CN104251849B - The application of the formic hydrazide of 4H [1] chromene [4,3 b] thiophene 2 and its derivative in the detection of glycoprotein specificity fluorescent - Google Patents
The application of the formic hydrazide of 4H [1] chromene [4,3 b] thiophene 2 and its derivative in the detection of glycoprotein specificity fluorescent Download PDFInfo
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Abstract
The present invention relates to glycoprotein specificity fluorescent detection technique, the application of the formic hydrazide of specifically 4H [1] chromene [4,3 b] thiophene 2 and its derivative in the detection of glycoprotein specificity fluorescent.Present invention also offers the method that the application formic hydrazide of 4H [1] chromene [4,3 b] thiophene 2 carries out glycoprotein specificity fluorescent detection, including step:Gel containing protein example after electrophoresis is fixed in fixer;Periodic acid solution oxide;Acetic acid aqueous solution is rinsed;Dyeing;Elution.The present invention has the advantages that sensitivity is high, selectivity it is good, simple to operate it is rapid, favorable reproducibility, linear relationship are good, mass spectrum is good, using safety, with low cost, research that can preferably suitable for high throughput protein group.
Description
Technical field
The present invention relates to glycoprotein specificity fluorescent detection technique, specifically 4H- [1]-chromene [4,3-b] thiophene
The application of fen -2- formic hydrazides and its derivative in the detection of glycoprotein specificity fluorescent.
Background technology
Modification of protein glycosylation has vital effect in life science field, as main and universal
One of protein post-translational modification, be currently known in mammalian proteins at least 1/2nd and glycosylated, this
A little glycoprotein are distributed widely in tissue, cell, body fluid, the rich content particularly in cell membrane surface, body fluid etc., glycosylation
Folding, transport and positioning for protein etc. all play an important role, and it is attached to participate in receptor activation, signal transduction, cell load
Etc. all more important bioprocess.The change of glycoprotein number change or sugar chain structure, may all cause disease.Not only mesh
The diagnosis marker of preceding known many diseases is glycoprotein, and in the medicine by international standard certification, glycoprotein is also accounted for
To higher ratio.
Current glycoprotein detection field kit focuses primarily upon the biological reagent company of foreign country, and most products are with height
The price of added value is in market sale, and price is high, is unfavorable for the development of biotechnology basic research.It is especially domestic using existing
Biological reagent carry out Bioexperiment cost and remain high, it is impossible to realize economic benefit and experiment joint development.Present invention exploitation
4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides and its sensitivity of derivatives fluorescent colouring method close to Pro-Q
Emerald488 decoration methods, are a kind of sensitive, convenient, special glycoprotein detection technique of fluorescences, there is preferably basis and clinic
Meaning.
The content of the invention
Exist it is an object of the invention to provide 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid acyl hydrazine and its derivative
Application in the detection of glycoprotein specificity fluorescent.
4H- [1] of the present invention-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides related compound refers to 4H-
[1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides anion is sodium salt, sylvite and ammonium salt of parent nucleus etc..
4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides parent nucleus is:
In order to realize the purpose of the present invention, present invention also offers apply 4H- [1]-chromene [4,3-b] thiophene -2-
The method that formic hydrazide carries out glycoprotein specificity fluorescent detection comprises the following steps:
1) by after SDS-PAGE electrophoresis the gel containing protein example be placed in fixer and fix 10~60min, abandon solid
Determine liquid.It is preferred that set time be 30min, fixer can be the aqueous solution containing 40% ethanol and 10% acetic acid;
2) 10~40min is aoxidized in periodic acid solution, its meso-periodic acid solution is containing w/v 0.1~1%
Periodic acid and by volume 0.5~5% acetic acid aqueous solution.Then 2 are rinsed with by volume 0.5~5% acetic acid aqueous solution
~5 times, every time 1~10min;It is preferred that oxidization time be 20min, preferably oxidation solution composition be containing w/v 0.5%
Periodic acid and 1% acetic acid aqueous solution by volume;
3) add 1~40min of dyeing liquor, wherein dyeing liquor be the 4H- [1] containing w/v 0.0001~0.001%-
Chromene [4,3-b] thiophene -2-carboxylic acid hydrazides or derivatives thereof and by volume 0.5~5% acetic acid aqueous solution.It is preferred that
Dyeing time is 10min, and dyeing liquor composition preferably is 4H- [1]-chromene [4,3- containing w/v 0.0002%
B] thiophene -2-carboxylic acid hydrazides and 1% acetic acid aqueous solution by volume;
4) add eluent, eluent composition for volume ratio 0.5~5.0% acetic acid, 10~70% ethanol and 0.5~
2% aqueous ethanolamine, is eluted 1~3 time, 1~10min every time.It is preferred that elution time be 20min, eluent group preferably
As 1.0% acetic acid by volume, 40% ethanol and 1% aqueous ethanolamine;
5) detect, the glycoprotein after dyeing can be observed under laser scanner.
Early-stage Study shows that the technology has the following advantages:
1) sensitivity is high:4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein specificity fluorescent detection
Method sensitivity is suitable with Pro-Q Emerald488 sensitivity;
2) it is simple to operate rapid:Operating procedure is few, can be completed within 2 hours;
3) favorable reproducibility:Influenceed smaller by external conditions such as temperature, shaking table hunting frequencies;
4) good reversibility:Easily decolourize;
5) mass spectrum is good:Due to 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides fluorescence detection not
Protein structure is influenceed, so can be with the mass spectrograph highly compatible such as MALDI-TOF;
6) using safety:Using the low dyestuff of toxicity, the security of experimental implementation is improved, environmental pollution is small;
7) cost is low.
Brief description of the drawings
The chemical constitution of Fig. 1 .4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides;
Fig. 2 are one on SDS-PAGE glue, and 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein is glimmering
Light decoration method detects the comparison of Protein standards effect with other decoration methods.(A) 4H- [1]-chromene [4,3-b] thiophene-
2- formic hydrazide glycoprotein fluorescence colours, (B) Pro-Q Emerald glycoprotein fluorescence colours, (C) SYPRO Ruby shell eggs
White fluorescence colour.Pro-Q Emerald glycoprotein fluorescence colours and SYPRO Ruby holoproteins fluorescence colour according to
Document records operation.8 kinds of various criterion protein of Sigma companies are used for sample:Transferrin (glycoprotein), BSA
(non-glycoprotein), IgG (glycoprotein), OVA (glycoprotein), α 1-acid glycoprotein (glycoprotein), α-casein (non-saccharide
Albumen), β-casein (non-glycoprotein), avidin (glycoprotein) represent glycoprotein in figure with italics.Band 1,1000ng;
Band 2,500ng;Band 3,250ng;Band 4,125ng;Band 5,64ng;Band 6,32ng;Band 7,16ng;Band 8,8ng;Band 9,4ng;Band
10,2ng.
Fig. 3 are one on SDS-PAGE glue, and 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein is glimmering
Light decoration method detects the comparison of actual sample effect with other colouring methods.(A) 4H- [1]-chromene [4,3-b] thiophene -2-
Formic hydrazide glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colours, (C) SYPRO Ruby holoproteins
Fluorescence colour.Pro-Q Emerald glycoprotein fluorescence colours and SYPRO Ruby holoproteins fluorescence colours are according to text
Offer record operation;The human serum total protein of extraction is used for sample.Band 1,5000ng;Band 2,2500ng;Band 3,1250ng;Band 4,
625ng;Band 5,312ng;Band 6,160ng;Band 7,80ng;Band 8,40ng;Band 9,20ng;Band 10,10ng.
Fig. 4 are one on SDS-PAGE glue, and 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein is glimmering
Light decoration method is investigated with other colouring methods for glyprotein stain is specific.(A) 4H- [1]-chromene [4,3-b] thiophene
Fen -2- formic hydrazide glycoprotein fluorescence colours, (B) Pro-Q Emerald glycoprotein fluorescence colours, (C) SYPRO Ruby
Holoprotein fluorescence colour.N- in standard protein α 1-acid glycoprotein and human serum total protein is removed with PNGase F
Chain sugar.Band 1, human serum total protein;Band 2, removes the sugared descendant's total serum protein of N- chains;Band 3, α 1-acid glycoprotein;Band
4, remove α l-acid glycoprotein after N- chains sugar;Band 5, PNGase F enzymes.
Fig. 5 are two on SDS-PAGE glue, and 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein is glimmering
Light decoration method detects the comparison of actual sample effect with other colouring methods.(A) 4H- [1]-chromene [4,3-b] thiophene -2-
Formic hydrazide glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colours, (C) SYPRO Ruby holoproteins
Fluorescence colour.Separation sample is rat liver total protein;IPG adhesive tape long 13cm, pH4-7;The concentration of separation gel is 11.4%;
Sample applied sample amount is 25 μ g/ adhesive tape.
Embodiment
The present invention is expanded on further with reference to specific embodiment.It should be appreciated that these embodiments are merely to illustrate
The present invention, and can not limit the scope of the invention.
The 4H- of experimental example 1 [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein specificity fluorescent dyeing
Fig. 1 is the chemical structural formula of 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides.
4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour is used as following step is entered
OK:
1) the protein example gel after SDS-PAGE electrophoresis is placed in 40% ethanol and 10% acetic acid aqueous solution solid
Determine 30min, abandon fixer;
2) aoxidize 20min in periodic acid solution, its meso-periodic acid solution for containing w/v 0.5% periodic acid and
1% acetic acid aqueous solution by volume.Then rinsed 3 times with by volume 1% acetic acid aqueous solution, each 5min;
3) dyeing liquor dyeing 10min is added, wherein dyeing liquor is 4H- [1]-benzo pyrrole containing w/v 0.0002%
Mutter [4,3-b] thiophene -2-carboxylic acid hydrazides and 1% acetic acid aqueous solution by volume;
4) eluent of the acetic acid containing volume ratio 1%, 40% ethanol and 1% aqueous ethanolamine is added, 20min is eluted;
5) detect, the albumen after dyeing can be observed under laser scanner.
Use the sodium salt, sylvite, ammonium of 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides respectively according to the method described above
Salt carries out protein staining, as a result shows be obtained being similar to 4H- [1]-chromene [4,3-b] thiophene with these derivatives
The testing result of fen -2- formic hydrazides.
The 4H- of experimental example 2 [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour and other dyes
Color method is contrasted to standard protein Detection results.
(A) 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q
Emerald glycoprotein fluorescence colours, (C) SYPRO Ruby holoprotein fluorescence colours.Pro-Q Emerald glycoprotein fluorescence
Decoration method and SYPRO Ruby holoproteins fluorescence colours are recorded according to document;Using 8 kinds of various criterion eggs of Sigma companies
White matter is sample.Band 1,1000ng;Band 2,500ng;Band 3,250ng;Band 4,125ng;Band 5,64ng;Band 6,32ng;Band 7,
16ng;Band 8,8ng;Band 9,4ng;Band 10,2ng.As a result as shown in Fig. 2 showing 4H- [1]-chromene [4,3-b] thiophene -2-
Formic hydrazide glycoprotein fluorescence colour detection sensitivity is close to Pro-Q Emerald488 glycoprotein fluorescence colours.
The 4H- of experimental example 3 [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour and other dyes
Color method is contrasted to human serum total protein Detection results.
(A) 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q
Emerald glycoprotein fluorescence colours, (C) SYPRO Ruby holoprotein fluorescence colours.Pro-Q Emerald glycoprotein fluorescence
Decoration method and SYPRO Ruby holoproteins fluorescence colour are recorded according to document and operated;Use the human serum total protein of extraction for
Sample.Band 1,5000ng;Band 2,2500ng;Band 3,1250ng;Band 4,625ng;Band 5,312ng;Band 6,160ng;Band 7,80ng;
Band 8,40ng;Band 9,20ng;Band 10,10ng.As a result as shown in figure 3, showing 4H- [1]-chromene [4,3-b] thiophene -2- first
Sour hydrazides glycoprotein fluorescence colour detection sensitivity is close to Pro-Q Emerald488 glycoprotein fluorescence colours.
The 4H- of experimental example 4 [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour and other dyes
Color method is contrasted to rat liver total protein Detection results.
Rat liver is taken, last, 0.02g packing of being pulverized in liquid nitrogen is added, the lysates of 500 μ 1 is added into every pipe,
Cell Ultrasonic Cell Disruptor crushes 3 times (1min/ times), centrifuges 15000g/min, 15min, takes part supernatant to be tried with Bradford
Agent box determines protein concentration, and remaining supernatant is sub-packed in Eppendorf pipes, and -80 DEG C preserve with standby.
After above-mentioned rat liver is separated through two to gel electrophoresis, (A) 4H- [1]-chromene [4,3-b] is respectively adopted
Thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colours, (C) SYPRO
Ruby holoprotein fluorescence colours are dyed, as a result as shown in Figure 4.Show 4H- [1]-chromene [4,3-b] thiophene -2-
Formic hydrazide glycoprotein fluorescence colour can detect the overwhelming majority that Pro-Q Emerald glycoprotein fluorescence colours are detected
Protein spotses, and the spot that part Pro-Q Emerald glycoprotein fluorescence colours can't detect can be detected, show 4H-
[1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour is a kind of simple to operate, and sensitivity can
The method matched in excellence or beauty with Pro-Q Emerald glycoprotein fluorescence colours.
The 4H- of experimental example 5 [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour is directed to sugared egg
The investigation of white specific detection.
N- chains sugar in standard protein α 1-acid glycoprotein and human serum total protein is removed with PNGase F.Will be upper
State after albumen separates through one to gel electrophoresis, (A) 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides sugar is respectively adopted
Protein fluorescence decoration method, (B) Pro-Q Emerald glycoprotein fluorescence colours and (C) SYPRO Ruby holoprotein fluorescent stainings
Method is dyed.As a result it is as shown in Figure 5.Remove standard protein α 1-acid glycoprotein and human serum after N- chains sugar total
Albumen can detect by SYPRO Ruby holoproteins fluorescence colour, but can hardly be by 4H- [1]-chromene [4,3-b] thiophene
Fen -2- formic hydrazide glycoprotein fluorescence colours and the identification of Pro-Q Emerald glycoprotein fluorescence colour.Further illustrate
4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides glycoprotein fluorescence colour has high special to glycoprotein detection
Property.
Reference colouring method and its pertinent literature in Fig. 2-5
Pro-Q Emerald488 glyprotein stain method operating methods are referring to document:Courtenay Hart., Birte
Schulenberg., Thomas H.Steinberg., Wai-Yee Leung., Wayne F.Patton, R. (2003)
Detection of glycoproteins in polyacrylamide gels and on electroblots using
Pro-Q Emerald488dye, a fluorescent periodate Schiff-base
Stain.Electrophoresis24,588-598.
SYPRO Ruby fluorescence colour operating methods are referring to document:Malone, J., Radabaugh, M.,
Leimgruber, R., Gerstenecker, G (2001) Practical aspects of fluorescent staining
For proteomic application.Electrophoresis22,919-932.
Claims (8)
1. glycoprotein specificity fluorescent detection method on a kind of gel, it includes step:
1) by after SDS-PAGE electrophoresis the gel containing protein example be placed in fixer and fix 10~60min, abandon fixer;
2) 10~40min is aoxidized in periodic acid solution, its meso-periodic acid solution is the high iodine containing w/v 0.1~1%
Acid and by volume 0.5~5% acetic acid aqueous solution, then rinse 2~5 with by volume 0.5~5% acetic acid aqueous solution
It is secondary, 1~10min every time;
3) 1~40min of dyeing liquor is added, wherein dyeing liquor is 4H- [1]-benzo containing w/v 0.0001~0.001%
Pyrans [4,3-b] thiophene -2-carboxylic acid hydrazides or derivatives thereof and by volume 0.5~5% acetic acid aqueous solution, wherein described spread out
Biology is the sodium salt, sylvite or ammonium salt of 4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides;
4) eluent is added, eluent is the acetic acid, 10~70% ethanol and 0.5~2% monoethanolamine of volume ratio 0.5~5%
The aqueous solution, is eluted 1~3 time, 1~10min every time;
5) detect.
2. the method as described in claim 1, it is characterised in that step 1) in the gel set time be 30min, fixer be by
The solution of the ethanol of volume ratio 40% and 10% acetic acid.
3. the method as described in claim 1, it is characterised in that step 2) in gel oxidization time in periodic acid solution be
20min, its meso-periodic acid solution 1% acetic acid aqueous solution for the periodic acid containing w/v 0.5% and by volume, then
Rinsed 3 times with by volume 1% acetic acid aqueous solution, each 5min.
4. the method as described in claim 1, it is characterised in that step 3) dyeing liquor composition is for volume ratio by weight
0.0002%4H- [1]-chromene [4,3-b] thiophene -2-carboxylic acid hydrazides or derivatives thereof and by volume 1% acetic acid water
Solution.
5. the method as described in claim 1, it is characterised in that step 4) eluent composition is 1% acetic acid, 40% by volume
The aqueous solution of ethanol and 1% monoethanolamine.
6. the method as described in claim 1, it is characterised in that step 3) dyeing time is 15min.
7. the method as described in claim 1, it is characterised in that step 4) elution time is 20min.
8. the method as described in claim 1, it is characterised in that step 5) it is that the sugared egg after dyeing is observed under laser scanner
In vain.
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