CN106771230A - Using the method for ADAMTS13 enzymatic activitys in SELDI TOF MS detection serum - Google Patents
Using the method for ADAMTS13 enzymatic activitys in SELDI TOF MS detection serum Download PDFInfo
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- CN106771230A CN106771230A CN201611038248.2A CN201611038248A CN106771230A CN 106771230 A CN106771230 A CN 106771230A CN 201611038248 A CN201611038248 A CN 201611038248A CN 106771230 A CN106771230 A CN 106771230A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
- G01N33/6851—Methods of protein analysis involving laser desorption ionisation mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention discloses the method for ADAMTS13 enzymatic activitys in a kind of utilization SELDI TOF MS detection serum, comprise the following steps：(1) expression and purification of ADAMTS13 digestions substrate GST vWF73 6XHis recombinant proteins；(2) endonuclease reaction；(3) by SELDI TOF MS recognition detections after ADAMTS13 digestion products are enriched with by IMAC chips, and the digestion activity of ADAMTS13 in serum sample is obtained by analysis software.Rapidly effectively, detection is accurate for detection method；All TMs (TMA) suspected case is detected, case of all ADAMTS13 enzymatic activitys less than 10% is filtered out, these patients will be treated as thrombotic thrombocytopenic purpura (TTP).For all TTP cases over the course for the treatment of and in later long-term monitoring of leaving hospital, the enzymatic activity of periodic detection ADAMTS13 is all needed, so as to preferably correctly be treated to patient, reduce treatment cost, effectively improve the survival rate and quality of life of patient.
Description
Technical field
The invention belongs to Medical Molecular Biology, polypeptide detection technique field, more particularly to one kind can be utilized
SELDI-TOF-MS is applied to the TTP diseases sieve in TMA diseases come the method for detecting metalloproteinases ADAMTS13 enzymatic activitys
Look into, and TTP disease treatments monitoring.
Background technology
SELDI-TOF-MS (surface enhanced laser desorption ionization flight time mass spectrum) is by Ciphergen companies of the U.S.
The new mass spectrometer developed, it is mainly made up of three parts：Part I is protein-chip (Protein Chip),
It is its core technology；Part II is time of-flight mass spectrometer (TOF MS), it is also possible to referred to as chip-reader (protein
chip reader)；Part III is system-specific analysis software.
Protein-chip is the core technology of SELDI-TOF-MS, and this detection method will be using the affine absorption chip of metal
(Immobilized Metal Affinity Capture, IMAC), IMAC chip surfaces can chelate positive bivalent metal ion
Ni, can be with specific protein and peptide of the capture with histidine group.
Chip-reader, is exactly MALDI-TOF-MS instrument.It is radiated by laser pulse and turns sample
Turn to the gas ion of motion and separated by matter lotus (M/Z) size, the flight time is long in the electric field according to different mass-to-charge ratioes
It is short to differ, so as to draw out a mass spectrogram.
System-specific analysis software is used for processing the data obtained by experiment.It is analyzed by given data, from
And find there is the peak change for differentiating meaning.
SELDI-TOF-MS technical operations step can be divided into the following steps：
1st, the analyzed direct point sample of sample is reacted to chip array, and the polypeptide in sample is specifically bound to
Reacted on chip；
2nd, chip is rinsed, and the small-molecule substance of uncombined and non-specific binding is washed away, so that in removing sample
" noise ", reduces background；
3rd, energy absorbing substance (energy absorbing molecule, EAM) is added on chip, when laser irradiates core
During piece, the energy of laser can be converted into heat energy by it；
4th, after the polypeptide energy absorption in sample, it is desorbed and ionizes, ion is flown to by vacuum tube and is examined
Device is surveyed, such mass-to-charge ratio its flight time of difference is different, so different polypeptides can be separated according to mass-to-charge ratio.
TM (thrombotic microangiopathy, TMA) is one group has common pathological characters
Actual clinical pathological syndrome, be divided into heredity and acquired, be mainly shown as endotheliocytic swelling come off, subendothelial fine hair
Shape electrodeposition substance and Endovascular platelet aggregation form the Microvasculatures such as microthrombus, Endovascular embolism and plasmorrhysis
It is abnormal.
TMA is clinically mainly shown as decrease of platelet, hemolytic anemia and the device that platelet thrombus is caused in microcirculation
Official is involved, the extent of disease of its clinical manifestation and TMA and to involve the dysfunction that Different Organs cause relevant.
Widely, patient is often possible in different section office, such as kidney internal medicine, blood for the clinical department that the disease is related to
Section, Neurology, gynemetrics, dept. of dermatology, Internal Medicine-Cardiovascular Dept. and division of respiratory disease etc. are gone to a doctor, and the doctor that such as sees and treat patients shortage is recognized the disease
Know, often cause it is serious fail to pinpoint a disease in diagnosis by mistake, therefore improve has turned at present that relevant speciality is led both at home and abroad to the cognition degree of such disease
The focus of attention in domain.
TMA mainly includes two class diseases：Thrombotic thrombocytopenic purpura (thrombotic throm-
Bocytopenic purpura, TTP), hemolytic uremic syndrome (hemolyticuremlc syndrome, HUS).This
The clinical manifestation of two kinds of diseases is closely similar, but the difference that both have a maximum is exactly metalloproteinases (ADAMTS13)
Enzymatic activity has significant difference.The ADAMTS13 enzymatic activitys of either heredity or acquired patient TTP are generally less than
10%, and the morbidity of HUS is with ADAMTS13 enzymatic activitys missing, and it doesn't matter, so the ADAMTS13 enzymatic activitys of patient HUS are normal.
The content of the invention
It is an object of the invention to provide the method using ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, its
Timely and effective and accurate detection.
To achieve the above object, the present invention uses following technical scheme：
Using the method for ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, the technology will be applied to TTP diseases
Diagnosis and treatment monitoring, it is specific as follows：(1) expression of ADAMTS13 digestions substrate GST-vWF73-6XHis recombinant proteins
Purifying；(2) endonuclease reaction；(3) by SELDI-TOF-MS recognition detections after ADAMTS13 digestion products are enriched with by IMAC chips,
And the digestion activity of ADAMTS13 in serum sample is obtained by analysis software.
As shown in Figure 1：This technology vWF73 eggs that great expression is marked with 6 histidines (6XHis) in bacterium BL21
White tiles section, the fragment carries ADAMTS13 specific cleavage sites.Later protein fragments will be purified and be incubated one with patients serum
After the section time, the ADAMTS13 digestion products with 6XHis will be caught by IMAC protein chips and be enriched with, and then by SELDI-
The detection identification of TOF-MS instruments.After being contrasted with normal human serum, the digestion of the ADAMTS13 of the patient can be calculated
Activity.
After adopting the above technical scheme, the invention has the advantages that：
1st, the detection method is effective rapidly, and detection is accurate；
2nd, all TM (TMA) suspected cases are detected, all ADAMTS13 enzymatic activitys is filtered out and is less than
10% case, these patients will be treated as thrombotic thrombocytopenic purpura (TTP).It is more than for enzymatic activity
10% case will carry out other further diagnosis, and start different treatment completely；
3rd, periodic detection is all needed over the course for the treatment of and in later long-term monitoring of leaving hospital for all TTP cases
The enzymatic activity of ADAMTS13, so as to preferably correctly be treated to patient, reduces treatment cost, effectively improves patient's
Survival rate and quality of life.
Brief description of the drawings
Fig. 1 is step schematic diagram of the invention.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The invention discloses the method using ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, including following step
Suddenly：
Comprise the following steps：
(1) expression and purification of ADAMTS13 digestions substrate GST-vWF73-6XHis recombinant proteins：
1.1.vWF 6 histidines (6XHis) of 73 amino acid fragment D1596~R1668 and C-terminal are inserted into N
On bacterial expression vector pGEX6p-1 of the end with GST albumen；
1.2. positive colony is selected, and transfects BL21 expression bacteriums；
The BL21 strains can be with high efficient expression purpose recombinant protein, and the albumen is soluble protein, it is easy to purify,
GST labels are easily sheared simultaneously.
1.3. mass propgation bacterium, collects thalline, bacteria lysis are carried out using ultrasound, and supernatant is collected after high speed centrifugation,
And add isometric PBS to dilute；
1.4. by GST affinity column purifying proteins, eluted using reductive glutathione, so as to the weight for being purified
The digestion substrate that histone GST-vWF73-6XHis needs to use as this technology；
1.5. take a certain amount of GST-vWF73-6XHis and scinderin enzyme Prescission Protease to be incubated, obtain
To the internal reference (internal control, IC) that this technology needs to use in detection-phase；
(2) endonuclease reaction：
Need to use following reagent and instrument：One group of serum standard, mixing human normal plasma (pooled normal
Plasma, PNP) 100%, 50%, 20%, 10%, 5% and 2.5% is serially diluted into, for the solution formula that dilutes such as
Under：150mM NaCl, 0.1%BSA, 1XPBS；
Height is compareed, and PNP is diluted into 50% and 5% compares as height；
Reaction buffer：50mM Tris-Cl pH8.0,1mM BaCl2, 50mM NaCl；
Chip activates buffer solution：50mM NiSO4；
Chip combination buffer：1XPBS；
Chip elution buffer：1XPBS, 300mM NaCl, 0.1%Tween20；The buffer solution of living again can be effective
Wash away with reference to the polypeptide on IMAC chips, and do not influence the binding ability of chip.
Energy absorbing substance (EAM)：SPA (5mg is dissolved in 200ul 50%ACN, 200ul 0.5%TFA).
IMAC chips are lived again buffer solution：50mM EDTA, 50%ACN, 0.5%TFA.
Pure water；
Concussion instrument；
Various model liquid feeding rifles.
Digestion substrate GST-vWF73-6XHis reaction buffers are diluted to 0.1ug/ul by 2.1, take 10ul serum standards
Substrate solution after thing, height control and patients serum's sample dilute with 25ul mixes；
2.2 after 37 degree of water-baths are incubated 1 hour, are heated to 95 degree of terminating reactions.At the same time internal reference is configured, will
Internal reference is diluted to 0.01ug/ul with PBS；
(3) by SELDI-TOF-MS recognition detections after ADAMTS13 digestion products are enriched with by IMAC chips, and by analyzing
Software obtains the digestion activity of ADAMTS13 in serum sample：
3.1IMAC chips are activated, and take the IMAC chips of needs, add 100ul activation buffer solutions, oscillation incubation 5 minutes two
It is secondary；
3.2 pure washing chips add combination buffer after twice, oscillation incubation 5 minutes is twice；
3.3 suck residual liquid on chip；
3.4 mixing 35ul internal references shine endonuclease reaction system, and vibration mixes, takes 60ul and be added on chip, oscillation incubation 30
Minute；
3.5 elution buffers are eluted 3 times, 5 minutes every time, are finally washed with water twice；
3.6 take out chip, carefully blot residual solution, and each chip point adds 1ulSPA, are put once again after air-drying；
3.7 feeding SELDI instruments carry out chip reading；
3.8 software analysis purpose peaks and internal reference peak area, using purpose peak divided by internal reference peak area value as X
Axle, a curve is done by Y-axis of standard serum concentration, calculated by peak area its digestion activity according to patient.
3.9 used IMAC chips are processed 30 minutes with buffer solution of living again, and are then rinsed repeatedly with pure water, after drying
It is standby.
The affine absorption chip IMAC surfaces of metal can chelate positive bivalent metal ion Ni, can be with specific capture
Protein and peptide with histidine group.
More than, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto, any
Those familiar with the art the invention discloses technical scope in, the change or replacement that can be readily occurred in, all should
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be defined by scope of the claims.
Claims (5)
1. using the method for ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, it is characterised in that：Comprise the following steps：
(1) expression and purification of ADAMTS13 digestions substrate GST-vWF73-6XHis recombinant proteins；
(2) endonuclease reaction；
(3) by SELDI-TOF-MS recognition detections after ADAMTS13 digestion products are enriched with by IMAC chips, and by analysis software
Obtain the digestion activity of ADAMTS13 in serum sample.
2. as claimed in claim 1 using the method for ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, its feature
It is：Comprise the following steps：
(1) expression and purification of ADAMTS13 digestions substrate GST-vWF73-6XHis recombinant proteins：
1.1.vWF 6 histidines (6XHis) of 73 amino acid fragment D1596~R1668 and C-terminal are inserted into N-terminal band
Have on the bacterial expression vector pGEX6p-1 of GST albumen；
1.2. positive colony is selected, and transfects BL21 expression bacteriums；
1.3. mass propgation bacterium, collects thalline, bacteria lysis are carried out using ultrasound, supernatant are collected after high speed centrifugation, and add
Enter isometric PBS dilution；
1.4. by GST affinity column purifying proteins, eluted using reductive glutathione, so as to the restructuring egg for being purified
The digestion substrate that white GST-vWF73-6XHis needs to use as this technology；
1.5. take a certain amount of GST-vWF73-6XHis and scinderin enzyme Prescission Protease to be incubated, obtain this
The internal reference that technology needs to use in detection-phase；
(2) endonuclease reaction：
Need to use following reagent and instrument：
One group of serum standard, mixing human normal plasma be serially diluted into 100%, 50%, 20%, 10%, 5% and
2.5%, the solution formula for diluting is as follows：150mM NaCl, 0.1%BSA, 1XPBS；
Height is compareed, and PNP is diluted into 50% and 5% compares as height；
Reaction buffer：50mM Tris-Cl pH8.0,1mM BaCl2, 50mM NaCl；
Chip activates buffer solution：50mM NiSO4；
Chip combination buffer：1XPBS；
Chip elution buffer：1XPBS, 300mM NaCl, 0.1%Tween20；
Energy absorbing substance EAM：SPA (5mg is dissolved in 200ul 50%ACN, 200ul 0.5%TFA)；
IMAC chips are lived again buffer solution：50mM EDTA, 50%ACN, 0.5%TFA；
Pure water；
Concussion instrument；
Various model liquid feeding rifles；
Digestion substrate GST-vWF73-6XHis reaction buffers are diluted to 0.1ug/ul by 2.1, take 10ul serum standards,
Substrate solution after height control and patients serum's sample dilute with 25ul mixes；
2.2 after 37 degree of water-baths are incubated 1 hour, are heated to 95 degree of terminating reactions；At the same time internal reference is configured, by internal reference
0.01ug/ul is diluted to according to PBS；
(3) by SELDI-TOF-MS recognition detections after ADAMTS13 digestion products are enriched with by IMAC chips, and by analysis software
Obtain the digestion activity of ADAMTS13 in serum sample：
3.1 IMAC chips are activated, and take the IMAC chips of needs, add 100ul activation buffer solutions, and oscillation incubation 5 minutes is twice；
3.2 pure washing chips add combination buffer after twice, oscillation incubation 5 minutes is twice；
3.3 suck residual liquid on chip；
3.4 mixing 35ul internal references shine endonuclease reaction system, and vibration mixes, takes 60ul and be added on chip, oscillation incubation 30 minutes；
3.5 elution buffers are eluted 3 times, 5 minutes every time, are finally washed with water twice；
3.6 take out chip, carefully blot residual solution, and each chip point adds 1ulSPA, are put once again after air-drying；
3.7 feeding SELDI instruments carry out chip reading；
3.8 software analysis purpose peaks and internal reference peak area, using purpose peak divided by internal reference peak area value as X-axis, with
Standard serum concentration does a curve for Y-axis, calculated by peak area its digestion activity according to patient；
3.9 used IMAC chips are processed 30 minutes with buffer solution of living again, and are then rinsed repeatedly with pure water, are dried with standby
With.
3. as claimed in claim 2 using the method for ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, its feature
It is：The BL21 strains can be with high efficient expression purpose recombinant protein, and the albumen is soluble protein, it is easy to purify, together
When GST labels be easily sheared.
4. as claimed in claim 2 using the method for ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, its feature
It is：The affine absorption chip IMAC surfaces of metal can chelate positive bivalent metal ion Ni, can be with specific capture zone
There is the protein and peptide of histidine group.
5. as claimed in claim 2 using the method for ADAMTS13 enzymatic activitys in SELDI-TOF-MS detection serum, its feature
It is：The buffer solution of living again can be washed away effectively with reference to the polypeptide on IMAC chips, and does not influence chip
Binding ability.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110627913A (en) * | 2019-11-06 | 2019-12-31 | 浙江壹晨生物科技有限公司 | ADAMTS13 substrate with lysine tag and preparation method and application thereof |
| CN110702922A (en) * | 2019-10-18 | 2020-01-17 | 晏妮 | Regeneration liquid formula of IMAC chip |
| CN110724202A (en) * | 2019-11-06 | 2020-01-24 | 浙江壹晨生物科技有限公司 | ADAMTS13 substrate with histidine tag as well as preparation method and application thereof |
| CN110760563A (en) * | 2019-10-10 | 2020-02-07 | 北京东西分析仪器有限公司 | Method for measuring enzyme activity |
| US10858689B2 (en) | 2016-10-11 | 2020-12-08 | Laboratory Corporation Of America Holdings | Methods and systems for determining ADAMTS13 enzyme activity |
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| US20080206787A1 (en) * | 2006-02-27 | 2008-08-28 | Wu Haifeng M | Process to determine enzyme activity |
| CN105384818A (en) * | 2015-12-17 | 2016-03-09 | 中国药科大学 | Anti-human Delta like 4 monoclonal antibody and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10858689B2 (en) | 2016-10-11 | 2020-12-08 | Laboratory Corporation Of America Holdings | Methods and systems for determining ADAMTS13 enzyme activity |
| US11959124B2 (en) | 2016-10-11 | 2024-04-16 | Laboratory Corporation Of America Holdings | Methods and systems for determining ADAMTS13 enzyme activity |
| CN110760563A (en) * | 2019-10-10 | 2020-02-07 | 北京东西分析仪器有限公司 | Method for measuring enzyme activity |
| CN110702922A (en) * | 2019-10-18 | 2020-01-17 | 晏妮 | Regeneration liquid formula of IMAC chip |
| CN110627913A (en) * | 2019-11-06 | 2019-12-31 | 浙江壹晨生物科技有限公司 | ADAMTS13 substrate with lysine tag and preparation method and application thereof |
| CN110724202A (en) * | 2019-11-06 | 2020-01-24 | 浙江壹晨生物科技有限公司 | ADAMTS13 substrate with histidine tag as well as preparation method and application thereof |
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