CN109444287A - A kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein - Google Patents
A kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein Download PDFInfo
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Abstract
The invention discloses a kind of LC-MS/MS quantitative detecting methods of alpha-fetoprotein, include the following steps: S1 sample preprocessing: sampling is originally, ammonium bicarbonate soln, internal standard stock solution, dithiothreitol (DTT) solution is added, it restores in a water bath, then iodoacetamido amine aqueous solution is added, is stood at room temperature dark, is subsequently added into trypsin digestion, it is eventually adding formic acid, enzymolysis reaction；S2: enzymolysis product liquid phase chromatographic tandem mass spectroscopy；S3: it is calculated according to formula and obtains α-Fetoprotein.The accuracy that the present invention detects is high, highly reliable, reproducible, stability is good.
Description
Technical field
The present invention relates to a kind of detection method of alpha-fetoprotein, in particular to a kind of LC-MS/MS of alpha-fetoprotein is quantitatively examined
Survey method.
Background technique
Currently, malignant tumour has come the first place of China resident principal disease death rate, becoming influences health of people
Number one killer.According to the Ministry of Public Health, China statistical data, the annual new cancer cases in China about 3,370,000, death about 2,110,000.Liver cancer
Have become the second largest cancer for being only second to gastric cancer.As a kind of disease of high mortality, the cause of disease of liver cancer, is answered at occurrence and development
Hair, transfer and diagnosing and treating all have attracted more and more attention from people.The symptom of liver cancer is in very unobvious or even patient in early days
The long period has no to feel after illness, just can gradually generate under some hepatalgias, appetite to a certain extent to progression of the disease
Drop, the powerless, symptoms such as day is gradually thin tired out.To advanced stage, there will be the performances such as jaundice, ascites, spitting blood, stupor.Hepatocarcinoma patient it is upper
Abdomen can touch huge lump, but arrive middle and advanced stage at this time, or even shift to lung etc..The total course of disease of liver cancer about 2
Half time of year, wherein 2 years, which were at, does not have Symptomatic early stage, when there is the symptom just only survival of half a year
Between.So early diagnosis becomes one of the most effective means of prevention and treatment liver cancer.
World Health Organization's statistical data shows: effective control can be obtained in 80% or more primary malignant tumour.It is more early to make a definite diagnosis,
The cure rate of tumour is higher.
The method of clinical diagnosis tumour can be divided into physics, histocytology and biochemistry three categories.Conventional physics
Diagnosis such as X-ray, CT, nuclear magnetic resonance, B ultrasound, infrared scan can only find the lump of diameter 1-2cm or more, and a tumour is again
Increase to such size about and need 5 years even longer times.On the basis of the inspection of cell pathology needs Physical test to find, lead to
Operation acquirement sample is crossed to test.Because physical diagnosis and histocytology diagnosis can only often find middle and advanced stage pathology, difficult
To achieve the purpose that early detection.Biochemical method is the molecular labelings such as the multiple protein for detecting tumour growth, this method pair
Early diagnosis, observation evaluation therapeutic effect and the judging prognosis of tumour have very big meaning.
Currently, alpha-fetoprotein is unique goldstandard of hepatocarcinoma early diagnosis, periodic detection alpha-fetoprotein can play pre-
The effect of anti-liver cancer.Meanwhile can be used as the index before and after operation of liver cancer, carry out the tracking in later period.In large and medium-sized hospital, first
The detection of fetoprotein is mostly based on the immunological method based on antigen-antibody reaction.This method flux is big, detection automation journey
Degree is high, but a disadvantage is that accuracy in detection is relatively low, is subjected to the interference of other materials in blood and generates false negative or false sun
Property is as a result, lead to hepatocarcinoma early diagnosis result mistake.This method needs antibody as primary raw material, stability between antibody producing is criticized
Difference, and an important factor for lead to unstable result.
Summary of the invention
The purpose of the present invention is to provide a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein, accuracy is high, reliable
Property is strong, reproducible.
The technical solution adopted by the present invention to solve the technical problems is:
A kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein, includes the following steps:
S1 sample preprocessing: originally, ammonium bicarbonate soln, internal standard stock solution, dithiothreitol (DTT) solution is added in sampling, in a water bath also
Then iodoacetamido amine aqueous solution is added in original, stand at room temperature dark, be subsequently added into trypsin digestion, be eventually adding formic acid,
Enzymolysis reaction；
S2: enzymolysis product liquid phase chromatographic tandem mass spectroscopy；
S3: according to formulaIt calculates and obtains α-Fetoprotein；Wherein first tire in G representative sample
Protein content, C are special peptide concentration in sample, MAlpha-fetoproteinRepresent the molecular weight of alpha-fetoprotein, MSpecial peptideRepresent the molecule of special peptide
Amount.
Method of the invention is first to digest to alpha-fetoprotein, and the special peptide generated using enzymatic hydrolysis is special by measuring
The concentration of peptide, to calculate acquisition α-Fetoprotein.Method of the invention is different from traditional immunological method, the standard of detection
Really property is high, highly reliable, reproducible, stability is good.
The internal standard stock solution is the aqueous solution of the special peptide of isotope labelling, the ammonia of the special peptide of the isotope labelling
Base acid sequence are as follows: NI*FL*ASFVHEYSR, wherein I* represents the isoleucine of isotope labelling, and L* represents isotope labelling
Leucine.
The amino acid sequence of the special peptide is NIFLASFVHEYSR.
The concentration of the internal standard stock solution is 1 μ g/mL.
S1 sample process specifically: take 0.5mL sample, be added 0.665mL ammonium bicarbonate soln, 100 μ L internal standard stock solutions,
10 μ L dithiothreitol (DTT) solution, restore 30 minutes in 50 DEG C of water-baths, and 10 μ L iodoacetamido amine aqueous solutions are then added, black in room temperature
Dark place stands 30 minutes, and 10 μ L trypsase are added, and digests overnight in 37 DEG C of water-baths, is eventually adding the 5 dense formic acid of μ L, terminates enzyme
Solution reaction.
The dense formic acid is concentration 95% or more.
Ammonium bicarbonate soln concentration is 500mmol/L, and dithiothreitol (DTT) solution concentration is 100mmol/L, iodo-acetamide
Solution concentration is 300mmol/L.
The liquid-phase condition of Liquid Chromatography-Tandem Mass Spectrometry in S2 are as follows:
Chromatographic column: Waters BEH 300C18, specification 100mm × 2.1mm, 1.7 μm of partial size；
Column temperature: 40 DEG C；Sample temperature: room temperature；
Mobile phase: mobile phase A: the aqueous solution containing 0.1% formic acid；Mobile phase B: the acetonitrile solution containing 0.1% formic acid；
Chromatographic isolation gradient condition: Mobile phase B content is promoted to 32% from 3% in 5 minutes；
Flow velocity: 0.3mL/min；
Sampling volume: 5 μ L.
Mass Spectrometer Method condition is as follows: ESI+ ion source, capillary voltage: 3.0kv, orifice potential: 15V, desolventizing temperature:
500 DEG C, desolventizing gas flow: 900L/min, cone hole backflow airflow amount: 150L/hr, collision chamber pressure: 3.0 × 10-3mbar；It is low
Hold resolution ratio 1:2.5V, high-end resolution ratio 1:15.0V, ion energy 1:0.5eV；Low side resolution ratio 2:2.8V, high-end resolution ratio
2:15.0V, ion energy 2:1.0eV；Ion source temperature: 150 DEG C, extractor voltage: 3.0V, 300V；Monitoring pattern is using more
Reaction detection mode MRM.
The beneficial effects of the present invention are: accuracy is high, highly reliable, reproducible.
Detailed description of the invention
Fig. 1 is the standard chromatogram of the special peptide of special peptide of the invention and isotope labelling.
Specific embodiment
Below by specific embodiment, technical scheme of the present invention will be further explained in detail.
In the present invention, if not refering in particular to, used raw material and equipment etc. are commercially available or commonly used in the art.
Method in following embodiments is unless otherwise instructed the conventional method of this field.
Embodiment:
Reagent and material
Dithiothreitol (DTT) solution: 100mmol/L, iodoacetamido amine aqueous solution: 300mmol/L, ammonium bicarbonate soln: 500mmol/L；
Special peptide stock solution: artificial synthesized NIFLASFVHEYSR polypeptide (SEQ ID No.1) is weighed, is configured to 1 μ g/mL's with water
Stock solution,
Internal standard stock solution: weighing artificial synthesized NI*FL*ASFVHEYSR polypeptide, and the stock solution of 1 μ g/mL is configured to water；Its
Middle I* represent [15N,13C6]-isoleucine, L* representative [15N,13C6]-leucine；Special peptide and the special peptide of isotope labelling
Standard chromatogram is shown in Fig. 1；
Trypsase: 1mg/mL, dense formic acid concn 95%, liquid chromatography tandem triple quadrupole bar mass spectrum is as detection device.
A kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein, includes the following steps:
S1 sample preprocessing: taking 0.5mL sample, and 0.665mL ammonium bicarbonate soln, 100 μ L internal standard stock solutions, 10 μ L, bis- sulphur is added
Threose alcoholic solution restores 30 minutes in 50 DEG C of water-baths, and 10 μ L iodoacetamido amine aqueous solutions are then added, and stands at room temperature dark
30 minutes, 10 μ L trypsase are added, is digested overnight in 37 DEG C of water-baths, is eventually adding the 5 dense formic acid of μ L, enzymolysis reaction；
S2: enzymolysis product liquid phase chromatographic tandem mass spectroscopy；
S3: according to formulaIt calculates and obtains α-Fetoprotein；Wherein first tire in G representative sample
Protein content (ng/mL), C are special peptide concentration (ng/mL), M in sampleAlpha-fetoproteinRepresent the molecular weight of alpha-fetoprotein, MSpecial peptideIt represents
The molecular weight of special peptide, 0.5: sample sampling amount (mL).
The liquid-phase condition of Liquid Chromatography-Tandem Mass Spectrometry in S2 are as follows:
Chromatographic column: Waters BEH 300C18, specification 100mm × 2.1mm, 1.7 μm of partial size；
Column temperature: 40 DEG C；Sample temperature: room temperature；
Mobile phase: mobile phase A: the aqueous solution containing 0.1% formic acid；Mobile phase B: the acetonitrile solution containing 0.1% formic acid；
Chromatographic isolation gradient condition: Mobile phase B content is promoted to 32% from 3% in 5 minutes；
Flow velocity: 0.3mL/min；
Sampling volume: 5 μ L.
Mass Spectrometer Method condition is as follows: ESI+ ion source, capillary voltage: 3.0kv, orifice potential: 15V, desolventizing temperature:
500 DEG C, desolventizing gas flow: 900L/min, cone hole backflow airflow amount: 150L/hr, collision chamber pressure: 3.0 × 10-3mbar；It is low
Hold resolution ratio 1:2.5V, high-end resolution ratio 1:15.0V, ion energy 1:0.5eV；Low side resolution ratio 2:2.8V, high-end resolution ratio
2:15.0V, ion energy 2:1.0eV；Ion source temperature: 150 DEG C, extractor voltage: 3.0V, 300V；Monitoring pattern is using more
Reaction detection mode MRM.
MRM analytical model
Performance verification the method for the present invention is with reference to internationally recognizable methodology validation method and the practical feelings of the combination industry
Condition has formulated the guideline of performance verification.
1, day to day precision experiment purpose: the stability of method is investigated.
Experimental method takes 0.5mL patients serum, and it is (special that 0.665mL ammonium bicarbonate soln, 100 μ L standard working solutions are added
Peptide stock solution), 100 μ L internal standard working solutions (internal standard stock solution), 10 μ L dithiothreitol (DTT) solution, in 50 DEG C of water-baths restore 30 points
Clock.Then 10 μ L iodoacetamido amine aqueous solutions are added, stand 30 minutes at room temperature dark.10 μ L trypsase are added, at 37 DEG C
It is digested overnight in water-bath.It is eventually adding the 5 dense formic acid of μ L, enzymolysis reaction.Daily every part of sample is five times parallel, repeats 4 days.
Experimental result: 2 are shown in Table
The experiment of table 1:AFP day to day precision
Experiment conclusion experimental result RSD≤15%, the correlation for meeting U.S. clinical laboratory standards institute CLSIC62-A are wanted
It asks.
2, the standard of result accuracy/rate of recovery experiments experiment purpose: is investigated by the rate of recovery test to mark-on sample
True property.
Experimental method takes 0.5mL patients serum, and the appropriate special poly saccharide peptide standard product of addition distinguishes sample spiked levels respectively
For 10,20,50ng/mL, the sample of three concentration points each parallel five times, repeat 4 days.Concrete operation method and Precision Experiment phase
Together.
Experimental result: 2 are shown in Table
The experiment of the table 2:AFP rate of recovery
The rate of recovery of experiment conclusion three spiked levels according to the experimental results is between 80-120%, and relative standard
Deviation RSD < 15% meets the related request of U.S. clinical laboratory standards institute CLSIC62-A.
3, standard curve experiment purpose: the range of linearity of this method is verified.
Special peptide standard solution is diluted to 1 with 0.1% formic acid water/acetonitrile solution by experimental method respectively, 5,10,20,50,
80,100ng/mL series of concentrations, while the special peptide of isotope is added, each concentration point is made to be 5ng/mL, sample introduction is analyzed, weight
It is 4 days multiple.
Experimental result: 3 are shown in Table
Table 3: standard curve verification result
| Time | Linearly (r2) | Curvilinear equation |
| First day | 0.9979 | Y=1.283*X+3.862 |
| Second day | 0.9989 | Y=1.208*X-3.510 |
| Third day | 0.9957 | Y=1.227*X+0.025 |
| 4th day | 0.9965 | Y=1.277*X+0.720 |
The linear r of experiment conclusion standard curve2It is all larger than 0.99, meets U.S. clinical laboratory standards institute CLSIC62-A
Related request.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form
Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.
SEQUENCE LISTING
<110>Laboratory of medical test Co., Ltd, Hangzhou Tontru
<120>the LC-MS/MS quantitative detecting method of a kind 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 (9)
1. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein, which comprises the steps of:
S1 sample preprocessing: originally, ammonium bicarbonate soln, internal standard stock solution, dithiothreitol (DTT) solution is added in sampling, in a water bath also
Then iodoacetamido amine aqueous solution is added in original, stand at room temperature dark, be subsequently added into trypsin digestion, be eventually adding formic acid,
Enzymolysis reaction；
S2: enzymolysis product liquid phase chromatographic tandem mass spectroscopy；
S3: according to formulaIt calculates and obtains α-Fetoprotein；Wherein first tire egg in G representative sample
Bai Hanliang, C are special peptide concentration in sample, MAlpha-fetoproteinRepresent the molecular weight of alpha-fetoprotein, MSpecial peptideRepresent the molecular weight of special peptide.
2. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 1, it is characterised in that: in described
Mark the aqueous solution for the special peptide that stock solution is isotope labelling, the amino acid sequence of the special peptide of the isotope labelling are as follows: NI*
FL*ASFVHEYSR, wherein I* represents the isoleucine of isotope labelling, and L* represents the leucine of isotope labelling.
3. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 2, it is characterised in that: the spy
The amino acid sequence of different peptide is NIFLASFVHEYSR.
4. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 1 or 2, it is characterised in that: institute
The concentration for stating internal standard stock solution is 1 μ g/mL.
5. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 1, it is characterised in that: S1 sample
Processing specifically: take 0.5mL sample, 0.665mL ammonium bicarbonate soln, 100 μ L internal standard stock solutions, 10 μ L dithiothreitol (DTT)s is added
Solution restores 30 minutes in 50 DEG C of water-baths, and 10 μ L iodoacetamido amine aqueous solutions are then added, and 30 points are stood at room temperature dark
10 μ L trypsase are added in clock, digest overnight in 37 DEG C of water-baths, are eventually adding the 5 dense formic acid of μ L, enzymolysis reaction.
6. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 5, it is characterised in that: described dense
Formic acid is concentration 95% or more.
7. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 1, it is characterised in that: bicarbonate
Ammonium salt solution concentration is 500mmol/L, and dithiothreitol (DTT) solution concentration is 100mmol/L, and iodo-acetamide solution concentration is
300mmol/L。
8. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 1, it is characterised in that: liquid in S2
The mass spectrographic liquid-phase condition of phase chromatographic tandem are as follows:
Chromatographic column: Waters BEH 300C18, specification 100mm × 2.1mm, 1.7 μm of partial size；
Column temperature: 40 DEG C；Sample temperature: room temperature；
Mobile phase: mobile phase A: the aqueous solution containing 0.1% formic acid；Mobile phase B: the acetonitrile solution containing 0.1% formic acid；
Chromatographic isolation gradient condition: Mobile phase B content is promoted to 32% from 3% in 5 minutes；
Flow velocity: 0.3mL/min；
Sampling volume: 5 μ L.
9. a kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein according to claim 1, it is characterised in that: mass spectrum inspection
Survey condition is as follows: ESI+ ion source, capillary voltage: 3.0kv, orifice potential: 15V, desolventizing temperature: 500 DEG C, desolventizing gas
Flow: 900L/min, cone hole backflow airflow amount: 150L/hr, collision chamber pressure: 3.0 × 10-3mbar；Low side resolution ratio 1:
2.5V, high-end resolution ratio 1:15.0V, ion energy 1:0.5eV；Low side resolution ratio 2:2.8V, high-end resolution ratio 2:15.0V, from
Sub- energy 2:1.0eV；Ion source temperature: 150 DEG C, extractor voltage: 3.0V, 300V；Monitoring pattern uses more reaction detection moulds
Formula MRM.
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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 |