CN113281442A - Method for valuing standard substance rich in leucine alpha-2 glycoprotein 1 - Google Patents
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Abstract
The invention discloses a method for valuing a standard substance rich in leucine alpha-2 glycoprotein 1. The invention has the beneficial effects that: the method for determining the value of the Leucine-rich alpha-2 glycoprotein 1(LRG1) standard substance uses an isotope dilution mass spectrometry to determine the content of the LRG1 standard substance, fills the blank of the prior art, ensures the accuracy, reliability and traceability of a value determination result, and has important significance for establishing a quality control and evaluation system of the LRG1 standard substance.
Description
Technical Field
The invention relates to a protein analysis and detection technology, in particular to a method for determining a value of a leucine-rich alpha-2 glycoprotein 1(LRG1) standard substance.
Background
With the vigorous development of the life science field, protein analysis and detection technology is widely applied to the fields of clinical medical examination, biomedical analysis and the like. In clinical medical examination, many disease markers such as alpha-fetoprotein, carcinoembryonic antigen, C-reactive protein and the like belong to proteins, and therefore, the accuracy of the quantitative result of the protein markers has a great influence on the diagnosis and prognosis monitoring of diseases.
At present, for the quantitative analysis of target protein markers, immunological analysis methods based on antigen-antibody reactions are mostly adopted. The immunological analysis method has the advantages of high detection speed, high sensitivity and good specificity, but the accuracy and the reliability of the immunological analysis method depend on standard substances (antigen proteins, namely target protein markers) provided by in vitro diagnostic reagent manufacturers, so that the detection results of products of different manufacturers on the same sample are greatly different. Therefore, to obtain accurate and comparable clinical test results, the most effective method is to quantify the protein standards and the results are traceable to SI units. The current internationally accepted method of quantifying protein standards is isotope dilution mass spectrometry.
The physiological role of LRG1 protein is to influence biological functions by modulating the transforming growth factor- β pathway in vivo. It participates in the generation and development of tumors by promoting the processes of abnormal angiogenesis, regulating apoptosis, promoting the transformation of epithelial cells to interstitial cells and the like, and is an important biomarker for early diagnosis and prognosis of cancers.
In actual detection work, the LRG1 is measured by an immunoassay method, and LRG1 protein standard substances are used, but no report of magnitude traceability analysis research on LRG1 standard substances exists at present.
Disclosure of Invention
The main purpose of the present application is to provide a method for valuing a leucine-rich alpha-2 glycoprotein 1(LRG1) standard substance with good accuracy, reliability and traceability.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for valuing a leucine-rich alpha-2 glycoprotein 1 standard substance, comprising the following steps:
the method comprises the following steps: screening a characteristic peptide fragment and a stable isotope labeled internal standard peptide fragment of LRG 1;
step two: optimizing the nanoliter liquid chromatography-mass spectrometry analysis conditions of the characteristic peptide segment and the internal standard peptide segment;
step three: mixing the characteristic peptide segments with different concentrations and the internal standard peptide segments with fixed concentrations in equal volume, analyzing by adopting the optimized nano-liter liquid chromatography-mass spectrometry method in the second step, and establishing a standard curve;
step four: and (3) carrying out enzymolysis on the LRG1 standard substance, adding an internal standard peptide segment into the reaction solution after the enzymolysis, analyzing by adopting the nano-liter liquid chromatography-mass spectrometry method optimized in the second step, and substituting the peak area ratio of the selected characteristic peptide segment and the internal standard peptide segment into the standard curve established in the third step to obtain the concentration of the LRG1 standard substance.
The method for valuing the leucine-rich alpha-2 glycoprotein 1 standard substance is a preferred embodiment, and comprises the following steps: the method for screening the characteristic peptide fragment and the stable isotope labeled internal standard peptide fragment of LRG1 comprises the following steps:
(1) sequentially adding NH into LRG1 recombinant protein solution4HCO3Heating to perform reduction reaction with TCEP, reducing the disulfide bond of LRG1 recombinant protein, adding iodoacetamide to perform alkylation reaction, adding trypsin solution for enzymolysis, and adding TFA to stop enzymolysis after enzymolysis is finished;
(2) filtering the reaction liquid obtained in the step (1), carrying out nanoliter liquid chromatography-mass spectrometry on the filtered liquid, and collecting Data in a Data Dependent scanning DDA (Data Dependent Acquisition, DDA) mode;
(3) comparing the data obtained in the step (2) with protein amino acid sequences in a Uniprot database by using Proteome resolver software and the Uniprot database, and identifying and screening 2 characteristic peptide fragments for quantification;
(4) based on the selected characteristic peptide fragment, the use13C and15and N, carrying out stable isotope labeling on the selected characteristic peptide segment to be used as an internal standard peptide segment.
Preferably, in step (1), 25ul of NH with a concentration of 50mmol/L is sequentially added to 20ul of LRG1 recombinant protein solution4HCO3And 3ul of TCEP with a concentration of 0.5mol/L, and reacting at 37 ℃ for 30min, reducing the disulfide bond of the LRG1 recombinant protein, then adding 2uL of iodoacetamide with the concentration of 20mmol/L for alkylation reaction, adding 10uL of trypsin solution with the concentration of 100ng/uL for enzymolysis, and adding 5uL of TFA with the concentration of 10% to terminate the enzymolysis after the enzymolysis is finished;
preferably, in the step (2), the filtration is performed by using a 0.22um filter membrane;
preferably, in step (3), the two characteristic peptide fragments screened for quantification are LRG1a and LRG1b, and the amino acid sequences are VAAGAFQGLR and LHLEGNK, respectively.
The principle of characteristic peptide fragment screening is as follows:
the peptide segment amino acid composition does not contain methionine (Met) and cysteine (Cys);
the peptide section does not contain a miscut site;
③ the mass spectrum carries 2+ or 3+ charge;
the length of the peptide segment is 6-20 amino acids;
the mass spectrum peak intensity of the peptide segment is at the first 3 position in the candidate peptide segment satisfying the condition I-IV.
Preferably, in step (4), a stable isotope is used13C and15labeling arginine of LRG1a with N label to obtain corresponding internal standard peptide segment LRG1a _ IS with amino acid sequence of VAAGAFQGLR: (13C6,15N4) (ii) a Using stable isotopes13C and15marking lysine of LRG1b by N marker to obtain corresponding internal standard peptide segment LRG1b _ IS with amino acid sequence of LHLEGNK: (L-lysine)13C6,15N2)；
In the above method for quantifying the leucine-rich α -2 glycoprotein 1 standard substance, as a preferred embodiment, in step two, the conditions for the nanoliter liquid chromatography-mass spectrometry analysis of the optimized characteristic peptide fragment and the internal standard peptide fragment are as follows:
(1) respectively carrying out nanoliter liquid chromatography-mass spectrometry on the selected characteristic peptide segment and the internal standard peptide segment, and acquiring data in a Parallel Reaction Monitoring (PRM) mode;
(2) and analyzing mass spectrum data obtained in the first step by Skyline software, and selecting 5 fragment ions with the highest peak intensity after the fragmentation of each peptide fragment parent ion as corresponding target peptide fragment child ions.
In the above method for quantifying the leucine-rich alpha-2 glycoprotein 1 standard substance, as a preferred embodiment, in step three, LRG1a lyophilized powder and LRG1b lyophilized powder are dissolved in 5% acetonitrile aqueous solution to prepare a mixed standard solution with equal concentration, and the mixed standard solution is diluted with 0.1% formic acid aqueous solution to 0.5ng/mL, 2.0ng/mL, 5.0ng/mL, 20.0ng/mL and 50.0ng/mL respectively to obtain a mixed standard series solution;
dissolving LRG1a _ IS freeze-dried powder and LRG1b _ IS freeze-dried powder in 5% acetonitrile water solution to prepare mixed internal standard solution with equal concentration, and diluting the mixed internal standard solution into 5.0ng/mL mixed internal standard working solution by using 0.1% formic acid water solution;
mixing the mixed standard series solutions with different concentrations and the mixed internal standard working solution in equal volume to prepare a standard series solution, analyzing according to an optimized nano-liter liquid chromatography-mass spectrometry analysis method, collecting data in a PRM scanning mode, and quantitatively analyzing through Skyline software; and (3) drawing a standard curve by taking the concentration of the characteristic peptide as an abscissa, taking the peak area ratio of the characteristic peptide and the internal standard peptide as an ordinate and taking the weight as 1/x.
The above-described method for valuing a leucine-rich alpha-2 glycoprotein 1 standard substance, as a preferred embodiment,
the LRG1 standard substance 20uL was taken and the volume V was recorded accuratelysample(ii) a Adding 0.5mol/L TCEP to make the final concentration be 25mM, and reacting for 30min at 37 ℃; adding 2uL of iodoacetamide with the concentration of 20mmol/L for alkylation reaction, adding 40uL of trypsin solution, and performing enzymolysis at 37 ℃ overnight;
after enzymolysis, adding 4uL 10% TFA to terminate the enzymolysis reaction; freeze drying the sample, re-dissolving with 0.1% formic acid water solution, and filtering with 0.22um filter membrane; mixing the filtrate with 5.0ng/mL mixed internal standard working solution in equal volume, and performing nanoliter liquid chromatography-mass spectrometry on 5uL according to an optimized PRM method;
quantitative analysis of collected mass spectra data by Skyline softwareSubstituting the peak area ratio of the characteristic peptide segment and the internal standard peptide segment in the on-machine sample into a standard curve to obtain the concentration C of the characteristic peptide segment in the on-machine samplepeptideThen, the protein concentration C of LRG1 standard substance was calculated according to the following formulaprotein；
Protein concentration C of LRG1 standard substanceproteinThe calculation formula of (2) is as follows:
Cprotein＝Cpeptide×VDilution×Rmol×Mprotein/Vsample×Mpeptide；
in the formula:
Cproteinrepresenting the protein concentration in the original protein sample;
Cpeptiderepresenting the concentration of the characteristic peptide fragment measured in the sample on the computer;
VDilutionrepresents the sample volume after freeze-drying and reconstitution;
Rmolrepresenting the molar ratio of the determined protein to the determined characteristic peptide fragment;
Mproteinindicating the molecular weight of the protein to be determined;
Vsamplerepresenting the volume of the original protein sample;
Mpeptiderepresenting the measured characteristic peptide molecular weight;
the protein concentrations obtained by respectively converting the two characteristic peptide fragments LRG1a and LRG1b are averaged to be used as the protein concentration of the LRG1 standard substance.
Compared with the prior art, the invention has the beneficial effects that:
1. the method for valuing the standard substance rich in the leucine alpha-2 glycoprotein 1 uses an isotope dilution mass spectrometry to measure the content of the LRG1 standard substance, fills the blank of the prior art, ensures the accuracy, reliability and traceability of a valuing result, and has important significance for establishing a quality control and evaluation system of the LRG1 standard substance.
2. The invention adopts the nanoliter liquid chromatography for tracing analysis for the first time, compared with the conventional liquid chromatography, the sample loading amount is less, the using amount of a mobile phase is reduced, the sensitivity is improved, the flow of the mobile phase (the inner diameter of a chromatographic column is 4.6mm) of the conventional liquid chromatography is 300 plus one 1000mL/min, and the flow of the nanoliter liquid chromatography (the inner diameter of the chromatographic column is 0.075mm) adopted by the invention is 300 nL/min; compared with the conventional liquid chromatography-mass spectrometry, the sensitivity of the nano-liter liquid chromatography-mass spectrometry can be improved by about 3800 times theoretically.
3. The invention carries out the characteristic peptide segment after LRG1 enzymolysis13C and15the N isotope labeling avoids the isotope effect caused by inconsistent retention time of the deuterated labeled peptide in reversed-phase chromatographic elution, and solves the problem of reproducibility of the intensity signal of the target peptide ion caused by factors such as co-elution matrix, sample composition, instrument state and the like.
Drawings
FIG. 1 is a standard curve of the characteristic peptide segment LRG1 a;
FIG. 2 is a standard curve of the characteristic peptide segment LRG1 b;
FIG. 3 is a chromatogram of the characteristic peptide fragment LRG1 a;
FIG. 4 IS a chromatogram of internal standard peptide segment LRG1a _ IS;
FIG. 5 is a chromatogram of the characteristic peptide fragment LRG1 b;
FIG. 6 IS a chromatogram of internal standard peptide segment LRG1b _ IS.
Detailed Description
In order to make the technical solutions in the embodiments of the present application better understood, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1
A method for valuing a leucine-rich alpha-2 glycoprotein 1 standard substance, comprising the following steps:
the method comprises the following steps: screening a characteristic peptide fragment and a stable isotope labeled internal standard peptide fragment of LRG 1;
step two: optimizing the nanoliter liquid chromatography-mass spectrometry analysis conditions of the characteristic peptide segment and the internal standard peptide segment;
step three: mixing the characteristic peptide segments with different concentrations and the internal standard peptide segments with fixed concentrations in equal volume, analyzing by adopting the optimized nano-liter liquid chromatography-mass spectrometry method in the second step, and establishing a standard curve;
step four: and (3) carrying out enzymolysis on the LRG1 standard substance, adding an internal standard peptide segment into the reaction solution after the enzymolysis, analyzing by adopting the nano-liter liquid chromatography-mass spectrometry method optimized in the second step, and substituting the peak area ratio of the selected characteristic peptide segment and the internal standard peptide segment into the standard curve established in the third step to obtain the concentration of the LRG1 standard substance.
The specific method for screening the characteristic peptide fragment of LRG1 and the stable isotope labeled internal standard peptide fragment comprises the following steps:
(1) to 20uL of LRG1 recombinant protein solution (purchased from Nanjing Kingsler Biotech Co., Ltd.) was added 25uL of NH of 50mmol/L concentration in sequence4HCO3Reacting with 3uL of TCEP (TCEP is a high-efficiency disulfide bond reducing agent) with the concentration of 0.5mol/L for 30min at 37 ℃ to reduce the disulfide bond of the LRG1 recombinant protein, then adding 2uL of iodoacetamide with the concentration of 20mmol/L for alkylation reaction (the reaction condition is that the iodoacetamide is protected from light at room temperature for 30min), adding 10uL of trypsin solution with the concentration of 100ng/uL for overnight enzymolysis at 37 ℃, and after the enzymolysis, adding 5uL of TFA (trifluoroacetic acid) with the concentration of 10% to stop the enzymolysis;
(2) filtering the reaction liquid obtained in the step (1) by adopting a 0.22um filter membrane, carrying out nanoliter liquid chromatography-mass spectrometry on the filtered liquid, and collecting data in a data dependent scanning DDA mode;
(3) comparing the data obtained in the step (2) with protein amino acid sequences in a Uniprot database by using Proteome resolver software and the Uniprot database, and identifying and screening 2 characteristic peptide fragments for quantification; two characteristic peptide segments for quantification are LRG1a and LRG1b, and the amino acid sequences are VAAGAFQGLR and LHLEGNK respectively;
(4) based on the selected characteristic peptide fragment, the use13C and15n, stable isotope labeling is carried out on the selected characteristic peptide segment to obtainTo corresponding internal standard peptide fragments LRG1a _ IS and LRG1b _ IS, the amino acid sequences are VAAGAFQGLR × (13C6,15N4) And LHLEGNK: (13C6,15N2)；
The nano-liter liquid chromatography-mass spectrometry analysis conditions for optimizing the characteristic peptide segment and the internal standard peptide segment are as follows:
(1) respectively carrying out nanoliter liquid chromatography-mass spectrometry on the screened characteristic peptide segment and the internal standard peptide segment, and acquiring data in a parallel reaction monitoring mode;
(2) analyzing the obtained mass spectrum data by Skyline software, selecting 5 fragment ions with the highest peak intensity after the fragmentation of each peptide fragment parent ion as corresponding target peptide fragment child ions, wherein the scanning result is shown in Table 1:
TABLE 1 PRM Scan information of LRG1
The nanoliter liquid chromatography conditions were: the analytical column adopts C18 capillary chromatographic column (3um, 75um 15cm), the column temperature is 55 deg.C, mobile phase A is 0.1% formic acid water solution, and mobile phase B is 80% acetonitrile water solution (containing 0.1% formic acid); the loading solution is 0.1% formic acid aqueous solution, and the volume is 15 uL; gradient elution is adopted for 22min, the sample injection amount is 5uL, the temperature of an autosampler is kept at 7 ℃, and a detection system is quadrupole-orbital ion trap tandem mass spectrometry. Before each sample analysis, the analytical column was equilibrated with mobile phase a for 5 uL.
The gradient elution procedure included: 0min, 300nL/min, 5% B; 16min, 300nL/min, 80% B; 17min, 1000nL/min, 100% B; 22min, 1000nL/min, 100% B;
the mass spectrometry conditions were: capillary voltage was 2.1kV, PRM scan mode, resolution was 30000, isolation window was 1.6m/z, and collision energy NCE was 27.
The specific method for establishing the standard curve comprises the following steps:
dissolving LRG1a lyophilized powder and LRG1b lyophilized powder in 5% acetonitrile water solution to prepare mixed standard solution with equal concentration, and diluting with 0.1% formic acid water solution to 0.5ng/mL, 2.0ng/mL, 5.0ng/mL, 20.0ng/mL and 50.0ng/mL respectively to obtain mixed standard series solution;
dissolving LRG1a _ IS freeze-dried powder and LRG1b _ IS freeze-dried powder in 5% acetonitrile water solution to prepare mixed internal standard solution with equal concentration, and diluting the mixed internal standard solution into 5.0ng/mL mixed internal standard working solution by using 0.1% formic acid water solution;
mixing the mixed standard series solutions with different concentrations with 5.0ng/mL mixed internal standard working solution in equal volume to prepare a standard series solution, analyzing according to an optimized nano-liter liquid chromatography-mass spectrometry analysis method, collecting data in a PRM scanning mode, and quantitatively analyzing through Skyline software; and (3) drawing a standard curve by taking the concentration of the characteristic peptide as an abscissa, the peak area ratio of the characteristic peptide and the internal standard peptide as an ordinate and the weight of the characteristic peptide and the internal standard peptide as 1/x, as shown in fig. 1 and 2.
Sample preparation and quantitative analysis of LRG1 standard:
the LRG1 standard substance 20uL was taken and the volume V was recorded accuratelysample. 0.5mol/L TCEP was added to give a final concentration of 25mM, and the reaction was carried out at 37 ℃ for 30 min. 2uL iodoacetamide at 20mmol/L was added for alkylation and 40uL trypsin solution (50ng/uL in 50mM NH) was added4HCO3) Enzymatic hydrolysis was carried out overnight at 37 ℃. After the enzymatic hydrolysis, 4uL 10% TFA was added to stop the enzymatic reaction. The samples were freeze dried, reconstituted with 0.1% aqueous formic acid and filtered through a 0.22um filter. And mixing the filtrate with 5.0ng/mL mixed internal standard working solution in equal volume, taking 5uL to perform nanoliter liquid chromatography-mass spectrometry according to an optimized PRM method, and recording a chromatogram, wherein the chromatogram is specifically shown in figures 3-6.
Carrying out quantitative analysis on the acquired mass spectrum data through Skyline software, substituting the peak area ratio of the characteristic peptide segment and the internal standard peptide segment in the on-machine sample into a standard curve, and obtaining the concentration C of the characteristic peptide segment in the on-machine samplepeptideThen, the protein concentration C of LRG1 standard substance was calculated according to the following formulaprotein。
Remarking: when calculating the protein concentration, a plurality of enzyme digestion peptide fragment quantitative results are selected for conversion. The embodiment of the invention simultaneously selects the average value of the quantitative results of two enzyme-digested peptide fragments, and can also select the quantitative result of any one peptide fragment.
The formula for calculating the protein concentration Cprotein of LRG1 standard substance is:
Cprotein＝Cpeptide×VDilution×Rmol×Mprotein/Vsample×Mpeptide；
in the formula:
Cproteinrepresenting the protein concentration in the original protein sample;
Cpeptiderepresenting the concentration of the characteristic peptide fragment measured in the sample on the computer;
VDilutionrepresents the sample volume after freeze-drying and reconstitution;
Rmolrepresenting the molar ratio of the determined protein to the determined characteristic peptide fragment;
Mproteinindicating the molecular weight of the protein to be determined;
Vsamplerepresenting the volume of the original protein sample;
Mpeptiderepresenting the measured characteristic peptide molecular weight;
the protein concentrations obtained by respectively converting the two characteristic peptide fragments LRG1a and LRG1b are averaged to be used as the protein concentration of the LRG1 standard substance.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (7)
1. A method for valuing a leucine-rich alpha-2 glycoprotein 1 standard substance, which is characterized by comprising the following steps:
the method comprises the following steps: screening a characteristic peptide fragment and a stable isotope labeled internal standard peptide fragment of LRG 1;
step two: optimizing the nanoliter liquid chromatography-mass spectrometry analysis conditions of the characteristic peptide segment and the internal standard peptide segment;
step three: mixing the characteristic peptide segments with different concentrations and the internal standard peptide segments with fixed concentrations in equal volume, analyzing by adopting the optimized nano-liter liquid chromatography-mass spectrometry method in the second step, and establishing a standard curve;
step four: and (3) carrying out enzymolysis on the LRG1 standard substance, adding an internal standard peptide segment into the reaction solution after the enzymolysis, analyzing by adopting the nano-liter liquid chromatography-mass spectrometry method optimized in the second step, and substituting the peak area ratio of the selected characteristic peptide segment and the internal standard peptide segment into the standard curve established in the third step to obtain the concentration of the LRG1 standard substance.
2. The method for valuing the leucine rich alpha-2 glycoprotein 1 standard substance according to claim 1, wherein in step one: the method for screening the characteristic peptide fragment and the stable isotope labeled internal standard peptide fragment of LRG1 comprises the following steps:
(1) sequentially adding NH into LRG1 recombinant protein solution4HCO3Heating to perform reduction reaction with TCEP, reducing the disulfide bond of LRG1 recombinant protein, adding iodoacetamide to perform alkylation reaction, adding trypsin solution for enzymolysis, and adding TFA to stop enzymolysis after enzymolysis is finished;
(2) filtering the reaction liquid obtained in the step (1), carrying out nano-liter liquid chromatography-mass spectrometry on the filtered liquid, and collecting data in a data dependent scanning DDA mode;
(3) comparing the data obtained in the step (2) with protein amino acid sequences in a Uniprot database by using Proteome resolver software and the Uniprot database, and identifying and screening 2 characteristic peptide fragments for quantification;
(4) based on the selected characteristic peptide fragment, the use13C and15and N, carrying out stable isotope labeling on the selected characteristic peptide segment to be used as an internal standard peptide segment.
3. The method for valuing a leucine rich alpha-2 glycoprotein 1 standard according to claim 2,
in the step (3), the two characteristic peptide fragments for quantification obtained by screening are LRG1a and LRG1b, and the amino acid sequences are VAAGAFQGLR and LHLEGNK respectively.
4. The method for valuing the standard substance of leucine-rich alpha-2 glycoprotein 1 of claim 2, wherein the two internal standard peptide fragments in step (4) are LRG1a _ IS and LRG1b _ IS, and the amino acid sequences are VAAGAFQGLR x respectively13C6,15N4And LHLEGNK13C6,15N2。
5. The method for valuing the standard substance of the leucine-rich alpha-2 glycoprotein 1 according to claim 1, wherein in the second step, the conditions for the nanoliter liquid chromatography-mass spectrometry analysis of the characteristic peptide fragment and the internal standard peptide fragment are optimized as follows:
(1) respectively carrying out nanoliter liquid chromatography-mass spectrometry on the characteristic peptide segment and the internal standard peptide segment, and acquiring data in a parallel reaction monitoring mode;
(2) analyzing the obtained mass spectrum data by Skyline software, and selecting 5 fragment ions with the highest peak intensity after the fragmentation of each peptide fragment parent ion as corresponding target peptide fragment daughter ions.
6. The method for valuing the standard substance rich in the leucine alpha-2 glycoprotein 1 according to claim 1, characterized in that in the third step, the characteristic peptide fragment solutions with different concentrations and the internal standard peptide fragment solution with fixed concentration are mixed in equal volume to prepare a standard series solution, the standard series solution is analyzed according to the optimized nanoliter liquid chromatography-mass spectrometry method, data are collected in a PRM scanning mode, and quantitative analysis is performed through Skyline software; and (3) drawing a standard curve by taking the concentration of the characteristic peptide as an abscissa, taking the peak area ratio of the characteristic peptide and the internal standard peptide as an ordinate and taking the weight as 1/x.
7. The method for valuing the leucine rich alpha-2 glycoprotein 1 standard substance according to claim 1, wherein in step four,
the LRG1 standard substance 20uL was taken and the volume V was recorded accuratelysample(ii) a Adding 0.5mol/L TCEP to make the final concentration be 25mM, and reacting for 30min at 37 ℃; 2ul of a concentration ofPerforming alkylation reaction on 20mmol/L iodoacetamide, adding 40uL trypsin solution, and performing enzymolysis at 37 ℃ overnight;
after enzymolysis, adding 4uL 10% TFA to terminate the enzymolysis reaction; freeze drying the sample, re-dissolving with 0.1% formic acid water solution, and filtering with 0.22um filter membrane; mixing the filtrate with 5.0ng/mL mixed internal standard working solution in equal volume, and performing nanoliter liquid chromatography-mass spectrometry on 5uL according to an optimized PRM method;
carrying out quantitative analysis on the acquired mass spectrum data through Skyline software, substituting the peak area ratio of the characteristic peptide segment and the internal standard peptide segment in the on-machine sample into a standard curve, and obtaining the concentration C of the characteristic peptide segment in the on-machine samplepeptideThen, the protein concentration C of LRG1 standard substance was calculated according to the following formulaprotein；
Protein concentration C of LRG1 standard substanceproteinThe calculation formula of (2) is as follows:
Cprotein＝Cpeptide×VDilution×Rmol×Mprotein/Vsample×Mpeptide；
in the formula:
Cproteinrepresenting the protein concentration in the original protein sample;
Cpeptiderepresenting the concentration of the characteristic peptide fragment measured in the sample on the computer;
VDilutionrepresents the sample volume after freeze-drying and reconstitution;
Rmolrepresenting the molar ratio of the determined protein to the determined characteristic peptide fragment;
Mproteinindicating the molecular weight of the protein to be determined;
Vsamplerepresenting the volume of the original protein sample;
Mpeptiderepresenting the measured characteristic peptide molecular weight;
the protein concentrations obtained by respectively converting the two characteristic peptide fragments LRG1a and LRG1b are averaged to be used as the protein concentration of the LRG1 standard substance.
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