CN102313773B - Method for identifying quantity of cysteine in protein and application thereof - Google Patents
Method for identifying quantity of cysteine in protein and application thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying quantity of cysteine in protein, especially a method for identifying quantity of cysteine in high molecular weight wheat glutenin subunits. The method enables composition of high molecular weight wheat glutenin subunits and alleles to be identified rapidly and accurately and quantity of cysteine residues in each high molecular weight glutenin subunits to be identified. The method provided in the invention is applicable to improvement of wheat quality and rapid detection of the quantity of cysteine residues in high molecular weight glutenin subunits, thereby enabling high-quality subunits to be explored and screened and efficiency of quality improvement to be improved. Furthermore, the method can also be used for identifying quantity of cysteine residues in salt-soluble protein and water-soluble protein of lupin, and therefore, a complete technological system for identification of quantity of cysteine residues is formed.
Description
Technical field
The present invention relates to method and the application thereof of halfcystine quantity in a kind of identification of protein, particularly relate to the method for halfcystine quantity in a kind of Rapid identification wheat high-molecular-weight glutelin subunit and the application in Wheat Quality Improvement thereof.
Background technology
Wheat is one of most important Three major grain crops in the world, and its output is only second to corn and is number two, and wheat planting area, total production and the consumption figure of China occupy first place in the world.Wheat seed is nutritious, is rich in starch, protein, fat, mineral matter, calcium, iron, thiamine, lactochrome, nicotinic acid and vitamin A etc., meets very much the Human Physiology needs, and is very useful to health.35% population is approximately arranged in the world take wheat as staple food, wheat gluten is the important phytoprotein sources of the mankind, accounts for 38.4% of corn protein; And wheat flour can be made bread, steamed bun, biscuit, cake, noodles, deep-fried twisted dough sticks, oil cake, baked wheaten cake, sesame seed cake, thin pancake, boiled dumpling, decoct the foods such as dumpling, steamed stuffed bun, won ton, egg roll, instant noodles, rice cake, Italian type wheaten food; Can be made into beer, alcohol, vodka etc. after fermentation.Wheat this special and face processing characteristics be mainly the structure and characteristics by the wheat seed storage protein determine (Wrigley, Giant proteins with flour power.Nature, 1996,381:738-739).Storage Proteins in Wheat refers to alcohol soluble protein (gliadins) and glutenin (glutenins), the content of this two albuminoid is larger, account for 85% of Seed Storage Protein, wherein, the elasticity of glutenin major decision dough, alcohol soluble protein determines the ductility (Payne of dough, Geneticsof wheat storage proteins and the effect of allelic variation on bread making quality.Ann.Rev.Plant Physiol., 198738:141-153).
Occurring in nature, most protein all contains cysteine residues, can form disulfide bond between cysteine residues, and disulfide bond is very important (Thornton to three grades of protein and the stability of quaternary structure, Disulphidebridges in globular proteins.J.Mol.Biol., 1981,151:261-287; Wetzel, Harnessingdisulfide-bonds using protein engineering.Trends Biochem.Sci., 1987,12:478-482).Disulfide bond can make protein avoid damaging and can increasing the half life period of protein, therefore disulfide bond can be kept the stability (Hogg of albumen, Disulfide bonds as switches for protein function.Trends Biochem.Sci., 2003,28:210-214).Because disulfide bond has very important effect to the 26S Proteasome Structure and Function of glutelin, crop science the research worker pay special attention to disulfide bond.the high low-molecular-weight glutenin subunit of wheat forms polymeric (the Masci S of glutelin by disulfide bond exactly, D ' Ovidio R, Lafiandra D, Kasarda DD, Characterization of alow-molecular-weight glutenin subunit gene from bread wheat and the correspondingprotein that represents a major subunit of the glutenin polymer.Plant Physiol., 1998, 118:1147-1158), and polymeric size is huge (Masci S on the impact of flower characters, Rovelli L, KasardaDD, Vensel WH, Lafiandra D, Characterisation and chromosomal localization of C-typelow-molecular-weight glutenin subunits in the bread wheat cultivar Chinese Spring.Theor.Appl.Genet., 2002, 104:422-428).The high quality subunit 1Dx5 that generally acknowledges is exactly because thereby many extra cysteine residues cause condensate to become the quality that has greatly improved flour.Therefore, the cysteine residues number has very important impact to the quality of wheat.Detect the cysteine residues number quality-improving of finding high quality subunit and wheat is all had a very important role, but also lack the method for wheat high-molecular-weight glutelin subunit halfcystine quantity of fast, efficiently identifying both at home and abroad at present.
Summary of the invention
In recent years, development along with the biological mass spectrometry technology, different mass-spectrometric techniques have become a kind of powerful tool (the Zhang Y that identifies wheat glutenin subunits, Li X, Yan Y, Xiong X, An X, Zhang Q, Pei Y, Gao L, HsamSLK, Zeller FJ, Molecular characterization of two novel x-type HMW glutenin genesfrom Aegilops tauschii and implications for the evolution of Glu-D1-1 alleles.Genetics, 2008,178:23-33; Qian Zhang, Yanmin Dong, Xueli An, Aili Wang, Yanzhen Zhang, Xiaohui Li, Xianchun Xia, Zhonghu He, Yueming Yan:Characterization of HMWglutenin subunits in common wheat and related species by matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) .Journal ofCereal Science, 2008,47:252-261; Li Liu, Aili Wang, Rudi Appels, Junhong Ma, Xianchun Xia, Ping Lan, Zhonghu He, Frank Bekes, Yueming Yan, Wujun Ma, AMALDI-TOF based analysis of high molecular weight glutenin subunits for wheatbreeding.Journal ofCereal Science, 2009,50:295-301).Classic method is in the past compared, and mass spectrum is more accurate, sensitiveer fast.
The object of the invention is to set up a kind of stable, mass spectrometry method fast, thus can be to the number of the cysteine residues in testing protein, the method for particularly number of the cysteine residues in wheat high-molecular-weight glutelin subunit being identified., by the method, can identify fast and accurately wheat high-molecular-weight glutelin subunit and allelic composition, and can determine the number of the cysteine residues that contains in each high-molecular-weight glutelin subunit.
Above-mentioned testing protein is from wheat or lupin.
The principle of the inventive method: thus the rare pyridine of 4-second can stop halfcystine to form disulfide bond in conjunction with halfcystine, therefore, rare pyridine of 4-second can with a halfcystine combination.When extracting gluten subunit, one group of sample adds the rare pyridine of 4-second, and another group sample does not add the rare pyridine of 4-second, then carries out Mass Spectrometric Identification.The contained halfcystine number of the molecular weight that gluten subunit increases and this subunit is relevant, be that high-molecular-weight glutelin subunit often contains a cysteine residues, its molecular weight can increase about 105.14Da (i.e. the molecular weight of a rare pyridine of 4-second) accordingly.
Therefore, at first to obtain the molecular weight of same albumen under two kinds of processing (rare pyridine is processed and not rare pyridine processing with 4-second with 4-second) by mass spectrum and what be respectively, then deduct with the molecular weight that the rare pyridine of 4-second was processed the molecular weight of with the rare pyridine of 4-second, not processing, finally again divided by 105, the result that obtains is exactly the number of cysteine residues contained in this testing protein.
Described testing protein is during from wheat, and the inventive method comprises that sample preparation, Mass Spectrometric Identification and Data Management Analysis and halfcystine number determine, specifically comprises the following steps:
(1) sample preparation
Get two parts of the flour 15mg of wheat Bumper, put into respectively the 1.5ml centrifuge tube; Adding volumn concentration is 70% ethanol 1ml, vortex 30min, and the centrifugal 10min of 12000rpm, remove supernatant.Adding respectively volumn concentration is 55% isopropyl alcohol 1ml again, 65 ℃ of water-bath 30min, and the centrifugal 10min of 12000g, remove supernatant.Then blot remaining supernatant with filter paper, above-mentioned steps repeats 3 times.Add again 0.1ml to contain the solution A (DTT and solution A mass percent are 1: 100) of DTT, consisting of of solution A wherein, isopropyl alcohol: 1M Tris-HCl (pH8): distilled water=50: 8: 42 (volume ratio); Vortex mixes, 65 ℃ of water-bath 30min.
Then, the centrifugal 10min of 12000g, the first duplicate samples is got supernatant, with the acetone precipitation at room temperature of 40% volumn concentration, spends the night; The second duplicate samples adds 0.1ml to contain the solution A (percent by volume of 4-vinylpridine and solution A is 1.4: 98.6) of 4-vinylpridine, and vortex mixes, 65 ℃ of water-bath 30min; The centrifugal 10min of 12000rpm; Getting supernatant spends the night with the acetone precipitation at room temperature of 40% volumn concentration; Two duplicate samples centrifugal 10min of 12000rpm after the precipitation of spending the night, abandon supernatant, adds 0.1ml to contain the solution A (DTT and solution A mass percent are 1: 100) of DTT, 65 ℃ of water-bath 30min; The first duplicate samples is got supernatant and is spent the night with the acetone precipitation at room temperature of 40% volumn concentration; The second duplicate samples adds 0.1ml to contain the solution A (percent by volume of 4-vinylpridine and solution A is 1.4: 98.6) of 4-vinylpridine again, and vortex mixes, 65 ℃ of water-bath 30min; The centrifugal 10min of 12000rpm; Getting supernatant spends the night with the acetone precipitation at room temperature of 40% volumn concentration; Rear two duplicate samples of the precipitation of spending the night are the centrifugal 10min of 12000rpm all, abandon supernatant, drying precipitated 10min, add 70 μ l chromatographic grade acetonitrile solutions (wherein contain final concentration be 0.1% TFA), dissolve more than 4 hours, adopt mixing loading method to carry out mass spectrophotometry the supernatant that obtains.
(2) Mass Spectrometric Identification
The mass spectrometer 4800 that adopts AppliedBiosystems company to produce, the mass spectrum parameter is laser intensity 2,500, mass range 65-95kDa, accelerating potential 25kV, plate voltage 92%, yardstick 0.3%, time delay 1,000ns.Utilize software to carry out Data Management Analysis to the protein quality spectrum that obtains.
The calculating of cysteine residues number: utilize the rare pyridine of 4-second in sample preparation to process and the variation of untreated protein molecular weight, the number of cysteine residues in the calculating high-molecular-weight glutelin subunit.
The using value of the inventive method:
The cysteine residues number that (1) not only can obtain containing in albumen, and can obtain the molecular weight of this albumen, thereby be used for identifying allele composition and wheat breed and the rapid screening and the selection that realize hybridization early generation high quality subunit, greatly improve the efficiency of Wheat Quality Improvement.
(2) can carry out to the high-molecular-weight glutelin subunit of common wheat and sibling species thereof the detection of cysteine residues number, thereby the function of further research disulfide bond is offered help.
(3) can be used for detecting on a large scale in wheat and sibling species thereof the number of cysteine residues in gluten subunit, thus the new glutelin candidate high quality subunit of rapid screening, and the inventive method is quicker, more accurate than identified for genes.
The present invention has utilized mass spectral:mass spectrographic accuracy just, thereby invented a kind of method of utilizing mass-spectrometric technique to identify halfcystine quantity in wheat high-molecular-weight glutelin subunit, can be applicable to the fast detecting of halfcystine quantity in Wheat Quality Improvement and high-molecular-weight glutelin subunit, thereby excavate and the screening high-quality subunit, improve the efficiency of quality-improving.In addition, the inventive method can also be identified cysteine residues number contained in salting-in-protein in lupin and aqueous soluble protein, and then form the complete technical system of cover that the cysteine residues number is identified.
Description of drawings
Fig. 1 is that in Common Wheat Varieties Bumper, high-molecular-weight glutelin subunit is processed and untreated mass spectrum Comparative map with the rare pyridine of 4-second;
Fig. 2 is that in Common Wheat Varieties Shan229, high-molecular-weight glutelin subunit is processed and untreated mass spectrum Comparative map with the rare pyridine of 4-second;
Fig. 3 is that lupin is identified collection of illustrative plates with the cysteine residues number in the rare pyridine processing of 4-second and untreated salting-in-protein;
Fig. 4 is that lupin is identified collection of illustrative plates with the cysteine residues number in the rare pyridine processing of 4-second and untreated aqueous soluble protein.
Embodiment
The experiment material that relates in the present invention is as follows: Common Wheat Varieties Ajana, Banks, Bullaring, Bumper, Calingiri, Chara, EGA Blanco, Endure, IGW2944, IGW3240, Pugsley, M12, M25, M26, Shan 229, Wanmai 33, Halberd, lupin (Wang Ke, the molecular cloning of common wheat and sibling species glutelin encoding gene thereof and phyletic evolution research, Capital Normal University's doctorate paper, 2011), above-mentioned biomaterial is bred and is preserved with biotech lab by Capital Normal University's heredity.
The evaluation of cysteine residues quantity in embodiment 1, wheat high-molecular-weight glutelin subunit
1, the extraction of wheat high-molecular-weight glutelin subunit and sample preparation
Get two parts of the flour 15mg of wheat Bumper, put into respectively the 1.5ml centrifuge tube; Adding volumn concentration is 70% ethanol 1ml, vortex 30min, and the centrifugal 10min of 12000rpm, remove supernatant.Adding respectively volumn concentration is 55% isopropyl alcohol 1ml again, 65 ℃ of water-bath 30min, and the centrifugal 10min of 12000g, remove supernatant.Then blot remaining supernatant with filter paper, above-mentioned steps repeats 3 times.Add again 0.1ml to contain the solution A (mass percent of DTT and solution A is 1: 100) of DTT, consisting of of solution A wherein, isopropyl alcohol: 1M Tris-HCl (pH8): distilled water=50: 8: 42 (volume ratio); Vortex mixes, 65 ℃ of water-bath 30min.
Then, the centrifugal 10min of 12000g, the first duplicate samples is got supernatant, with the acetone precipitation at room temperature of 40% volumn concentration, spends the night; The second duplicate samples adds 0.1ml to contain the solution A (percent by volume of 4-vinylpridine and solution A is 1.4: 98.6) of 4-vinylpridine, and vortex mixes, 65 ℃ of water-bath 30min; The centrifugal 10min of 12000rpm; Getting supernatant spends the night with the acetone precipitation at room temperature of 40% volumn concentration; Two duplicate samples centrifugal 10min of 12000rpm after the precipitation of spending the night, abandon supernatant, adds 0.1ml to contain the solution A (DTT and solution A mass percent are 1: 100) of DTT, 65 ℃ of water-bath 30min; The first duplicate samples is got supernatant and is spent the night with the acetone precipitation at room temperature of 40% volumn concentration; The second duplicate samples adds 0.1ml to contain the solution A (percent by volume of 4-vinylpridine and solution A is 1.4: 98.6) of 4-vinylpridine again, and vortex mixes, 65 ℃ of water-bath 30min; The centrifugal 10min of 12000rpm; Getting supernatant spends the night with the acetone precipitation at room temperature of 40% volumn concentration; Rear two duplicate samples of the precipitation of spending the night are the centrifugal 10min of 12000rpm all, abandon supernatant, drying precipitated 10min, add 70 μ l chromatographic grade acetonitrile solutions (wherein contain final concentration be 0.1% TFA), dissolve more than 4 hours, adopt mixing loading method to carry out mass spectrophotometry the supernatant that obtains.
The mass spectrum result as shown in Figure 1.Wherein, Figure 1A is the mass spectrum that in Common Wheat Varieties Bumper, high-molecular-weight glutelin subunit is processed with the rare pyridine of 4-second, and Figure 1B is the mass spectrum that in Common Wheat Varieties Bumper, high-molecular-weight glutelin subunit is not processed with the rare pyridine of 4-second.
2, the evaluation of cysteine residues quantity in gluten subunit
(1) instrument and equipment: the mass spectrometer 4800 that adopts AppliedBiosystems company to produce.
(2) mass spectrum parameter: laser intensity 2,500, mass range 65-95kDa, accelerating potential 25kV, plate voltage 92%, yardstick 0.3%, time delay 1,000ns.
(3) data are processed with cysteine residues quantity and are determined: utilize software to carry out Data Management Analysis.The calculating of cysteine residues number: as can be seen from Figure 1, after in wheat Bumper, the 1Dx5 subunit adds the rare pyridine of 4-second, molecular weight has increased 517.67Da; And other x-type and y-type HMW-GS ratio with the rare pyridine of 4-second is processed do not increase respectively about 400Da and 700Da with the molecular weight that the rare pyridine of 4-second is processed, these presentation of results, the 1Dx5 subunit contains 5 cysteine residues, and the number of the cysteine residues in other x-type and y-type HMW-GS is respectively 4 and 7.These results are consistent with known result, and the reliability of the method has been described.
Embodiment 2, the wheat breed that utilizes 17 known subunits to form are verified the inventive method
1, the extraction of wheat high-molecular-weight glutelin subunit and sample preparation
Get Ajana, Banks, Bullaring, Bumper, Calingiri, Chara, EGA Blanco, Endure, IGW2944, IGW3240, Pugsley, M12, M25, M26, Shan229, Wanmai33, the wheat seed of 17 different cultivars such as Halberd, sampling and sample preparation and Mass Spectrometric Identification are equal to embodiment 1.
The mass spectrum result of wheat breed Shan229 as shown in Figure 2.Wherein, Fig. 2 A is the mass spectrum that in Common Wheat Varieties Shan229, high-molecular-weight glutelin subunit is processed with the rare pyridine of 4-second, and Fig. 2 B is the mass spectrum that in Common Wheat Varieties Shan229, high-molecular-weight glutelin subunit is not processed with the rare pyridine of 4-second.
The mass spectrum result of 17 Wheat Cultivars as shown in Table 1 and Table 2.
Cysteine residues number testing result in the high-molecular-weight glutelin of Glu-A1 and Glu-B1 site coding in the wheat breed that table 117 a known higher molecular weight gluten subunit forms
Cysteine residues number testing result in the high-molecular-weight glutelin of Glu-D1 site coding in the wheat breed that table 217 a known higher molecular weight gluten subunit forms
2, data analysis
As can be seen from Table 1, no matter be special 1Bx20 subunit and the 1Dx5 subunit that contains 2 and 5 cysteine residues, still other all conventional high-molecular-weight glutelin subunits that contain 4 cysteine residues, the molecular weight that the high-molecular-weight glutelin subunit of processing with the rare pyridine of 4-second increases is all corresponding with himself contained cysteine residues number, and namely high-molecular-weight glutelin subunit often contains its molecular weight of cysteine residues will increase about 105.14Da accordingly.
1, the extraction of sample and preparation
Salting-in-protein: the powder of getting two parts of 15mg lupins, a copy of it sample adds the NaCl solution of 0.2ml 0.5M, vortex vibration 30 minutes, then add 0.2ml to contain the NaCL solution (percent by volume of the NaCl solution of 4-vinylpridine and 0.5M is 1.4: 98.6) of the 0.5M of 4-vinylpridine in another duplicate samples, vortex mixes, 65 ℃ of water-bath 30min, the centrifugal 10min of 12000rpm; Getting supernatant spends the night with the acetone precipitation at room temperature of 80% volumn concentration; Rear two duplicate samples of the precipitation of spending the night are the centrifugal 10min of 12000rpm all, abandon supernatant, drying precipitated 10min, add 70 μ l chromatographic grade acetonitrile solutions (wherein contain final concentration be 0.1% TFA), dissolve more than 4 hours, adopt mixing loading method to carry out mass spectrophotometry the supernatant that obtains.
Aqueous soluble protein: the powder of getting two parts of 15mg lupins, a copy of it sample adds 0.2ml distilled water, vortex vibration 30 minutes, then be 1.4% 4-vinylpridine aqueous solution to adding the 0.2ml concentration of volume percent in another duplicate samples, vortex mixes, 65 ℃ of water-bath 30min, the centrifugal 10min of 12000rpm; Getting supernatant spends the night with the acetone precipitation at room temperature of 80% volumn concentration; Rear two duplicate samples of the precipitation of spending the night are the centrifugal 10min of 12000rpm all, abandon supernatant, drying precipitated 10min, add 70 μ l chromatographic grade acetonitrile solutions (wherein contain final concentration be 0.1% TFA), dissolve more than 4 hours, adopt mixing loading method to carry out mass spectrophotometry the supernatant that obtains.
2, atlas analysis
Calculate the contained cysteine residues number of each subunit according to the difference with the rare pyridine processing of 4-second and untreated protein molecular weight.Result as shown in Figure 3 and Figure 4.Wherein, Fig. 3 A is the mass spectrum of lupin with the salting-in-protein of the rare pyridine processing of 4-second, and Fig. 3 B is the mass spectrum that lupin is used the salting-in-protein of the rare pyridine processing of 4-second.Fig. 4 A is that lupin is processed the mass spectrum of aqueous soluble protein with the rare pyridine of 4-second, and Fig. 4 B is the aqueous soluble protein mass spectrum that lupin is not processed with the rare pyridine of 4-second.
Only have the difference of the molecular weight of the subunit that marks out with square frame to be about 100Da between Fig. 3 A and 3B, and the variation of other molecular weight subunit is very little in error range, so only have first subunit to contain 1 cysteine residues, other two subunits do not contain cysteine residues.
Between Fig. 4 A and 4B, the difference of the molecular weight of corresponding all subunits all is about 100Da, so all subunits all contain 1 cysteine residues.
Claims (4)
1. method of identifying cysteine residues quantity in testing protein, be to utilize the variation of biological mass spectrometry method according to the testing protein molecular weight of processing with 4-vinylpyridine and not processing with 4-vinylpyridine, determines the quantity of cysteine residues in testing protein;
Described testing protein is from wheat, and the extracting method of described testing protein comprises the steps:
Get two parts of 15mg wheat flours, two parts all add the 1ml volumn concentration is 70% ethanol, and vortex is centrifugal, removes supernatant; Adding respectively volumn concentration is 55% isopropyl alcohol 1ml again, and 65 ℃ of water-bath 30min, remove supernatant; Add 0.1ml to contain the solution A of DTT, DTT and solution A mass percent are 1:100 again, consisting of of solution A wherein, and the 1M Tris-HCl of isopropyl alcohol: pH8: the volume ratio of distilled water is 50:8:42; Vortex mixes, 65 ℃ of water-bath 30min, then, the centrifugal 10min of 12000g;
The first duplicate samples is got supernatant, with the acetone precipitation at room temperature of 40% volumn concentration, spends the night; The second duplicate samples adds 0.1ml to contain the solution A of 4-vinylpridine, and the percent by volume of 4-vinylpridine and solution A is 1.4:98.6, and vortex mixes, 65 ℃ of water-bath 30min; Centrifugal; Getting supernatant spends the night with the acetone precipitation at room temperature of 40% volumn concentration; After the precipitation of spending the night, two duplicate samples are all centrifugal, abandon supernatant, add 0.1ml to contain the solution A of DTT, and DTT and solution A mass percent are 1:100,65 ℃ of water-bath 30min; The first duplicate samples is got supernatant and is spent the night with the acetone precipitation at room temperature of 40% volumn concentration; The second duplicate samples adds 0.1ml to contain the solution A of 4-vinylpridine again, and the percent by volume of 4-vinylpridine and solution A is 1.4:98.6, and vortex mixes, 65 ℃ of water-bath 30min; Centrifugal; Getting supernatant spends the night with the acetone precipitation at room temperature of 40% volumn concentration; After the precipitation of spending the night, two duplicate samples are all centrifugal, abandon supernatant, and are drying precipitated, add 70 μ l chromatographic grade acetonitrile solutions, wherein contain final concentration and be 0.1% TFA, dissolve more than 4 hours, and the supernatant that obtains is adopted and mixes the loading method and carry out mass spectrophotometry.
2. method according to claim 1, it is characterized in that: the condition determination of described biological mass spectrometry method is: adopt AppliedBiosystems 4800 mass spectrometers, the mass spectrum parameter is laser intensity 2,500, mass range 65-95 kDa, accelerating potential 25 kV, plate voltage 92%, yardstick 0.3%, time delay 1,000 ns.
3. the application of the described method of claim 1 or 2 in the cysteine residues number is identified.
4. the application of the described method of claim 1 or 2 in wheat breed evaluation and quality-improving.
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