CN109444287B - LC-MS/MS quantitative detection method for alpha fetoprotein - Google Patents
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Abstract
the invention discloses an LC-MS/MS quantitative detection method of alpha fetoprotein, which comprises the following steps: s1 sample pretreatment: taking a sample, adding an ammonium bicarbonate solution, an internal standard stock solution and a dithiothreitol solution, reducing in a water bath, then adding an iodoacetamide solution, standing in a dark place at room temperature, then adding trypsin for enzymolysis, and finally adding formic acid to terminate the enzymolysis reaction; s2: performing liquid chromatography tandem mass spectrometry on the enzymolysis product; s3: and (5) calculating according to a formula to obtain the alpha-fetoprotein content. The invention has the advantages of high detection accuracy, strong reliability, good repeatability and good stability.
Description
Technical Field
The invention relates to a detection method of alpha fetoprotein, in particular to an LC-MS/MS quantitative detection method of alpha fetoprotein.
Background
at present, malignant tumors are ranked at the top of the death rate of main diseases of residents in China, and become the first killers affecting the health of people. According to the statistical data of Ministry of health in China, about 337 thousands of new cancer cases and 211 thousands of deaths occur every year in China. Liver cancer has become the second largest cancer to gastric cancer. As a disease with high mortality, the etiology, development, recurrence, metastasis, and diagnosis and treatment of liver cancer are attracting more and more attention. Liver cancer symptoms are not obvious in the early stage, even patients do not feel for a long time after the liver cancer is ill, and symptoms such as liver pain, appetite decrease, fatigue and weakness, and gradual emaciation can be gradually generated only after the disease condition develops to a certain extent. In late stage, jaundice, ascites, hematemesis, coma, etc. may occur. Huge masses can be seen in the upper abdomen of patients with liver cancer, but the mass reaches the middle and advanced stage and even has metastasized to the lung and the like. The overall course of liver cancer is approximately 2 and half years, with 2 years being the early stage without symptoms and half-year survival time once symptoms are present. Therefore, early diagnosis is one of the most effective means for preventing and treating liver cancer.
The world health organization statistics show that: more than 80% of early malignant tumors can be effectively controlled. The earlier the diagnosis is made, the higher the cure rate of the tumor.
Clinical diagnosis of tumors can be divided into three major categories, namely physics, histocytology and biochemistry. Conventional physical diagnosis such as X-ray, CT, nmr, B-ray, ir scan, etc. can only detect tumors with a diameter of 1-2cm, and it takes about 5 years or more for a tumor to double to such a size. The cytopathology examination needs physical examination findings, and the examination is carried out by taking a specimen through operations. Because only middle and late stage pathology can be found in physical diagnosis and tissue cytology diagnosis, early detection is difficult to achieve. The biochemical method is used for detecting molecular markers such as various proteins for tumor growth, and has great significance for early diagnosis, observation and evaluation of treatment effect and judgment of prognosis of tumors.
At present, alpha fetoprotein is the only gold standard for early diagnosis of liver cancer, and regular detection of alpha fetoprotein can play a role in preventing liver cancer. Meanwhile, the method can be used as an index before and after the liver cancer operation for later-stage tracking. In large and medium hospitals, detection of alpha-fetoprotein is mostly based on an immunological method based on antigen-antibody reaction. The method has the advantages of high flux and high detection automation degree, but has the defects of low detection accuracy and false negative or false positive results caused by the interference of other substances in blood, so that the early diagnosis result of the liver cancer is wrong. The method needs the antibody as a main raw material, and the poor stability among antibody production batches is also an important factor causing the instability of the result.
Disclosure of Invention
The invention aims to provide an LC-MS/MS quantitative detection method for alpha fetoprotein, which has the advantages of high accuracy, strong reliability and good repeatability.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an LC-MS/MS quantitative detection method of alpha fetoprotein comprises the following steps:
S1 sample pretreatment: taking a sample, adding an ammonium bicarbonate solution, an internal standard stock solution and a dithiothreitol solution, reducing in a water bath, then adding an iodoacetamide solution, standing in a dark place at room temperature, then adding trypsin for enzymolysis, and finally adding formic acid to terminate the enzymolysis reaction;
S2: performing liquid chromatography tandem mass spectrometry on the enzymolysis product;
S3: according to the formulacalculating to obtain the alpha fetoprotein content; wherein G represents the alpha-fetoprotein content in the sample, C is the specific peptide concentration in the sample, MAlpha-fetoproteinrepresents the molecular weight of alpha-fetoprotein, MSpecific peptidesrepresents the molecular weight of the specific peptide.
The method comprises the steps of carrying out enzymolysis on alpha fetoprotein, utilizing specific peptides generated by the enzymolysis, and calculating to obtain the content of the alpha fetoprotein by measuring the concentration of the specific peptides. The method is different from the traditional immunological method, and has the advantages of high detection accuracy, strong reliability, good repeatability and good stability.
the internal standard stock solution is an aqueous solution of isotope-labeled specific peptides, and the amino acid sequences of the isotope-labeled specific peptides are as follows: NI FL ASFVHEYSR, wherein I represents isotopically labelled isoleucine and L represents isotopically labelled leucine.
The amino acid sequence of the specific peptide is NIFLASFVHEYSR.
The concentration of the internal standard stock solution is 1 mug/mL.
The S1 sample processing specifically comprises: taking 0.5mL of sample, adding 0.665mL of ammonium bicarbonate solution, 100 mu L of internal standard stock solution and 10 mu L of dithiothreitol solution, reducing for 30 minutes in a water bath at 50 ℃, then adding 10 mu L of iodoacetamide solution, standing for 30 minutes in the dark at room temperature, adding 10 mu L of trypsin, carrying out enzymolysis overnight in a water bath at 37 ℃, finally adding 5 mu L of concentrated formic acid, and stopping the enzymolysis reaction.
The concentration of the concentrated formic acid is more than 95%.
the concentration of the ammonium bicarbonate solution is 500mmol/L, the concentration of the dithiothreitol solution is 100mmol/L, and the concentration of the iodoacetamide solution is 300 mmol/L.
The liquid phase conditions of the liquid chromatography tandem mass spectrum in the S2 are as follows:
a chromatographic column: waters BEH 300C18specification 100mm × 2.1mm, particle size 1.7 μm;
Column temperature: 40 ℃; sample temperature: room temperature;
Mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
Chromatographic separation gradient conditions: the mobile phase B content was increased from 3% to 32% in 5 minutes;
Flow rate: 0.3 mL/min;
Sample introduction volume: 5 μ L.
The mass spectrometric detection conditions were as follows: ESI + ion source, capillary voltage: 3.0kv, cone voltage: 15V, desolventizing temperature: 500 ℃, desolventizing gas flow: 900L/min, cone hole back-blowing gas flow: 150L/hr, collision chamber pressure: 3.0X 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 150 ℃, extractor voltage: 3.0V, 300V; the monitoring mode adopts a multi-reaction detection mode MRM.
The invention has the beneficial effects that: high accuracy, high reliability and good repeatability.
Drawings
FIG. 1 is a standard chromatogram of a specific peptide of the present invention and an isotope-labeled specific peptide.
Detailed Description
the technical solution of the present invention will be further specifically described below by way of specific examples.
in the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example (b):
Reagents and materials
Dithiothreitol solution: 100mmol/L, iodoacetamide solution: 300mmol/L, ammonium bicarbonate solution: 500 mmol/L; specific peptide stock solutions: weighing the synthetic NIFLASFVHEYSR polypeptide (SEQ ID No.1), preparing a stock solution of 1 mug/mL with water,
Internal standard stock solution: weighing artificially synthesized NI FL ASFVHEYSR polypeptide, and preparing a stock solution of 1 μ g/mL with water; wherein I represents15N,13C6]-isoleucine, L represents15N,13C6]-leucine; the standard chromatograms of the specific peptide and the isotope-labeled specific peptide are shown in FIG. 1;
Trypsin: 1mg/mL, the concentration of concentrated formic acid is 95%, and a liquid chromatogram is connected with a triple quadrupole mass spectrum as a detection device.
an LC-MS/MS quantitative detection method of alpha fetoprotein comprises the following steps:
S1 sample pretreatment: taking 0.5mL of sample, adding 0.665mL of ammonium bicarbonate solution, 100 mu L of internal standard stock solution and 10 mu L of dithiothreitol solution, reducing for 30 minutes in a water bath at 50 ℃, then adding 10 mu L of iodoacetamide solution, standing for 30 minutes in the dark at room temperature, adding 10 mu L of trypsin, carrying out enzymolysis overnight in a water bath at 37 ℃, finally adding 5 mu L of concentrated formic acid, and stopping the enzymolysis reaction;
S2: performing liquid chromatography tandem mass spectrometry on the enzymolysis product;
S3: according to the formulaCalculating to obtain the alpha fetoprotein content; wherein G represents the alpha-fetoprotein content (ng/mL) in the sample, C is the specific peptide concentration (ng/mL) in the sample, and M isAlpha-fetoproteinRepresents the molecular weight of alpha-fetoprotein, MSpecific peptidesRepresents the molecular weight of the specific peptide, 0.5: sample size (mL).
The liquid phase conditions of the liquid chromatography tandem mass spectrum in the S2 are as follows:
a chromatographic column: waters BEH 300C18Specification 100mm × 2.1mm, particle size 1.7 μm;
Column temperature: 40 ℃; sample temperature: room temperature;
Mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
chromatographic separation gradient conditions: the mobile phase B content was increased from 3% to 32% in 5 minutes;
Flow rate: 0.3 mL/min;
Sample introduction volume: 5 μ L.
The mass spectrometric detection conditions were as follows: ESI + ion source, capillary voltage: 3.0kv, cone voltage: 15V, desolventizing temperature: 500 ℃, desolventizing gas flow: 900L/min, cone hole back-blowing gas flow: 150L/hr, collision chamber pressure: 3.0X 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 150 ℃, extractor voltage: 3.0V, 300V; the monitoring mode adopts a multi-reaction detection mode MRM.
MRM analysis mode
The method of the invention refers to an internationally known methodology verification method and combines the actual situation of the industry to establish the guiding principle of performance verification.
1. Day precision experimental purpose: the stability of the method was investigated.
The experimental method takes 0.5mL of patient serum, and adds 0.665mL ammonium bicarbonate solution, 100 μ L of standard working solution (specific peptide stock solution), 100 μ L of internal standard working solution (internal standard stock solution) and 10 μ L of dithiothreitol solution, and reduces in 50 ℃ water bath for 30 minutes. Then, 10. mu.L of iodoacetamide solution was added and allowed to stand at room temperature in the dark for 30 minutes. 10 μ L of trypsin was added and the mixture was digested overnight in a 37 ℃ water bath. Finally, 5. mu.L of concentrated formic acid is added to terminate the enzymolysis reaction. Each sample was run in parallel five times per day for 4 days.
the experimental results are as follows: see Table 2
Table 1: AFP precision in day experiment
Experimental conclusion the experimental result RSD is less than or equal to 15 percent and meets the relevant requirements of the American clinical laboratory standards institute CLSIC 62-A.
2. Accuracy/recovery experimental objectives: the accuracy of the results was examined by recovery testing of the spiked samples.
the experimental method takes 0.5mL of patient serum, respectively adds a proper amount of specific peptide standard substance to ensure that the standard concentration of a sample is respectively 10, 20 and 50ng/mL, samples with three concentration points are respectively paralleled for five times, and the operation is repeated for 4 days. The specific operation method is the same as the precision experiment.
The experimental results are as follows: see Table 2
Table 2: AFP recovery test
Experimental conclusion from the experimental results, it can be seen that the recovery rates of the three spiked concentrations are all between 80-120%, and the relative standard deviation RSD is < 15%, which meets the requirements of the american clinical laboratory standards institute CLSIC 62-a.
3. standard curve experimental purpose: the linear range of the method was verified.
The experimental method comprises the steps of respectively diluting specific peptide standard solutions to 1, 5, 10, 20, 50, 80 and 100ng/mL series of concentrations by using 0.1% formic acid water/acetonitrile solutions, simultaneously adding isotope specific peptides to enable each concentration point to be 5ng/mL, carrying out sample injection analysis, and repeating for 4 days.
The experimental results are as follows: see Table 3
Table 3: standard curve verification results
| Time of day | Linearity (r)2) | Equation of the curve |
| day one | 0.9979 | y＝1.283*X+3.862 |
| the next day | 0.9989 | y＝1.208*X-3.510 |
| The third day | 0.9957 | y＝1.227*X+0.025 |
| the fourth day | 0.9965 | y＝1.277*X+0.720 |
Experimental conclusions Standard Curve Linearity r2are all larger than 0.99, and meet the requirements of the American clinical laboratory standards institute CLSIC 62-A.
the above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
SEQUENCE LISTING
<110> Hangzhou Co-invasive medical laboratory Co., Ltd
<120> LC-MS/MS quantitative detection method of alpha fetoprotein
<130> 2018.12
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 13
<212> PRT
<213> Artificial sequence (Artificial sequence)
<400> 1
Asn Ile Phe Leu Ala Ser Phe Val His Glu Tyr Ser Arg
1 5 10
Claims (5)
1. a LC-MS/MS quantitative detection method of non-diagnostic target alpha-fetoprotein is characterized by comprising the following steps:
S1 sample pretreatment: taking a sample, adding an ammonium bicarbonate solution, an internal standard stock solution and a dithiothreitol solution, reducing in a water bath, then adding an iodoacetamide solution, standing in a dark place at room temperature, then adding trypsin for enzymolysis, and finally adding formic acid to terminate the enzymolysis reaction;
s2: performing liquid chromatography tandem mass spectrometry on the enzymolysis product;
s3: according to the formulaCalculating to obtain the alpha fetoprotein content; wherein G represents the alpha-fetoprotein content in the sample, C is the specific peptide concentration in the sample, MAlpha-fetoproteinRepresents the molecular weight of alpha-fetoprotein, MSpecific peptidesrepresents the molecular weight of the specific peptide;
the internal standard stock solution is an aqueous solution of isotope-labeled specific peptides, and the amino acid sequences of the isotope-labeled specific peptides are as follows: NI FL ASFVHEYSR, wherein I represents isotopically labeled isoleucine and L represents isotopically labeled leucine; the amino acid sequence of the specific peptide is NIFLASFVHEYSR;
The liquid phase conditions of the liquid chromatography tandem mass spectrum in the S2 are as follows:
A chromatographic column: waters BEH 300C18Specification 100mm × 2.1mm, particle size 1.7 μm;
Column temperature: 40 ℃; sample temperature: room temperature;
mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
Chromatographic separation gradient conditions: the mobile phase B content was increased from 3% to 32% in 5 minutes;
flow rate: 0.3 mL/min;
Sample introduction volume: 5 muL;
The mass spectrometric detection conditions were as follows: ESI + ion source, capillary voltage: 3.0kv, cone voltage: 15V, desolventizing temperature: 500 ℃, desolventizing gas flow: 900L/min, cone hole back-blowing gas flow: 150L/hr, collision chamber pressure: 3.0X 10-3 mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 150 ℃, extractor voltage: 3.0V; the monitoring mode adopts a multi-reaction detection mode MRM.
2. The method for LC-MS/MS quantitative detection of alpha-fetoprotein of non-diagnostic interest according to claim 1, wherein: the concentration of the internal standard stock solution is 1 mug/mL.
3. The method for LC-MS/MS quantitative detection of alpha-fetoprotein of non-diagnostic interest according to claim 1, wherein: the S1 sample processing specifically comprises: taking 0.5mL of sample, adding 0.665mL of ammonium bicarbonate solution, 100 muL of internal standard stock solution and 10 muL of dithiothreitol solution, reducing for 30 minutes in a water bath at 50 ℃, then adding 10 muL of iodoacetamide solution, standing for 30 minutes in a dark place at room temperature, adding 10 muL of trypsin, carrying out enzymolysis overnight in a water bath at 37 ℃, finally adding 5 muL of concentrated formic acid, and stopping enzymolysis reaction.
4. the method for LC-MS/MS quantitative detection of alpha-fetoprotein of non-diagnostic interest according to claim 3, wherein: the concentration of the concentrated formic acid is more than 95%.
5. The method for LC-MS/MS quantitative detection of alpha-fetoprotein of non-diagnostic interest according to claim 1, wherein: the concentration of the ammonium bicarbonate solution is 500mmol/L, the concentration of the dithiothreitol solution is 100mmol/L, and the concentration of the iodoacetamide solution is 300 mmol/L.
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| CN102590413A (en) * | 2012-01-18 | 2012-07-18 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
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| CN108398503A (en) * | 2018-03-27 | 2018-08-14 | 北京市营养源研究所 | A kind of liquid chromatography mass detection method of lactoferrin |
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| CN102590413A (en) * | 2012-01-18 | 2012-07-18 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
| CN103616454A (en) * | 2013-12-06 | 2014-03-05 | 浙江贝因美科工贸股份有限公司 | Method and kit for quantitatively detecting human beta-casein content |
| CN108398503A (en) * | 2018-03-27 | 2018-08-14 | 北京市营养源研究所 | A kind of liquid chromatography mass detection method of lactoferrin |
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Denomination of invention: A LC-MS/MS quantitative detection method for alpha fetoprotein Effective date of registration: 20231123 Granted publication date: 20191217 Pledgee: China Construction Bank Corporation Hangzhou Qiantang sub branch Pledgor: HANGZHOU TONGCHUANG MEDICAL EXAMINATION LABORATORY Co.,Ltd. Registration number: Y2023330002725 |