CN104251849A - Application of 4 H-[1]-benzo pyran [4, 3-b] thiophene-2-formic acid hydrazide and derivative thereof in glycoprotein specific fluorescence detection - Google Patents
Application of 4 H-[1]-benzo pyran [4, 3-b] thiophene-2-formic acid hydrazide and derivative thereof in glycoprotein specific fluorescence detection Download PDFInfo
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Abstract
The invention relates to glycoprotein specific fluorescence detection technology, and particularly relates to application of 4 H-[1]-benzo pyran [4, 3-b] thiophene-2-formic acid hydrazide and a derivative thereof in glycoprotein specific fluorescence detection. The invention also provides a method for the application of the 4 H-[1]-benzo pyran [4, 3-b] thiophene-2-formic acid hydrazide for glycoprotein specific fluorescence detection, and the method comprises the steps of: fixation of after-electrophoresis gel containing a protein sample in a fixing solution; oxidation with periodic acid solution; washing with acetic acid aqueous solution; dyeing; and elution. The invention has the advantages of high sensitivity, good selectivity, fast and simple operation, good reproducibility, good linear relationship, good mass spectrum compatibility, safe use, low cost, and the like, and can be better suitable for the study of high-throughput proteomics.
Description
Technical field
The present invention relates to glycoprotein specificity fluorescent detection technique, the application of specifically 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid acyl hydrazine and its derivative in glycoprotein specificity fluorescent detects.
Background technology
Modification of protein glycosylation has vital effect in life science field, as one of main and general protein post-translational modification, 1/2nd are had at least to there occurs glycosylation in current known mammalian proteins matter, these glycoprotein are distributed widely in tissue, cell, body fluid, particularly rich content in surface of cell membrane, body fluid etc., glycosylation all plays an important role for folding, the transport of protein and location etc., and participate in receptor activation, signal transduction, cell carry all more important bioprocess such as attached.The change of glycoprotein number change or sugar chain structure, all may cause disease to occur.The diagnosis marker of not only known at present a lot of diseases is glycoprotein, and in medicine by international standard certification, glycoprotein also accounts for higher ratio.
Current glycoprotein detection field kit mainly concentrate on foreign country biological reagent company, and most product all with the price of high added value in market sale, price is high, is unfavorable for the development of biotechnology fundamental research.Especially domestic utilize existing biological reagent carry out Bioexperiment cost remain high, cannot realize economic benefit with experiment common development.4H-[the 1]-chromene [4 of the present invention's exploitation, 3-b] thiophene-2-carboxylic acid hydrazides and the sensitivity of derivatives fluorescent colouring method thereof is close to Pro-Q Emerald488 decoration method, be a kind of sensitive, convenient, special glycoprotein detection technique of fluorescence, have good Preclinic and clinic meaning.
Summary of the invention
The object of the present invention is to provide the application of 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid acyl hydrazine and its derivative in glycoprotein specificity fluorescent detects.
4H-of the present invention [1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides related compound refers to 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides negative ion to be sodium salt, the sylvite and ammonium salt etc. of parent nucleus.
4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides parent nucleus is:
In order to realize object of the present invention, present invention also offers the method that application 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides carries out the detection of glycoprotein specificity fluorescent and comprising the steps:
1) gel containing protein example after SDS-PAGE electrophoresis is placed in immobile liquid and fixes 10 ~ 60min, abandon immobile liquid.The preferred set time is 30min, and immobile liquid can be containing 40% ethanol and 10% second aqueous acid;
2) in periodic acid solution be oxidized 10 ~ 40min, its meso-periodic acid solution be containing w/v 0.1 ~ 1% periodic acid and by volume 0.5 ~ 5% acetic acid aqueous solution.Then the acetic acid aqueous solution of by volume 0.5 ~ 5% is used to rinse 2 ~ 5 times, each 1 ~ 10min; Preferred oxidization time is 20min, preferred oxidation solution consist of containing w/v 0.5% periodic acid and by volume 1% acetic acid aqueous solution;
3) add dyeing liquor 1 ~ 40min, wherein dyeing liquor be containing w/v 0.0001 ~ 0.001% 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides or derivatives thereof and by volume 0.5 ~ 5% acetic acid aqueous solution.Preferred dyeing time is 10min, preferred dyeing liquor consist of containing w/v 0.0002% 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides and by volume 1% acetic acid aqueous solution;
4) add eluent, eluent consists of the acetic acid of volume ratio 0.5 ~ 5.0%, 10 ~ 70% ethanol and 0.5 ~ 2% aqueous ethanolamine, wash-out 1 ~ 3 time, each 1 ~ 10min.Preferred elution time is 20min, and preferred eluent consists of 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 this technology has the following advantages:
1) highly sensitive: 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) rapidly simple to operate: operation steps is few, can complete in 2 hours;
3) favorable reproducibility: affect less by external conditions such as temperature, shaking table hunting frequencies;
4) good reversibility: easily decolour;
5) mass spectrum compatibility is good: because 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides fluorescence detection does not affect protein structure, thus can with the mass spectrometer highly compatibles such as MALDI-TOF;
6) use safety: adopt the dyestuff that toxicity is low, improve the security of experimental implementation, environmental pollution is little;
7) cost is low.
Accompanying drawing explanation
The chemical constitution of Fig. 1 .4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides;
Fig. 2. on one to SDS-PAGE glue, 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour detects comparing of Protein standards effect with other decoration method.(A) 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colour, (C) SYPRO Ruby holoprotein fluorescence colour.Pro-Q Emerald glycoprotein fluorescence colour and SYPRO Ruby holoprotein fluorescence colour all record operation according to document.The 8 kinds of various criterion protein adopting Sigma company are sample: Transferrin (glycoprotein), BSA (non-glycoprotein), IgG (glycoprotein), OVA (glycoprotein), α 1-acid glycoprotein (glycoprotein), α-casein (non-glycoprotein), β-casein (non-glycoprotein), avidin (glycoprotein), represents glycoprotein with italics in the drawings.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. on one to SDS-PAGE glue, 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour detects comparing of actual sample effect with other colouring method.(A) 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colour, (C) SYPRO Ruby holoprotein fluorescence colour.Pro-Q Emerald glycoprotein fluorescence colour and SYPRO Ruby holoprotein fluorescence colour all record operation according to document; The human serum total protein extracted is adopted to be 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. on one to SDS-PAGE glue, 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour and other colouring method are for the specific investigation of glyprotein stain.(A) 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colour, (C) SYPRO Ruby holoprotein fluorescence colour.N-chain sugar in standard protein α 1-acid glycoprotein and human serum total protein is removed with PNGase F.Band 1, human serum total protein; Band 2, removes N-chain sugar descendant total serum protein; Band 3, α 1-acid glycoprotein; Band 4, removes α l-acid glycoprotein after N-chain sugar; Band 5, PNGase F enzyme.
Fig. 5. on two to SDS-PAGE glue, 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour detects comparing of actual sample effect with other colouring method.(A) 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colour, (C) SYPRO Ruby holoprotein fluorescence colour.Sample separation 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 set forth further below in conjunction with specific embodiment.Should be appreciated that these embodiments are only for illustration of the present invention, and can not limit the scope of the invention.
Experimental example 1 4H-[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 adopts as following step is carried out:
1) the protein example gel after SDS-PAGE electrophoresis is placed in 40% ethanol and 10% acetic acid aqueous solution fixes 30min, abandons immobile liquid;
2) in periodic acid solution, be oxidized 20min, its meso-periodic acid solution be containing w/v 0.5% periodic acid and by volume 1% acetic acid aqueous solution.Then the acetic acid aqueous solution of by volume 1% is used to rinse 3 times, each 5min;
3) add dyeing liquor dyeing 10min, wherein dyeing liquor be containing w/v 0.0002% 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides and by volume 1% acetic acid aqueous solution;
4) add containing volume ratio 1% acetic acid, the eluent of 40% ethanol and 1% aqueous ethanolamine, wash-out 20min;
5) detect, the albumen after dyeing can be observed under laser scanner.
Use 4H-[1]-chromene [4 according to the method described above respectively, 3-b] sodium salt of thiophene-2-carboxylic acid hydrazides, sylvite, ammonium salt carry out protein staining, result shows the testing result that all can obtain being similar to 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides with these derivants.
Experimental example 2 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour and other decoration method contrast 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 colour, (C) SYPRO Ruby holoprotein fluorescence colour.Pro-Q Emerald glycoprotein fluorescence colour and SYPRO Ruby holoprotein fluorescence colour are all recorded according to document; The 8 kinds of various criterion protein adopting Sigma company are 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.Result as shown in Figure 2, shows that 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour detection sensitivity is close to Pro-Q Emerald488 glycoprotein fluorescence colour.
Experimental example 3 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour and other decoration method contrast 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 colour, (C) SYPRO Ruby holoprotein fluorescence colour.Pro-Q Emerald glycoprotein fluorescence colour and SYPRO Ruby holoprotein fluorescence colour all record operation according to document; The human serum total protein extracted is adopted to be 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.Result as shown in Figure 3, shows that 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour detection sensitivity is close to Pro-Q Emerald488 glycoprotein fluorescence colour.
Experimental example 4 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour and other decoration method contrast rat liver total protein Detection results.
Get rat liver, add in liquid nitrogen pulverize last, 0.02g packing, 500 μ 1 lysates are added, broken 3 times (1min/ time) of cell Ultrasonic Cell Disruptor, centrifugal 15000g/min in every pipe, 15min, get part supernatant Bradford kit measurement protein concentration, all the other supernatants are sub-packed in Eppendorf pipe, preserve with for subsequent use for-80 DEG C.
After above-mentioned rat liver is separated to gel electrophoresis through two, adopt (A) 4H-[1]-chromene [4 respectively, 3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colour, (C) SYPRO Ruby holoprotein fluorescence colour dyes, and result as shown in Figure 4.Display 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour can detect most protein spotses that Pro-Q Emerald glycoprotein fluorescence colour detects, and the spot that part Pro-Q Emerald glycoprotein fluorescence colour 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 the method that sensitivity can match in excellence or beauty with Pro-Q Emerald glycoprotein fluorescence colour.
Experimental example 5 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour is for the investigation of glycoprotein specific detection.
N-chain sugar in standard protein α 1-acid glycoprotein and human serum total protein is removed with PNGase F.After above-mentioned albumen is separated to gel electrophoresis through one, adopt (A) 4H-[1]-chromene [4 respectively, 3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour, (B) Pro-Q Emerald glycoprotein fluorescence colour and (C) SYPRO Ruby holoprotein fluorescence colour dye.Result as shown in Figure 5.Standard protein α 1-acid glycoprotein after removal N-chain sugar and human serum total protein can be detected by SYPRO Ruby holoprotein fluorescence colour, but almost can not by 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides glycoprotein fluorescence colour 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, to glycoprotein detection, there is high degree of specificity.
Reference colouring method in Fig. 2-5 and pertinent literature thereof
Pro-Q Emerald488 glyprotein stain method method of operating is see 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 method of operating is see document: Malone, J., Radabaugh, M., Leimgruber, R., Gerstenecker, G (2001) Practical aspects of fluorescent staining for proteomic application.Electrophoresis22,919-932.
Claims (10)
- The application of 1.4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid acyl hydrazine and its derivative in glycoprotein specificity fluorescent detects.
- 2. apply as claimed in claim 1, it is characterized in that described 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid acyl hydrazine and its derivative is the sodium salt of 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides, sylvite or ammonium salt.
- 3. a glycoprotein specificity fluorescent detection method on gel, it comprises step:1) gel containing protein example after SDS-PAGE electrophoresis is placed in immobile liquid and fixes 10 ~ 60min, abandon immobile liquid;2) in periodic acid solution be oxidized 10 ~ 40min, its meso-periodic acid solution be containing w/v 0.1 ~ 1% periodic acid and by volume 0.5 ~ 5% acetic acid aqueous solution.Then the acetic acid aqueous solution of by volume 0.5 ~ 5% is used to rinse 2 ~ 5 times, each 1 ~ 10min;3) add dyeing liquor 1 ~ 40min, wherein dyeing liquor be containing w/v 0.0001 ~ 0.001% 4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides or derivatives thereof and by volume 0.5 ~ 5% acetic acid aqueous solution;4) add eluent, eluent is the acetic acid of volume ratio 0.5 ~ 5%, 10 ~ 70% ethanol and 0.5 ~ 2% aqueous ethanolamine, wash-out 1 ~ 3 time, each 1 ~ 10min;5) detect.
- 4. method as claimed in claim 3, is characterized in that, step 1) in the gel set time be 30min, immobile liquid is 40% ethanol/10% acetic acid solution by volume.
- 5. method as claimed in claim 3, is characterized in that, step 2) in gel oxidization time in periodic acid solution be 20min, its meso-periodic acid solution be containing w/v 0.5% periodic acid and by volume 1% acetic acid aqueous solution.Then the acetic acid aqueous solution of by volume 1% is used to rinse 3 times, each 5min.
- 6. the method as described in any one of claim 3 ~ 5, it is characterized in that, step 3) dyeing liquor consist of volume ratio be by weight 0.0002%4H-[1]-chromene [4,3-b] thiophene-2-carboxylic acid hydrazides or derivatives thereof and by volume 1% acetic acid aqueous solution.
- 7. the method as described in any one of claim 3 ~ 5, is characterized in that, step 4) eluent consists of 1% acetic acid by volume, 40% ethanol and 1% aqueous ethanolamine.
- 8. the method as described in any one of claim 3 ~ 5, is characterized in that, step 3) dyeing time is 15min.
- 9. the method as described in any one of claim 3 ~ 5, is characterized in that, step 4) elution time is 20min.
- 10. the method as described in any one of claim 3 ~ 5, is characterized in that, step 5) glycoprotein after dyeing can be observed under laser scanner.
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