CN107064522B - The quantitative detecting method of fibronectin and application in a kind of acellular matrix material - Google Patents
The quantitative detecting method of fibronectin and application in a kind of acellular matrix material Download PDFInfo
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Abstract
The present invention provides the quantitative detecting method of fibronectin and application in a kind of acellular matrix material.The quantitative detecting method includes pretreatment, stepwise discretization method and the Mass Spectrometer Method process to acellular matrix, all polypeptide sequence mixtures of fibronectin are obtained by the pretreatment of acellular matrix, stepwise discretization method, feature polypeptide sequence in screen fibre connection albumen again, feature content of peptides is detected by mass spectrography, to determine fibronectin content in acellular matrix.This method solve be not available tectotype fibronectin content in mass spectrography quantitative detection acellular matrix in the past；Further, collagen component in acellular matrix is removed by way of stepwise discretization, reduces the interference of the non-targeted polypeptide sequence of test process, to save the testing time, improves working efficiency, detection accuracy and stability.
Description
Technical field
The present invention relates to a kind of protein detection method and its applications, in particular it relates to which a kind of utilize mass spectrum skill
The method that art detects fibronectin content in acellular matrix material by detection feature polypeptide.
Technical background
Submucous layer of small intestine matrix (Small Intestinal Submucosa, SIS) material refers to submucous layer of small intestine
Obtained biomaterial after removing immunogenic substance after taking off the processing of the methods of cell is widely used in various soft tissues and lacks
The reparation of damage has the function of inducing cell adherency growth, promotes regeneration and reconstruction etc..SIS material is as surgical implant
It is successfully used for the reparation of the tissue defects such as nerve, stomach wall, endocranium, tendon, the urinary tract, eardrum, is had safe and effective, postoperative
The advantages that few intercurrent disease, infection rate are low, patient satisfaction is high, it was confirmed that SIS material is that one kind is preferably repaired for tissue defect
The animal derived implantation material without immunological rejection that is multiple and rebuilding.
SIS host material is collagen as a kind of acellular matrix main component, and includes the structure of non-collagen class
Albumen and biological mucopolysaccharide.Wherein collagen is mainly made of I type and type III, and non-collagen albuminoid mainly includes that fiber connects
Connect albumen (Fibronectin, FN), laminin and growth factor etc..FN is a kind of glycoprotein, is by two molecular weight
The dimeric forms that the subunit of 220kDa is connected with disulfide bond exist, mainly by fibroblast, endothelial cell, cartilage cell,
The synthesis such as Deiter's cells and myocyte secretion, is divided into blood plasma type and tectotype.By comparing the FN gene core of different plant species
Nucleotide sequence, FN gene is highly conserved as the result is shown.The FN of different plant species different tissue sources, molecular weight, substantially sky
Between structure and biological property it is almost the same.FN is that mediated cell is viscous as its major function of main cell adhesion molecule
, promote the bonding of cell and extracellular matrix and enhance the endocytosis of macrophage, generate in embryo, wound healing and only
Key effect, the unconventionality expression and degradation process of FN and the generation of many diseases are played in many important physiology courses such as blood
It is closely related.Adherency of the FN contained in SIS to cell on the surface SIS has a great impact with migration.But do not have also at present
There is the detection method of tectotype FN in efficient, stable, accurate biomaterial.
Current existing FN measuring method include enzyme-linked immunization (ELISA), radioimmunology, Immunity transmission turbidity,
Unidirectional rocket electrophoresis, chemoluminescence method, Immunohistochemical Method, rate nephelometry, Micro BCA method etc..Wherein apply
Most often ELISA method is reacted in conjunction with antibody, then through chemical colour reaction using FN as antigen first in ELISA method, is led to
The absorbance value for crossing detection chromogenic substrate determines the content of FN, but this method is mainly used for the detection of blood plasma type FN.FN in SIS
Belong to tectotype and combining form exists, directlying adopt ELISA method, then antibody is difficult to directly in conjunction with FN, non-specific adsorption
Also the accuracy that will affect result, causing testing result with actual content, there are relatively large deviations.
In addition, although the quantitative analysis based on mass spectrographic protein detection method for low-abundance protein has sensitivity
The advantages that high, reproducible, can carry out unmarked quantitative detection according to the polypeptide abundance in protein degradation products.But make
The content in acellular matrix for acellular matrix ingredient FN is seldom, additionally due to the structure of acellular matrix makes sequence special
Heterozyme is difficult to these FN that directly degrade.Therefore, it is impossible to digest the FN in acellular matrix completely.It can be seen that even if obtaining some peptides
Section also also can just be available mass spectrum method quantitative measurement FN in acellular matrix as the basis of accurate detection FN content
Exact level.
Summary of the invention
In acellular matrix FN detection there are aiming at the problem that, the present invention provide it is a kind of based on enzymatic hydrolysis and mass spectral analysis fibre
The quantitative detecting method of dimension connection albumen, to overcome the above problem accurately to detect fibronectin in acellular matrix
In content.
A kind of method of fibronectin content in detection acellular matrix, which is characterized in that the detection method includes
Following steps：(1) it pre-processes：The acellular matrix for weighing constant weight is cut into the small pieces that size is less than 1cm × 1cm,
Powder is ground under the conditions of protection of liquid nitrogen, powder size is controlled at 50-300 μm；(2) 1 is digested：Cell is taken off after taking a certain amount of crushing
Matrix powder is added to the buffer of 500-10000 times of acellular matrix powder weight, cold in 100 DEG C of heat treatment 5-10min
But the clostridiopetidase A of acellular matrix weight 1/200~1/50 is added afterwards, in 37 DEG C of oscillation treatment 2-24h；(3) 2 are digested：By step
(2) pH value of solution obtained is adjusted to 7.5-8.5, and non-collagen albuminoid enzyme is added, and the additional amount of non-collagen albuminoid enzyme is de- thin
The 1/200~1/50 of cytoplasmic matrix weight, in 37 DEG C of reaction 8-24h；(4) Mass Spectrometer Method：By step (3) obtain enzymatic hydrolysis solution into
Row concentration, concentrate are directly analyzed by mass spectrometry, and the enzymolysis product with the fibronectin standard solution of known concentration is
Control detects feature polypeptide, calculates de- cell by fibronectin feature polypeptide in measurement acellular matrix catabolite
The content of fibronectin in matrix.
Clostridiopetidase A used in step (2) enzymolysis process can be I type, II type, type III, V-type or different type glue
The mixture of protoenzyme, additional amount are the 1/200~1/50 of acellular matrix weight.
Protease used in step (3) enzymolysis process can be trypsase, chemically modified trypsase
Or the trypsase of immobilization, additional amount are the 1/200~1/50 of acellular matrix weight.
Feature polypeptide in the step (4) for detecting fibronectin in acellular matrix includes：
FTQVTPTSLTAQWTAPNVQLTGYR、EINLAPDSSSVVVSGLMVATK、DTLTSR、PAQGVVTTLENVSPPR、
VTDATETTITISWR、SYTITGLQPGTDYK、SSPVVIDASTAIDAPSNLR、FLATTPNSLLVSWQPPR、PGSPPR、
One of EVVPR, NNQK or SEPLIGR or a variety of.
The present invention also provides a kind of feature polypeptides of fibronectin, which is characterized in that the fibronectin warp
The feature polypeptide can be obtained by crossing step as the aforementioned (1), (2) and (3), and the feature polypeptide can be used in detecting the fibre
The content of dimension connection albumen；The feature polypeptide is feature polypeptide above-mentioned：FTQVTPTSLTAQWTAPNVQLTGYR,
EINLAPDSSSVVVSGLMVATK、DTLTSR、PAQGVVTTLENVSPPR、VTDATETTITISWR、SYTITGLQPGTDYK、
SSPVVIDASTAIDAPSNLR, FLATTPNSLLVSWQPPR, PGSPPR, EVVPR, NNQK or SEPLIGR.
There is provided a kind of detection method of fibronectin, which is characterized in that the detection method uses special as mentioned
Levy polypeptide.
It provides a kind of for detecting the kit of fibronectin, which is characterized in that including one or more enzymes；It is described
One or more enzymes can digest fibronectin, and generate feature polypeptide as the aforementioned.
Kit further includes the standard items of fibronectin standard items or the feature polypeptide above-mentioned.
One or more enzymes include：Clostridiopetidase A and protease；The preferred I type of the clostridiopetidase A, II type, type III or V-type
Clostridiopetidase A；The trypsase of the preferred trypsase of the protease, chemically modified trypsase or immobilization.
The application in detection fibronectin of feature polypeptide described in one kind is also provided.
Detailed description of the invention
The dynamic change of Fig. 1 static state enzymatic hydrolysis and SIS residual quantity in enzymolysis process in batches
Fig. 2 is the total ion current figure of fibronectin enzymolysis polypeptide；
Fig. 3 is characterized the extraction ion flow graph and first mass spectrometric figure of polypeptide SSPVVIDASTAIDAPSNLR；
Fig. 4 is characterized the second order ms figure of polypeptide SSPVVIDASTAIDAPSNLR；
The extraction ion flow graph of the fibronectin feature polypeptide SSPVVIDASTAIDAPSNLR of Fig. 5 various concentration；
Fig. 6 fibronectin standard concentration and signal strength relation curve；
Fig. 7 and Fig. 8 is characterized the extraction ion flow graph and first mass spectrometric of polypeptide EINLAPDSSSVVVSGLMVATK respectively
Figure；
Fig. 9 and Figure 10 is characterized the extraction ion flow graph and level-one matter of polypeptide FTQVTPTSLTAQWTAPNVQLTGYR respectively
Spectrogram；
Figure 11 and Figure 12 is characterized the extraction ion flow graph and first mass spectrometric figure of polypeptide FLATTPNSLLVSWQPPR respectively；
Figure 13 and Figure 14 is characterized the extraction ion flow graph and first mass spectrometric figure of polypeptide PAQGVVTTLENVSPPR respectively；
Figure 15 and Figure 16 is characterized the extraction ion flow graph and first mass spectrometric figure of polypeptide DTLTSR respectively.
Above-mentioned attached drawing facilitates those skilled in the art understand that the present invention, and simultaneously for illustrating technical solution of the present invention
It is non-to be used to limit technical solution of the present invention.
Specific embodiment
The feature polypeptide for the fibronectin that the present invention is determined according to mass spectral analysis first simultaneously uses fibronectin
Reference substance establishes the linear relationship between concentration and mass spectrum peak area, then using feature polypeptide in mass spectrography detection sample to be tested
Peak value and containing for fibronectin in submucous layer of small intestine matrix is determined according to the relationship between concentration and mass spectrum peak area
Amount.It will hereafter be illustrated by specific embodiment.
Determinand is respectively the submucous layer of small intestine matrix of the pig through immunogene removal processing；The Type I collagen used
Enzyme (analysis is pure) is purchased from Beijing Xin Jingke Bioisystech Co., Ltd；Trypsase (sequence is pure) is purchased from U.S. Promega company；
Fibronectin is purchased from U.S. CALBIOCHEM company.High performance liquid chromatography-mass spectrometry system (HPLC-MS) is by the U.S.
1100 liquid chromatogram of Agilent company and Thermo Fisher company of U.S. LCQ DecaXPElectrospray ionization mass spectrum composition, data are adopted
Integrate with processing software as Xcalibur 1.3；The triple level four bars mass spectrometry systems of liquid chromatogram-are by U.S. Thermo Fisher
Company's U3000 liquid phase and TSQ Quantum ACCESS MAX mass spectrum composition, data acquisition and procession software are Xcalibur
1.3。
1, submucous layer of small intestine substrate degradation is tested
Static state enzymatic hydrolysis：Weigh 100mg submucous layer of small intestine matrix, be added 100mL buffer (50mM Tris-HCl,
0.36mM CaCl2, pH7.4), 10min is handled in 100 DEG C of thermal denaturations, is down to the collagenase solution (1mg/ of addition 1mL after room temperature
ML Hank's balanced salt solution (HBSS) buffer), 37 DEG C of water-baths are shaken to without visible solid content, and supernatant is collected after centrifugation.
It digests in batches：Weigh 100mg submucous layer of small intestine matrix, be added 8mL buffer (50mM Tris-HCl,
0.36mM CaCl2, pH7.4), 100 DEG C of thermal denaturations handle 10min, are down to after room temperature and the collagenase solution (1mg/ of 200 μ L is added
ML HBSS buffer), 37 DEG C of enzymatic hydrolysis 1h, 10000rpm are centrifuged 3min, take out supernatant, 200 μ L collagenase solutions are added in precipitating
With 8mL buffer, continues 37 DEG C and digest 1 hour, repeat the above process 5 times, merge supernatant.
2, trypsin digestion is tested
The degraded by collagenase product of submucous layer of small intestine matrix is freeze-dried, 50mM NH is used4HCO3Solution (pH
8.0) it is configured to the solution of 1mg/mL, trypsin solution (1mg/mL, the 50mM NH of 20 μ L is added4HCO3, pH 8.0), 37 DEG C
12h is digested, enzymolysis product directly carries out HPLC-MS detection.
3, amino acid composition analysis
Method for hydrolysis reference literature " the acid-soluble collagen extraction of wall of sea cucumber Stichopus japonicus of submucous layer of small intestine matrix and its enzymolysis product
And amino acid composition analysis " (Gao Yang, Dong Xiuping, Xiao Guihua, Jiang Huimin, Zhu Beiwei,《Food and fermentation industries》,2008,34
(11):The method recorded in 171-174).
Amino acid composition analysis：Chromatographic column is Zorbax C18 [150mm × 3.0mm (I.D.), 5 μm]；Mobile phase A is
0.05M sodium acetate solution, B are 50% acetonitrile；0~15min of gradient 30~55%B, 15~25min 55~100%B, 25~
100~30%B of 35min, 5 μ L of sample volume；UV is 360nm；Flow velocity 1mL/min.
4, HPLC-MS is analyzed
Chromatographic column is Zorbax SB C18 [150mm × 2.1mm (I.D.), 5 μm]；Mobile phase A is that water (contains 0.1% first
Acid), B is acetonitrile (containing 0.1% formic acid)；0~80min of gradient, 5%~50% acetonitrile；80~90min, 50%~90% acetonitrile,
50 μ L of sample volume；UV is 214nm and 254nm；Flow velocity 0.2mL/min.Mass Spectrometry Conditions：Ion source spray voltage 4.5kV, capillary
300 DEG C of temperature, the scanning of scanning range m/z 957-958. accurate mass number and second order ms scanning (MS/MS) are data dependence
Type scanning, MS/MS collision energy are 35%.
Triple quadrupole bar Mass Spectrometry Conditions：Ion source spray voltage 3.5kV, 300 DEG C of capillary temperature, evaporating temperature：200
DEG C, shell gas：35arb assists gas：8psi, scanning mode：Salbutamol Selected Ion Monitoring：M/z 957.5, sweep length：1.0.
It is gradually decreased using acellular matrix residual quantity in solution after static enzymatic isolation method addition clostridiopetidase A, digests 6h degradation rate
Reach 40%, rear degradation rate is 60% for 24 hours, and degradation still has 10% acellular matrix not to be degraded after 3 days, residual quantity after 4 days
It tends towards stability, the floccule that cannot be degraded, about 5% (Fig. 1) is remained in solution.Amino acid analysis the result shows that, remain owner
To become amino acid and some non-amino acid ingredients such as aspartic acid, glutamic acid, glycine, alanine, proline, leucine,
Hydroxyproline is not almost detected in residue, shows that the collagen component in acellular matrix is effectively degraded.It should but use
Method degrade acellular matrix process time-consuming, and there are easy microbiological contamination, enzyme inactivation and random degenerate rate it is high the deficiencies of.In order to
Enzymolysis efficiency is improved, the mode degraded in batches has been used, that is, has degraded and be separated by solid-liquid separation after a certain period of time, sediment enzyme again
Solution.Acellular matrix residual quantity change in preceding degradation process three times it is more apparent, when changing liquid for the first time residual quantity be 70%, second
Secondary residual quantity when changing liquid is 50%, and residual quantity is about 30% (Fig. 1) when changing liquid for the third time, which takes off cell base after repeating five times
Matter enzymatic hydrolysis is more complete.In order to utmostly reduce loss of proteins in sample, number of degrading in degradation process in batches is tested
It extends to 6 times.
Due to there is the binding site affine with collagen height on FN, collagen is first digested by it using clostridiopetidase A
Afterwards, then trypsase is used to digest FN for polypeptide, for determining the feature polypeptide of fibronectin.
Fibronectin generates a large amount of low molecular weight polypeptides after the pure trypsin treatment of sequence.Meet quantitative detection
Polypeptide should be met certain condition：Experiment carries out BLAST Multiple Sequence Alignment to polypeptide first, is only detected in fibronectin
Peptide fragment is the feature polypeptide of fibronectin；Polypeptide Information in Mass Spectra after enzymatic hydrolysis carries out database search with SEQUEST software,
The related coefficient Xcorr of mass spectrum and theoretical mass spectra is tested when 1 charge of polypeptid belt>1.5, Xcorr when 2 charges of band>2.0
Xcorr when 3 charges of band>2.5, while Deltacn>0.1, then generated peptide fragment is considered as positive findings；In HPLC-MS
The ion flow graph that feature polypeptide is extracted in the mass spectrogram of analysis, can extract low concentration sample and its abundance is higher, while noise
Compare S/N>10, that is, it is suitable as feature polypeptide.
Fibronectin after trypsin digestion is subjected to full scan analysis with ion trap mass spectrometry (LCQ), Fig. 2 is fibre
The total ion current figure of dimension connection proteolysis polypeptide.Polypeptide segment after enzymatic hydrolysis is subjected to BLAST Multiple Sequence Alignment, the results showed that fine
There are a certain number of feature polypeptides for dimension connection albumen.Using whole fibronectins as database, fibronectin is dropped
Polypeptide Information in Mass Spectra after solution carries out database search with Bioworks software, screens the polypeptide segment with positive findings, according to
Blast Multiple Sequence Alignment and Bioworks software search library, determine that the feature polypeptide of fibronectin is
FTQVTPTSLTAQWTAPNVQLTGYR、EINLAPDSSSVVVSGLMVATK、DTLTSR、PAQGVVTTLENVSPPR、
VTDATETTITISWR、SYTITGLQPGTDYK、SSPVVIDASTAIDAPSNLR、FLATTPNSLLVSWQPPR、PGSPPR、
EVVPR, NNQK or SEPLIGR
Fig. 3 is that the extraction ion flow graph of polypeptide SSPVVIDASTAIDAPSNLR and first mass spectrometric figure, Fig. 4 are characterized polypeptide
The second order ms figure of SSPVVIDASTAIDAPSNLR.Therefore, it is more as feature that polypeptide SSPVVIDASTAIDAPSNLR may be selected
Peptide, for the identification of Fibronectins type and quantitative detection.Take various concentration through being denaturalized the fibronectin of enzymolysis processing
Standard solution, the successively 50 μ L of sample introduction from low concentration to high concentration, by HPLC-MS quantitative detection condition analysis, monitoring objective from
Sub- m/z=957.5 obtains the extraction ion stream of various concentration fibronectin feature polypeptide SSPVVIDASTAIDAPSNLR, ginseng
See Fig. 5, wherein in A, B, C, D, E the concentration of fibronectin be respectively 0.0024,0.0048,0.0096,0.0192,
0.048mg/mL.Extracting ion flow graph peak area as ordinate, fibronectin standard concentration using selected feature polypeptide is
Abscissa carries out linear regression, as a result sees Fig. 6.Regression equation is y=4109.5x-12.82 (R2=0.995), 0.0024~
Linear relationship is good both in 0.048g/L concentration range.
Further determine that FN content in acellular matrix.It weighs 50mg acellular matrix and 5 enzymatic hydrolysis of clostridiopetidase A point is added, close
And the supernatant of 5 enzymatic hydrolysis, freeze-drying obtain 123mg lyophilized products (containing the salts substances in extracting solution), after taking 4mg to be lyophilized
Product redissolve, using trypsin digestion, enzymolysis product is more by feature in FN according to HPLC-MS quantitative detection condition analysis
The extraction ion concentration of peptide SSPVVIDASTAIDAPSNLR is 0.007mg/mL, calculates containing for FN in acellular matrix
Amount is 0.43%.
The range of linearity：Same sample is diluted to different concentration, respectively 2.4 μ g/mL, 4.8 μ g/mL, 9.6 μ g/mL,
19.2 μ g/mL, 48 μ g/mL, 96 μ g/mL, 192 μ g/mL, detect its fibronectin content by HPLC-MS, by concentration ladder
Degree and peak area do curve, determine the range of the linear concentration of peak area.Many experiments the result shows that, this method is in 2.4 μ g/
It is linear between the μ of mL~192 g/mL.
Precision：100 μ L of FN standard items accurately is weighed, is analyzed by quantitative HPLC-MS condition, is repeated sample introduction five times,
As a result the peak area relative deviation RSD of feature polypeptide SSPVVIDASTAIDAPSNLR is 2.58% in FN standard items, the results showed that
Method precision is good.
Fig. 7 and Fig. 8 is characterized the extraction ion flow graph and first mass spectrometric of polypeptide EINLAPDSSSVVVSGLMVATK respectively
Figure.Using EINLAPDSSSVVVSGLMVATK peptide fragment as feature polypeptide, fibronectin content is detected in the following manner：
(1) it pre-processes：The acellular matrix for taking 200mg is cut into the sheet of size about 0.5cm × 0.5cm, is placed in low
In warm pulverizer, liquid nitrogen is added, is crushed under the conditions of crushing temperature lower than -100 DEG C, powder diameter controlling value is at 100 microns.
(2) 1 is digested：100mg acellular matrix powder is accurately weighed, (50mM/L Tris- is added in 8ml buffer
HCl, 0.36mmol/L CaCl2, pH7.4), it is heated to 100 DEG C and keeps 10min, the clostridiopetidase A that 2mg is added after cooling (contains
80%I Collagenase Type and 20% type III clostridiopetidase A), be centrifuged 3min in 37 DEG C of oscillation treatment 1h, 10000rpm, take out supernatant
2mg clostridiopetidase A and 8mL buffer is added in liquid, precipitating, continues 37 DEG C and digests 1 hour, repeat the above process 5 times, merges supernatant.
(3) 2 are digested：The pH value of solution that step (2) obtains is adjusted to 8.0 using the NaOH solution of 0.05mol/L, is added
1mg trypsase, in 37 DEG C of reaction 16h.
(4) Mass Spectrometer Method：The enzymatic hydrolysis solution that step (3) obtain is subjected to nitrogen and blows method drying, after accurately weighing 1mg drying
Powder is dissolved in the NH that 1ml concentration is 0.5mol/L4HCO3Solution is directly analyzed by mass spectrometry, and detection fiber links protease
The corresponding band double-charge ion m/z1059 of feature polypeptide EINLAPDSSSVVVSGLMVATK in product is solved, peak area is
1.1×105, it is to compare and make matched curve, mass spectrography with the trypsin digestion product of the FN standard solution of 1mg/ml
The corresponding band double-charge ion m/z1059 peak area of feature polypeptide EINLAPDSSSVVVSGLMVATK is detected, extracellular base is measured
The content of fiber link albumen is 0.48% in matter.
Similarly, referring to attached drawing, wherein Fig. 9 and Figure 10 is characterized mentioning for polypeptide FTQVTPTSLTAQWTAPNVQLTGYR respectively
Take ion flow graph and first mass spectrometric figure；Figure 11 and Figure 12 is characterized the extraction ion stream of polypeptide FLATTPNSLLVSWQPPR respectively
Figure and first mass spectrometric figure；Figure 13 and Figure 14 is characterized the extraction ion flow graph and level-one matter of polypeptide PAQGVVTTLENVSPPR respectively
Spectrogram；Figure 15 and Figure 16 is characterized the extraction ion flow graph and first mass spectrometric figure of polypeptide DTLTSR respectively.Connected by enzymatic hydrolysis fiber
It connects protein standard object and draws concentration gradient and peak area matched curve for features described above polypeptide；To acellular matrix enzyme twice
Solution, mass spectrography detect concentration one or more in features described above polypeptide, to accurately measure cell connection in acellular matrix
The content of albumen.
The present invention is realized by the method for secondary enzymolysis and detects fibronectin in acellular matrix using mass-spectrometric technique
The problem of Bai Hanliang.Furthermore further using in batches enzymatic hydrolysis and LC-MS technology to FN content in acellular matrix material into
Quantitative detection is gone.It is optimized for the clostridiopetidase A enzymolysis scheme of SIS, it is long to eliminate the time, the drawbacks such as low efficiency, by collagen
Enzyme enzymolysis time is reduced to 6h by 96h, greatly increases enzymolysis efficiency.FN obtains multiple feature peptide fragments through enzymatic hydrolysis in experiment, can
For detecting the content of fibronectin.This method is easy to operate, and suitable for tissue FN detection.
In addition, it is based on above-mentioned enzyme solution, it can be using enzyme used in fibronectin reference substance and stepwise discretization
It is made as kit.Kit is combined with mass spectrometer to be used for quantitative detection acellular matrix fibronectin to be measured
White content.The reference substance of features described above polypeptide can also be directlyed adopt and digest enzyme used twice as kit.It will
Kit is combined with mass spectrometer with the content for quantitative detection acellular matrix fibronectin to be measured.
The present invention obtains all polypeptide sequences of fibronectin by the pretreatment of acellular matrix, stepwise discretization method
Feature polypeptide sequence in column mixture, then screen fibre connection albumen, detects feature content of peptides by mass spectrography, to survey
Make fibronectin content in acellular matrix.This method solve be not available mass spectrography quantitative detection in the past to take off cell
In matrix the problem of tectotype fibronectin content；Further, acellular matrix is removed by way of stepwise discretization
Middle collagen component, the interference for reducing the non-targeted polypeptide sequence of test process improve work to save the testing time
Make efficiency, detection accuracy and stability.
Claims (13)
1. it is a kind of detection acellular matrix in fibronectin content method, which is characterized in that the detection method include with
Lower step：
(1) it pre-processes：The acellular matrix for weighing constant weight is cut into the small pieces that size is less than 1cm × 1cm, in liquid nitrogen
Powder is ground under protective condition, powder size is controlled at 50-300 μm；
(2) 1 is digested：Acellular matrix powder after a certain amount of crushing is taken, 500-10000 times of acellular matrix powder weight is added to
Buffer the clostridiopetidase A of acellular matrix weight 1/200~1/50 is added after cooling in 100 DEG C of heat treatment 5-10min, in
37 DEG C of oscillation treatment 2-24h；
(3) 2 are digested：The pH value of solution that step (2) obtain is adjusted to 7.5-8.5, non-collagen albuminoid enzyme, non-collagen class egg is added
The additional amount of white enzyme is the 1/200~1/50 of acellular matrix weight, in 37 DEG C of reaction 8-24h；
(4) Mass Spectrometer Method：The enzymatic hydrolysis solution that step (3) obtain is concentrated, concentrate is directly analyzed by mass spectrometry, with known
The enzymolysis product of the fibronectin standard solution of concentration is control, detects feature polypeptide, by measuring acellular matrix
Fibronectin feature polypeptide calculates the content of fibronectin in acellular matrix in catabolite.
2. the method for fibronectin content, feature exist in a kind of detection acellular matrix according to claim 1
In clostridiopetidase A used in step (2) enzymolysis process is the mixed of I type, II type, type III, V-type or different collagen enzyme
Object is closed, additional amount is the 1/200~1/50 of acellular matrix weight.
3. the method for fibronectin content, feature exist in a kind of detection acellular matrix according to claim 1
In protease used in step (3) enzymolysis process is trypsase, and additional amount is the 1/200 of acellular matrix weight
~1/50.
4. the method for fibronectin content, feature exist in a kind of detection acellular matrix according to claim 1
In protease used in step (3) enzymolysis process is chemically modified trypsase, and additional amount is acellular matrix
The 1/200~1/50 of weight.
5. the method for fibronectin content, feature exist in a kind of detection acellular matrix according to claim 1
In protease used in step (3) enzymolysis process is the trypsase of immobilization, and additional amount is acellular matrix weight
1/200~1/50.
6. the method for fibronectin content, feature exist in a kind of detection acellular matrix according to claim 1
In the feature polypeptide in the step (4) for detecting fibronectin in acellular matrix includes：
FTQVTPTSLTAQWTAPNVQLTGYR、EINLAPDSSSVVVSGLMVATK、DTLTSR、PAQGVVTTLENVSPPR、
VTDATETTITISWR、SYTITGLQPGTDYK、SSPVVIDASTAIDAPSNLR、FLATTPNSLLVSWQPPR、PGSPPR、
One of EVVPR, NNQK or SEPLIGR or a variety of.
7. a kind of feature polypeptide of fibronectin, which is characterized in that the fibronectin is by as claimed in claim 1
Step (1), (2) and (3) can obtain the feature polypeptide, and the feature polypeptide can be used in detecting the fibronectin
Content；The feature polypeptide is：FTQVTPTSLTAQWTAPNVQLTGYR, DTLTSR, PGSPPR or EVVPR.
8. a kind of detection method of fibronectin, which is characterized in that the detection method uses as claimed in claim 7
Feature polypeptide.
9. a kind of for detecting the kit of fibronectin, which is characterized in that including one or more enzymes；It is described a kind of or
A variety of enzymes can digest fibronectin, and generate feature polypeptide as claimed in claim 7；
The kit further includes the standard items of the feature polypeptide according to claim 7.
10. according to the kit for being used to detect fibronectin in claim 9, which is characterized in that described one or more
Enzyme includes：Clostridiopetidase A and protease；The clostridiopetidase A is I type, II type, type III or collagen type v enzyme；The protease is pancreas egg
White enzyme.
11. according to the kit for being used to detect fibronectin in claim 9, which is characterized in that described one or more
Enzyme includes：Clostridiopetidase A and protease；The clostridiopetidase A is I type, II type, type III or collagen type v enzyme；The protease is chemistry
Trypsase after modification.
12. according to the kit for being used to detect fibronectin in claim 9, which is characterized in that described one or more
Enzyme includes：Clostridiopetidase A and protease；The clostridiopetidase A is I type, II type, type III or collagen type v enzyme；The protease is to fix
The trypsase of change.
13. a kind of feature polypeptide as claimed in claim 7 detects, in quality testing or quality control in fibronectin
Using.
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