remove non-specifically bound proteins on the magnetic beads;
(3) Sequentially adding a denaturing reagent, a reducing reagent and an alkylating reagent into the magnetic bead sample combined with the pro-SFTPB obtained in the step (2) to react with the pro-SFTPB in the sample; then adding trypsin solution for enzymolysis to obtain a sample containing characteristic peptide segment SFTPBb;
(4) Adding an isotope labeled internal standard peptide segment SFTPBb_IS into the sample containing the characteristic peptide segment SFTPBb obtained in the step (3), purifying by a desalting column, and collecting eluent;
(5) And (3) re-dissolving the eluent obtained in the step (4) after freeze drying, carrying out nano liter liquid chromatography-mass spectrometry analysis, obtaining the concentration of the characteristic peptide fragment in the on-line sample through the peak area ratio of the characteristic peptide fragment to the internal standard peptide fragment, and calculating the concentration of pro-SFTPB in the serum sample according to the concentration of the characteristic peptide fragment.
2. The method for determining pro-SFTPB in serum according to claim 1, wherein in step (1), the activated magnetic beads are: sequentially adding EDC and NHS reagents into the magnetic beads, and performing rotary incubation for 30min at room temperature for activation; after the magnetic beads are activated, removing EDC and NHS reagents, adding a pro-SFTPB monoclonal antibody into the activated magnetic beads, mixing uniformly, and performing rotary incubation at room temperature for 2 hours to perform coupling reaction, wherein the weight ratio of the added pro-SFTPB monoclonal antibody to the magnetic beads is as follows: 30ug to 1mg.
3. The method for determining pro-SFTPB in serum according to claim 1, wherein in step (1), the blocking agent is PBS buffer containing 1% BSA, the blocking agent is added to incubate for 2 hours at room temperature with rotation, and the mixture is allowed to stand at 4℃for 15-24 hours to block the non-specific binding sites on the beads.
4. The method of claim 1, wherein in step (1), the re-suspending is: PBS buffer containing 0.1% BSA was added to the beads to a concentration of 1-10mg/mL.
5. The method for the quantification of pro-SFTPB in serum according to claim 1, wherein in step (3), the denaturing agent is a 50mM ammonium bicarbonate solution containing 5M urea, and after the denaturing agent is added to the sample, the sample is incubated with shaking at 2000rpm at room temperature for 10min; adding a reducing reagent of 500mM TCEP solution, uniformly mixing, and carrying out shaking incubation for 60min at 37 ℃ and 2000 rpm; adding 500mM iodoacetamide solution as alkylating agent, mixing, incubating at 2000rpm for 60min at room temperature in dark condition, adding trypsin solution to reduce urea concentration in denaturing agent to below 1M, and incubating at 37deg.C and 1500rpm for 24-48 hr to obtain sample containing characteristic peptide SFTPBb.
6. The method for determining the pro-SFTPB in serum according to claim 5, wherein the sequence of the characteristic peptide segment SFTPBb is: LVLPVLPGALQAR.
7. The method for determining pro-SFTPB in serum according to claim 1, wherein in step (4), 10% aqueous formic acid solution and isotope labeled internal standard peptide segment SFTPBb_IS are sequentially added to the sample containing characteristic peptide segment SFTPBb, and after mixing, the mixture IS added to a pre-equilibrated desalting column, and the mixture IS washed with 15% aqueous acetonitrile solution for 2 times and then eluted with 0.1% aqueous acetonitrile solution containing 0.1% formic acid for 3 times, and the eluents are collected and combined.
8. The method for determining the pro-SFTPB in serum according to claim 7, wherein the sequence of the internal standard peptide segment SFTPBb_IS IS: LVLPVLPGALQAR x 13 C 6 ， 15 N 4 。
9. The method for determining the pro-SFTPB value in serum according to claim 1, wherein in step (5), the re-solubilization is: the reconstitution was performed with 15% acetonitrile in water containing 0.1% formic acid.
10. The method for determining the concentration C of pro-SFTPB in serum according to claim 1, wherein in the step (5), the concentration C of pro-SFTPB in the serum sample protein The calculation formula of (2) is as follows:
C protein ＝C peptide ×V Dilute ×P UV ×M protein /V sample ×M peptide
wherein:
C protein representing the protein concentration in the original blood sample;
C peptide representing the concentration of the characteristic peptide fragments measured in the sample of the machine;
V Dilute representing the reconstituted volume of the sample after desalting and drying;
P UV indicating the determined UV purity of the characteristic peptide fragment;
M protein indicating the molecular weight of the protein measured;
V sample representing the volume of the original serum sample;
M peptide representing the molecular weight of the characteristic peptide fragment measured.
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