Isotope dilution mass spectrometry valuing method of plasma gastrin-releasing peptide precursor

Abstract

The invention provides an isotope dilution mass spectrometry valuing method for a plasma gastrin-releasing peptide precursor. According to the method, two plasma pretreatment strategies of immunoaffinity capture and protein precipitation are adopted, and an isotope dilution mass spectrometry technology is combined, so that high-accuracy quantification of the ProGRP in the plasma is realized. Target protein is efficiently enriched through two complementary pretreatment methods respectively, after trypsin digestion, isotope-labeled characteristic peptide fragments are introduced to serve as internal labels, absolute quantification is conducted on ProGRP through isotope dilution mass spectrometry, and the measurement result is traceable to the international unit (SI). The method effectively overcomes measurement deviation caused by cross reaction in a traditional immunoassay method, has high recovery rate, high specificity and high sensitivity, is suitable for reliable and accurate value determination of ProGRP in plasma, and can be used as a candidate scheme of a protein reference measurement procedure.

Description

Technical Field

[0001] This invention belongs to the field of biological sample pretreatment and protein quantitative analysis technology, specifically relating to an isotope dilution mass spectrometry method for plasma progastrin-releasing peptide (ProGRP). Background Technology

[0002] Progastrin-releasing peptide (ProGRP) is a peptide hormone precursor secreted by neuroendocrine cells, with a molecular weight of approximately 14 kDa. As a stable precursor of gastrin-releasing peptide (GRP), ProGRP is highly expressed in the plasma of patients with small cell lung cancer (SCLC). Its detection sensitivity and specificity are superior to most traditional tumor markers, and it has been established as a key hematological indicator for the auxiliary diagnosis, efficacy assessment, and prognosis of SCLC. Unlike GRP, which is easily degraded in blood, ProGRP has excellent in vivo stability, making it more suitable for the detection and analysis of clinical samples. Currently, immunological methods are commonly used for detection in clinical practice; however, these methods carry the risk of cross-reactivity interference, which may affect the accuracy of the detection. In recent years, quantitative methods based on mass spectrometry have been extensively studied to achieve more accurate, specific, and standardized detection and measurement of ProGRP.

[0003] Isotope dilution mass spectrometry (IDMS) performs quantitative analysis by adding a known amount of isotopically labeled internal standard to the sample. After sample pretreatment, the peak area ratio of the target analyte to the internal standard is measured using a mass spectrometer, thereby achieving accurate quantification of the target analyte. This method utilizes the internal standard to compensate for systematic errors in sample processing and instrument analysis, accurately calculating the absolute content of the target analyte in the sample. However, applying IDMS to the detection of low concentrations of ProGRP in plasma matrices still faces significant challenges. Plasma contains a large amount of insoluble salts, lipids, and high-abundance proteins, which can not only damage the mass spectrometer but also generate high background noise during detection, severely masking the characteristic signal of ProGRP and leading to a decrease in the accuracy of its quantitative results. Therefore, for the accurate quantification of ProGRP in plasma, it is urgent to establish a reliable determination method that can effectively overcome matrix interference, possesses high specificity and sensitivity, and whose results are traceable to International Units (SI). Summary of the Invention

[0004] In view of the above-mentioned defects and needs of the prior art, the present invention aims to provide an isotope dilution mass spectrometry method for plasma gastrin-releasing peptide precursor (ProGRP).

[0005] The isotope dilution mass spectrometry method for determining plasma progastrin-releasing peptide (ProGRP) provided by this invention includes the following steps: 1) Plasma sample pretreatment (a) Add the magnetic bead antibody complex to the plasma sample to be tested, incubate, wash to remove plasma, elute the ProGRP bound to the magnetic beads with elution buffer, neutralize to pH=7, then reconstitute with enzyme digestion buffer, and calculate the enrichment efficiency f by enzyme-linked immunosorbent assay (ELISA). EE ; (b) Trypsin and an isotopic internal standard (labeled peptide) were added to the enriched sample for enzymatic digestion to obtain the digested sample. The digestion efficiency f was calculated by ELISA. DE ; or (a') Add pure acetonitrile to the plasma sample to be tested, precipitate, centrifuge the precipitated sample to obtain the supernatant, add an isotope internal standard (labeled peptide), heat dry and reconstitute with enzyme digestion buffer, and calculate the enrichment efficiency f by ELISA. EE '; (b') Add trypsin to perform enzymatic digestion and calculate the digestion efficiency f using ELISA. DE '; (c') The enzyme-digested plasma sample was diluted and desalted by passing it through an HLB column; the desalted plasma sample was then heat-dried and reconstituted, and finally filtered through a filter membrane to collect the filtrate to obtain the enzyme-digested and desalted sample; 2) Perform liquid chromatography-mass spectrometry analysis on the enzyme-digested sample obtained in step (b) or the enzyme-digested and desalted sample obtained in step (c'). Use multiple reaction monitoring to analyze the enzyme-digested peptides and the added isotope-labeled peptides. Quantitative NMR is used to quantify the peptides and labeling peptide solutions to prepare a standard curve. Calculate the concentration of ProGRP and incorporate the correction factor into the final measurement value to correct for losses caused by the enrichment and enzyme digestion processes.

[0006] In step (a) of the above method, the magnetic bead antibody complex is prepared by a method including the following steps: activating magnetic beads and adding ProGRP monoclonal antibody in a certain proportion to obtain the magnetic bead antibody complex; The ratio of antibody to magnetic beads can be 10-50 μg: 1 mg, specifically 20 μg: 1 mg. The ProGRP monoclonal antibody is required to react with human ProGRP, but not with human globulin; The ProGRP monoclonal antibody may specifically be ProGRP mAb; The resulting magnetic bead antibody complex is a magnetic bead antibody complex solution. The solvent can be phosphate buffer, borate buffer, or tris(hydroxymethyl)aminomethane hydrochloride solution, specifically 0.1M borate buffer.

[0007] The concentration of the magnetic bead antibody complex in the solution is 2-5 mg / mL, specifically 4 mg / mL; The operation of step (a) of the above method is as follows: add a certain amount of magnetic bead antibody complex to the plasma sample to be tested, enrich at 37°C for 2–4 h, so that ProGRP in the plasma binds to the magnetic bead antibody, remove the plasma on a magnetic rack; wash the magnetic beads thoroughly with detergent; add eluent, and vortex thoroughly to elute the ProGRP bound to the magnetic beads. The ratio of the magnetic bead antibody complex to the plasma sample to be tested can be 0.4-4 μg: 1 μL, specifically 1.6 μg: 1 μL. The detergent is a PBST solution and / or a PBS solution; The volume ratio of the detergent to the plasma sample to be tested can be 1-2:1, specifically 1.5:1; The eluent may be an aqueous solution of acetic acid with a volume percentage of 2-10%, specifically an aqueous solution of acetic acid with a volume percentage of 5%. The volume ratio of the eluent to the plasma sample to be tested can be 1:5-20, specifically 1:10; Step (a) further includes the following operations: collecting the elution solution on a magnetic rack and neutralizing it to pH=7 with ammonia water, reconstituted to 100 μL with a solution containing 20% ​​ACN·100 mM ABC (20% acetonitrile·100 mM ammonium bicarbonate solution), and then reconstituted to 200 μL with a solution containing 10% ACN·50 mM ABC; The operation of step (b) of the above method is as follows: add an isotope internal standard (labeled peptide) solution to the obtained sample, add trypsin in proportion, and carry out the enzymatic digestion reaction at 25-40℃ (specifically 37℃) to obtain the digested sample; The labeled peptide is NLLGLIEA-[K_ 13 C6 15 N2].

[0008] The amount of labeled peptide added was 5 μL (labeled peptide concentration was 40 ng / mL), based on a volume of 200 μL after reconstitution.

[0009] The mass ratio of trypsin to total protein can be 1:15.

[0010] The enzymatic digestion reaction is carried out under stirring, with a stirring rate of 800-1200 rpm, specifically 1000 rpm, and the reaction time is 12-48 hours, specifically 18 hours. In step (a') of the above method, pure acetonitrile is added to the plasma sample to be tested to make the final volume concentration of acetonitrile 50%; precipitation is carried out at 2-8℃ (specifically 4℃) for 15-45 minutes (specifically 30 minutes); the precipitated sample is centrifuged to obtain the supernatant; the supernatant is filtered through a filter membrane, labeled peptide solution is added, the resulting clarified solution is dried by heat and reconstituted with 10% ACN·50mM ABC solution; The labeled peptide is NLLGLIEA-[K_ 13 C6 15 N2].

[0011] The amount of labeled peptide added was 5 μL (labeled peptide concentration was 40 ng / mL), based on a volume of 200 μL after reconstitution.

[0012] The operation of step (b') of the above method is as follows: add trypsin to the obtained sample in a certain proportion, and carry out the enzymatic digestion reaction at 25℃-40℃ (specifically 37℃) to obtain the digested sample. 2.4 μg of trypsin was added based on the total protein content after plasma precipitation.

[0013] The enzymatic digestion reaction is carried out under stirring, with a stirring rate of 800-1200 rpm, specifically 1000 rpm, and the reaction time is 18-48 hours, specifically 18 hours. The procedure in step (c') of the above method is as follows: Dilute the protein precipitate and enzyme-digested sample obtained in (b') with water for later use; prepare activation solution, equilibration solution, washing solution, and elution solution separately for later use; install the HLB column on a vacuum filter; activation: add 2 mL of activation solution and dry under vacuum; equilibration: add 2 mL of equilibration solution and dry under vacuum; loading: add the diluted sample and dry under vacuum; washing: add 2 mL of washing solution and dry under vacuum; elution: add 800 μL of elution solution and dry under vacuum; repeat all steps 6 times; rehydrate the desalted sample by heat drying and filter it through a filter membrane into a new centrifuge tube. The activation solution was 1% FA·100% ACN solution (1% formic acid·100% pure acetonitrile), the equilibration solution was 1% FA·100% H2O (1% formic acid·100% aqueous solution), the cleaning solution was 1% FA·5% ACN (1% formic acid·5% acetonitrile aqueous solution), and the elution solution was 1% FA·80% ACN (1% formic acid·80% acetonitrile aqueous solution).

[0014] The reconstitution solution is 10% ACN·50mM ABC.

[0015] Step 2) of the above method involves: placing the sample into a liquid chromatography-mass spectrometry (LC-MS) instrument for detection, plotting a standard curve using the NLLGLIEAK characteristic peptide and its internal standard, and calculating the ProGRP concentration.

[0016] in The mass concentration of ProGRP in the plasma to be tested. The peak area of ​​the ProGRP characteristic peptide NLLGLIEAK. The peak area is the NLLGLIEAK isotope internal standard peptide. This represents the relative molecular mass of ProGRP. denoted as NLLGLIEAK, a characteristic peptide of ProGRP, b is the intercept of the standard curve, and k is the slope of the standard curve.

[0017] The final measured value is obtained by substituting the correction factors from both methods into the test results, i.e., by dividing the test results by the correction factors. Where, the correction factor f = f EE ×f DE Or f'=f EE '×f DE '; f represents the correction factor obtained through steps (a) and (b), and f' represents the correction factor obtained through steps (a') and (b'). The method for determining plasma progastrin-releasing peptide (ProGRP) by combining plasma sample pretreatment, trypsin digestion, isotope dilution mass spectrometry, and the introduction of a correction factor, provided by this invention, has the following beneficial results: (1) The plasma sample pretreatment method used in this invention can efficiently remove matrix interferences such as high-abundance proteins, lipids and salts in the sample, significantly reducing ion suppression and background noise in the mass spectrometry analysis process, thereby fundamentally improving the accuracy and reliability of plasma ProGRP quantification results.

[0018] (2) By introducing a correction factor, the present invention systematically compensates for the loss of target protein caused by sample pretreatment and enzymatic digestion, thereby further improving the accuracy of plasma ProGRP quantitative data.

[0019] (3) This invention provides a reliable and accurate detection method for ProGRP in plasma, which has important value in the fields of disease diagnosis and the development of ProGRP reference measurement procedures. Attached Figure Description

[0020] Figure 1 This is an experimental flowchart of the method for determining the plasma gastrin-releasing peptide precursor of the present invention.

[0021] Figure 2 This refers to the immunoaffinity enrichment efficiency in Example 1 of the present invention.

[0022] Figure 3 This refers to the protein precipitation and enrichment efficiency in Example 2 of the present invention.

[0023] Figure 4 This refers to the trypsin digestion efficiency in Example 3 of the present invention.

[0024] Figure 5 This is a comparison of the enzyme digestion efficiency of the three enzyme digestion systems in Example 3, Comparative Example 1, and Comparative Example 2 of the present invention.

[0025] Figure 6 The standard curve of NLLGLIEAK in Embodiment 5 of the present invention. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0028] Example 1: Enrichment of plasma samples using an immunoaffinity enrichment method The plasma sample pretreatment method based on antibody capture in this embodiment includes the following steps: Activate the magnetic beads and add monoclonal antibody (ProGRP mAb) at a ratio of antibody:magnetic beads = 20 μg: 1 mg to prepare a magnetic bead antibody complex solution. The solvent is 0.1 M borate solution, and the final concentration is 4 mg / mL. Take 200 μL of plasma sample containing ProGRP into a 1.5 mL centrifuge tube; add 320 μg of magnetic bead antibody complex to the patient's plasma and enrich at 37°C for 2 h to allow ProGRP in the plasma to bind to the magnetic bead antibody. Remove the plasma on a magnetic rack. Wash the magnetic beads three times with 300 μL of PBST solution and PBS solution, respectively. Add 20 μL of 5% acetic acid aqueous solution and vortex for 12 min to elute the ProGRP bound to the magnetic beads. Repeat twice. Collect the eluent on a magnetic rack and neutralize to pH 7 with 50% ammonia water. Use a solution containing 20% ​​ACN·100 mM Reconstitute the ABC solution to 100 μL, then add a 10% ACN·50 mM ABC solution to reconstitute to 200 μL.

[0029] The enriched plasma supernatant and the unenriched plasma were collected separately, and the immunoaffinity enrichment efficiency was assessed using ELISA. Results are as follows: Figure 2 As shown, the plasma samples to be tested contained different concentrations (1.2 ng / mL, 1.4 ng / mL, 3 ng / mL, 5 ng / mL) of ProGRP. After enrichment by the immunoaffinity enrichment method of the present invention, the average enrichment efficiency was 88.90% ± 2.88%.

[0030] Example 2: Plasma sample pretreatment based on acetonitrile precipitation The plasma sample pretreatment method based on acetonitrile precipitation in this embodiment includes the following steps: Add 200 μL of ACN to 200 μL of ProGRP-containing plasma sample to achieve a final ACN concentration of 50%. Incubate the precipitated sample at 4°C for 30 minutes. Centrifuge the precipitated sample at 12000 rpm for 10 minutes. Aspirate the supernatant using a 1 mL syringe and filter it through a 0.22 μm filter membrane into a new 1.5 mL centrifuge tube. Add the labeled peptide (NLLGLIEA-[K_ 13 C6 15 [N2]), heat the supernatant to dry at 60°C, and then reconstitute it with 200 μL of 10% (v / v) ACN·50 mMABC solution.

[0031] The enriched plasma was collected, and the original concentration was spiked with blank plasma after precipitation. The enrichment efficiency of protein precipitation was assessed using ELISA. Results are as follows: Figure 3As shown, the plasma samples to be tested contained different concentrations (0.5 ng / mL, 1 ng / mL, 5 ng / mL) of ProGRP. After protein precipitation enrichment according to the present invention, the average enrichment efficiency was 87.51% ± 1.96%.

[0032] Example 3: High-efficiency trypsin digestion based on 10% ACN·50mM ABC digestion buffer This embodiment describes a highly efficient trypsin digestion method based on 10% (v / v) ACN·50mM ABC digestion buffer, which includes the following steps: 200 μL of ProGRP protein solutions (1 ng / mL, 3 ng / mL, 5 ng / mL, and 10 ng / mL, respectively) were prepared using 10% ACN·50 mM ABC buffer. The ProGRP protein was purchased from Sangon Biotech. A labeled peptide (NLLGLIEA-[K_) was then added. 13 C6 15 The solution was prepared by adding trypsin (at a mass ratio of enzyme to total protein of 1:15) and placing the reaction system in a metal bath. The enzymatic digestion reaction was carried out at 37°C and 1000 rpm for 18 hours.

[0033] The digested solutions at concentrations of 1 ng / mL, 3 ng / mL, and 5 ng / mL, as well as the original solutions before digestion, were collected. The trypsin digestion efficiency was assessed using ELISA. Results are as follows: Figure 4 As shown, the average enzyme digestion efficiency was 97.68% ± 3.59%.

[0034] A 10 ng / mL ProGRP digestion solution was collected and analyzed by mass spectrometry. The results are as follows: Figure 5 As shown, the enzyme digestion efficiency in this embodiment is the highest under the buffer system of 10% ACN·50mMABC.

[0035] Comparative Example 1: Trypsin digestion based on 50 mM ABC digestion buffer The trypsin digestion method in this comparative example, based on 50 mM ABC digestion buffer, includes the following steps: Prepare 200 μL of a 10 ng / mL ProGRP protein solution using 50 mM ABC buffer, add labeled peptide solution, and then add trypsin (enzyme:total protein = 1:15). Place the reaction system in a metal bath and carry out the enzymatic digestion reaction at 37°C and 1000 rpm for 18 hours.

[0036] like Figure 5As shown, in the 50mM ABC buffer system, the mass spectrometry signal of the obtained enzyme-digested peptide could not be detected in this comparative example, proving that the solvent composition is difficult to meet the enzyme digestion requirements of ProGRP protein solution with a concentration not higher than 10ng / mL.

[0037] Comparative Example 2: Trypsin digestion based on 10mM CaCl2·50mM ABC digestion buffer This comparative example demonstrates a highly efficient trypsin digestion method based on 10 mM CaCl2·50 mM ABC digestion buffer, comprising the following steps: Prepare 200 μL of a 10 ng / mL ProGRP protein solution using 10 mM CaCl2·50 mM ABC buffer, add labeled peptide solution, and then add trypsin (enzyme:total protein = 1:15). Place the reaction system in a metal bath and carry out the enzymatic digestion reaction at 37°C and 1000 rpm for 18 hours.

[0038] like Figure 5 As shown, in the 10mM CaCl2·50mM ABC buffer system, the peak area ratio of the enzyme-digested peptide obtained in this comparative example to the internal standard peptide was significantly lower than that in Example 1 (10% ACN·50mM ABC).

[0039] like Figure 5 As shown, in the 10% ACN·50mM ABC buffer system, the peak area ratio of the enzyme-digested peptide obtained in Example 3 to the internal standard peptide was significantly higher than that of Comparative Example 1 (50mM ABC) and Comparative Example 2 (10mM CaCl2·50mM ABC), indicating that this solvent composition can effectively improve the enzyme digestion efficiency of ProGRP.

[0040] Example 4: Sample desalting based on HLB column The sample desalting method based on HLB columns in this embodiment includes the following steps: The protein-precipitated enzyme-digested sample (the sample obtained in Example 2 after enzyme digestion using the method in Example 3) was diluted with water to 1200 μL and set aside. 1% formic acid (FA) aqueous solution, 1% FA·5% ACN (v / v, the same below) aqueous solution, 1% FA·80% ACN solution, and 1% FA·100% ACN solution were prepared and set aside. The HLB column was mounted on a vacuum filter. Activation: 2 mL of 1% FA·100% ACN solution was added, and the solution was dried under vacuum. Equilibration: 2 mL of 1% FA aqueous solution was added, and the solution was dried under vacuum. Sample loading: 200 μL of the diluted sample was added, and the solution was dried under vacuum. Washing: 2 mL of 1% FA·5% ACN aqueous solution was added, and the solution was dried under vacuum. Elution: 800 μL of 1% FA·80% ACN solution was added, and the solution was dried under vacuum. All steps were repeated 6 times. The desalted sample was heat-dried, and 200 μL of 10% ACN·50 mM solution was added. ABC was reconstituted and filtered through a 0.22 μm filter membrane into a new centrifuge tube.

[0041] Example 5: Mass spectrometry quantification based on characteristic peptides The mass spectrometry quantification method based on characteristic peptides in this embodiment includes the following steps: The samples obtained from Example 1, after enzyme digestion in Example 3, and the protein precipitates, enzyme digestions, and desalting samples obtained from Example 2, after enzyme digestion in Example 3 and desalting in Example 4, were transferred to liquid chromatography vials containing insert tubes for mass spectrometry detection.

[0042] High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to analyze the enzyme-digested samples. In this embodiment, a Thermo Fisher Scientific Vanquish HPLC system tandem with an Orbitrap 240 mass spectrometer was used. The column specifications were a Thermo Scientific C18 analytical column (150 mm × 1 mm, 2 µm, 100 Å), the column temperature was 35 °C, and the injection volume was 15 μL. The mobile phase was a binary high-pressure gradient: phase A was 0.1% FA·H₂O, and phase B was 0.1% FA·ACN. The mobile phase gradient is shown in Table 1.

[0043] Table 1 Chromatographic conditions in Example 1

[0044] Mass spectrometry conditions: ESI was used as the ion source, ion transfer tube temperature was 320℃, spray voltage was +4.3 kV, sheath gas flow rate was 25 arb, auxiliary gas flow rate was 5 arb, and purge gas flow rate was 0 arb. For multiple reaction monitoring (MRM), the HCD collision energy was set to 30%, and the precursor ion isolation window was 0.4 kV. m / z The resolution is set to 90000.

[0045] The precursor ion of the characteristic peptide NLLGLIEAK is 485.8, and the daughter ion is 743.5.

[0046] The parent ion of the NLLGLIEAK isotope internal standard is 489.8, and the daughter ion is 751.5.

[0047] A standard curve was plotted using the characteristic peptide of NLLGLIEAK and its internal standard. The purity of the NLLGLIEAK characteristic peptide was determined by NMR, yielding a purity of 69.83% and an uncertainty expansion of 1.34%. A standard curve was established through linear fitting, with a slope k of 0.6384 and an intercept of -0.0376. Figure 6 The measured characteristic peptide NLLGLIEAK and its isotope internal standard were substituted into the calculation formula to accurately quantify plasma ProGRP.

[0048]

[0049] in The mass concentration of ProGRP in the plasma to be tested. The peak area of ​​the ProGRP characteristic peptide NLLGLIEAK. This represents the peak area of ​​the NLLGLIEAK isotope internal standard peptide.

[0050] Example 6: Calculation of Correction Factors Based on ELISA The calculation of the correction factor based on ELISA in this embodiment includes the following steps: The enrichment efficiency of immunoaffinity was measured to be 88.90% ± 2.88%; the enrichment efficiency of protein precipitation was 87.51% ± 1.96%; and the enzyme digestion efficiency was 97.68% ± 3.59%. The correction factor was calculated using the following formula: f=f EE ×f DE f is the correction factor; f EE For enrichment efficiency; f DE This represents the enzyme digestion efficiency.

[0051] The calculated correction factor for immunoaffinity capture-isotope dilution mass spectrometry was 86.84%, and for protein precipitation-isotope dilution mass spectrometry it was 85.48%. The results obtained from Example 5 were divided by the correction factor (by substituting the correction factor), and are shown in Tables 2 and 3, indicating consistency between the two methods.

[0052] Table 2 Mass spectrometry measurements before calibration

[0053] Table 3 Corrected mass spectrometry measurements

[0054] Comparative Example 3 The comparative example of ELISA-based quantitative plasma ProGRP levels includes the following steps: The ELISA kit was equilibrated at room temperature for 30 minutes. Then, 50 μL of blank control, standard, and test sample were added sequentially to each well, followed by 50 μL of enzyme-labeled antibody. The kit was incubated at 37°C for 1 hour. After incubation, the liquid in the wells was discarded. 300 μL of washing buffer was added to each well, and the buffer was allowed to stand for 5 minutes before discarding. This washing step was repeated five times. Next, 50 μL of chromogenic reagent A and 50 μL of chromogenic reagent B were added to each well sequentially, and the kit was incubated at 37°C in the dark for 15 minutes. Finally, 50 μL of stop solution was added to each well to terminate the reaction, and the absorbance of each well was immediately measured at 450 nm.

[0055] The experimental results are shown in Table 4. Comparing the results in Table 3 and Table 4, it can be seen that the results of ELISA are generally higher than those of mass spectrometry detection.

[0056] Comparative Example 4: Quantitative analysis of plasma ProGRP levels based on CLIA (chemiluminescence immunoassay) The quantitative plasma ProGRP content based on CLIA (chemiluminescence immunoassay) in this comparative example includes the following steps: A 50 μL sample was mixed with 66 μL of paramagnetic microparticles coated with ProGRP antibody, allowing the ProGRP in the sample to fully bind to the antibody immobilized on the microparticle surface. After the reaction, the microparticles were washed, and then 59 μL of acridine ester-labeled ProGRP antibody conjugate was added to form an immune complex. After washing again to remove unbound components, pre-excitation solution and excitation solution were added sequentially to initiate the chemiluminescence reaction. The relative luminescence unit (RLU) value was measured using an optical detection system, showing that the ProGRP concentration in the sample was directly proportional to the detected RLU signal value.

[0057] Table 4 ELISA and CLIA Measurement Values

[0058] The experimental results are shown in Table 4. Comparing the results in Tables 3 and 4, it can be seen that the CLIA results are generally higher than the mass spectrometry results. The reason why the results of the two traditional immunoassay methods are higher than those of the mass spectrometry method may be that traditional immunoassay methods cannot avoid cross-reactions between structural analogs and antibodies in the sample. In addition, another possible hypothesis is that the indirect detection strategy based on the principle of enzyme-catalyzed substrate luminescence may lead to an overestimation of the actual sample concentration due to signal amplification.

[0059] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for isotope dilution mass spectrometry determination of plasma gastrin-releasing peptide precursor ProGRP, comprising the following steps: 1) Plasma sample pretreatment (a) Add the magnetic bead antibody complex to the plasma sample to be tested, incubate, wash to remove plasma, elute the ProGRP bound to the magnetic beads with elution buffer, neutralize to pH=7, then reconstitute with enzyme digestion buffer, and calculate the enrichment efficiency f by enzyme-linked immunosorbent assay. EE ; (b) Trypsin and an isotopic internal standard (labeled peptide) were added to the enriched sample for enzymatic digestion to obtain the digested sample. The digestion efficiency f was calculated by ELISA. DE ; or (a') Add pure acetonitrile to the plasma sample to be tested, precipitate, centrifuge the precipitated sample to obtain the supernatant, add an isotope internal standard (labeled peptide), heat dry and reconstitute with enzyme digestion buffer, and calculate the enrichment efficiency f by ELISA. EE '; (b') Add trypsin to perform enzymatic digestion and calculate the digestion efficiency f using ELISA. DE '; (c') The enzyme-digested plasma sample was diluted and desalted by passing it through an HLB column; the desalted plasma sample was then heat-dried and reconstituted, and finally filtered through a filter membrane to collect the filtrate to obtain the enzyme-digested and desalted sample; 2) Perform liquid chromatography-mass spectrometry analysis on the enzyme-digested sample obtained in step (b) or the enzyme-digested and desalted sample obtained in step (c'). Use multiple reaction monitoring to analyze the enzyme-digested peptides and the added isotope-labeled peptides. Quantitative NMR is used to quantify the peptides and labeling peptide solutions to prepare a standard curve. Calculate the concentration of ProGRP and incorporate the correction factor into the final measurement value to correct for losses caused by the enrichment and enzyme digestion processes.

2. The method according to claim 1, characterized in that, In step (a), the magnetic bead antibody complex is prepared by a method including the following steps: activating magnetic beads and adding ProGRP monoclonal antibody in a certain proportion to obtain the magnetic bead antibody complex; The ratio of the antibody to the magnetic beads is 10-50 μg: 1 mg. The ProGRP monoclonal antibody is ProGRP mAb; The resulting magnetic bead antibody complex is a magnetic bead antibody complex solution, and the solvent is at least one of phosphate buffer, borate buffer and tris(hydroxymethyl)aminomethane hydrochloride solution; The concentration of the magnetic bead antibody complex in the solution is 2-5 mg / mL.

3. The method according to claim 1, characterized in that, Step (a) is performed as follows: a certain amount of magnetic bead antibody complex is added to the plasma sample to be tested, and enriched at 37°C for 2–4 hours to allow ProGRP in the plasma to bind to the magnetic bead antibody. The plasma is then removed on a magnetic rack. The magnetic beads are thoroughly washed with detergent. Eluent is added, and the mixture is vortexed thoroughly to elute the ProGRP bound to the magnetic beads. The ratio of the magnetic bead antibody complex to the plasma sample to be tested is 0.4-4 μg: 1 μL; The detergent is a PBST solution and / or a PBS solution; The volume ratio of the detergent to the plasma sample to be tested is 1-2:1; The eluent may be an aqueous solution of acetic acid with a volume percentage of 2-10%; The volume ratio of the eluent to the plasma sample to be tested is 1:5-20.

4. The method according to claim 1, characterized in that, The enzyme digestion buffer is a 10% ACN·50mM ABC solution.

5. The method according to claim 1, characterized in that, Step (b) involves adding an isotope internal standard (labeled peptide) solution to the obtained sample, adding trypsin in a specific ratio, and performing an enzymatic digestion reaction at 25-40℃ to obtain the digested sample. The labeled peptide is NLLGLIEA-[K_ 13 C6 15 N2]; The amount of labeled peptide added was 5 μL (labeled peptide concentration was 40 ng / mL), based on a reconstituted volume of 200 μL. The mass ratio of trypsin to total protein is 1:15; The enzymatic digestion reaction is carried out under stirring at a speed of 800-1200 rpm for 12-48 hours.

6. The method according to claim 1, characterized in that, In step (a'), pure acetonitrile is added to the plasma sample to be tested to make the final volume concentration of acetonitrile 50%; precipitation is carried out at 2-8℃ for 15-45 minutes; the precipitated sample is centrifuged to obtain the supernatant; the supernatant is filtered through a filter membrane, labeled peptide solution is added, the resulting clear solution is dried by heat and reconstituted with 10% ACN·50mM ABC solution; The labeled peptide is NLLGLIEA-[K_ 13 C6 15 N2]; The amount of labeled peptide added was 5 μL (labeled peptide concentration was 40 ng / mL), based on a volume of 200 μL after reconstitution.

7. The method according to claim 1, characterized in that, Step (b') involves adding trypsin to the obtained sample in a specific ratio and performing the enzymatic digestion reaction at 25℃-40℃ to obtain the digested sample. 2.4 μg of trypsin was added based on the total protein content after plasma precipitation; The enzymatic digestion reaction is carried out under stirring at a speed of 800-1200 rpm for 18-48 hours.

8. The method according to claim 1, characterized in that, Step (c') is as follows: dilute the protein precipitate and enzyme digested sample obtained in (b') with water for later use; prepare activation solution, equilibration solution, washing solution and elution solution for later use; install the HLB column on the vacuum filter; Activation: Add 2 mL of activation solution and dry under vacuum; Equilibration: Add 2 mL of equilibration buffer and dry under vacuum; Sample loading: Add the diluted sample and dry under vacuum; Cleaning: Add 2 mL of cleaning solution and vacuum dry; Elution: Add 800 μL of elution buffer, dry under vacuum, and repeat all steps 6 times; rehydrate the desalted sample by heat drying, and filter it through a filter membrane into a new centrifuge tube. The activation solution was 1% FA·100% ACN solution, the equilibration solution was 1% FA·100% H2O, the cleaning solution was 1% FA·5% ACN, and the elution solution was 1% FA·80% CAN. The reconstitution solution is 10% ACN·50mM ABC.

9. The method according to claim 1, characterized in that, Step 2) involves placing the sample into a liquid chromatography-mass spectrometry (LC-MS) instrument for detection, plotting a standard curve using the NLLGLIEAK characteristic peptide and its internal standard, and calculating the ProGRP concentration. in The mass concentration of ProGRP in the plasma to be tested. The peak area of ​​the ProGRP characteristic peptide NLLGLIEAK. The peak area is the NLLGLIEAK isotope internal standard peptide. This represents the relative molecular mass of ProGRP. denoted as NLLGLIEAK, a characteristic peptide of ProGRP, b is the intercept of the standard curve, and k is the slope of the standard curve.

10. The method according to claim 1, characterized in that, The final measured value is obtained by substituting the correction factors from both methods into the test results, i.e., by dividing the test results by the correction factors. Where, the correction factor f = f EE ×f DE Or f'=f EE '×f DE '; f represents the correction factor obtained through steps (a) and (b), and f' represents the correction factor obtained through steps (a') and (b').

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