CN102313773B - Method for identifying quantity of cysteine in protein and application thereof - Google Patents

Abstract

The invention discloses a method for identifying the amount of cysteine in proteins, in particular discloses a method for identifying the amount of cysteine in wheat high molecular weight glutenin subunits. The method can quickly and accurately identify the composition of wheat high molecular weight glutenin subunits and alleles, and can determine the number of cysteine residues contained in each high molecular weight glutenin subunit. The method of the invention can be applied to wheat quality improvement and rapid detection of the number of cysteine residues in high-molecular-weight glutenin subunits, so as to discover and screen high-quality subunits and improve the efficiency of quality improvement. In addition, the method of the present invention can also identify the number of cysteine residues contained in the salt-soluble protein and water-soluble protein in lupine, and then form a complete technical system for identifying the number of cysteine residues.

Description

A method for identifying the number of cysteine in proteins and its application

technical field

The present invention relates to a method for identifying the amount of cysteine in protein and its application, in particular to a method for quickly identifying the amount of cysteine in wheat high molecular weight glutenin subunits and its application in wheat quality improvement .

Background technique

Wheat is one of the three most important food crops in the world, and its output ranks second only to corn. my country's wheat planting area, total output and consumption rank first in the world. Wheat seeds are rich in nutrition, rich in starch, protein, fat, minerals, calcium, iron, thiamine, riboflavin, niacin and vitamin A, etc., which meet the physiological needs of the human body and are very beneficial to human health. About 35% of the world's population takes wheat as a staple food, and wheat protein is an important source of plant protein for humans, accounting for about 38.4% of grain protein; Burning, sesame seed cakes, pancakes, dumplings, fried dumplings, buns, wontons, egg rolls, instant noodles, rice cakes, pasta and other foods; after fermentation, it can be made into beer, alcohol, vodka, etc. The special dough processing characteristics of wheat are mainly determined by the structure and characteristics of wheat seed storage proteins (Wrigley, Giant proteins with flour power. Nature, 1996, 381: 738-739). Wheat storage protein refers to gliadin (gliadins) and glutenin (glutenins), the content of these two types of protein is relatively large, accounting for about 85% of seed protein, among them, glutenin mainly determines the elasticity of dough, while gliadin is Determine the extensibility of dough (Payne, Genetics of wheat storage proteins and the effect of allelic variation on bread making quality. Ann. Rev. Plant Physiol., 198738: 141-153).

In nature, most proteins contain cysteine residues, and disulfide bonds can be formed between cysteine residues, and disulfide bonds are very important for the stability of the tertiary and quaternary structures of proteins ( Thornton, Disulphidebridges in globular proteins. J. Mol. Biol., 1981, 151: 261-287; Wetzel, Harnessing disulfide-bonds using protein engineering. Trends Biochem. Sci., 1987, 12: 478-482). Disulfide bonds can protect proteins from damage and increase the half-life of proteins, so disulfide bonds can maintain protein stability (Hogg, Disulfide bonds as switches for protein function. Trends Biochem. Sci., 2003, 28: 210-214 ). Because disulfide bonds play a very important role in the structure and function of glutenin, researchers in crop science pay great attention to disulfide bonds. Wheat high and low molecular weight glutenin subunits form glutenin polymers through disulfide bonds (Masci S, D'Ovidio R, Lafiandra D, Kasarda DD, Characterization of alow-molecular-weight glutenin subunit gene from bread wheat and the corresponding protein that represents a major subunit of the glutenin polymer.Plant Physiol., 1998, 118:1147-1158), and the size of the aggregate has a great influence on the flour quality (Masci S, Rovelli L, KasardaDD, Vensel WH, Lafiandra D , Characterization 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 well-recognized high-quality subunit 1Dx5 is due to the addition of an extra cysteine residue, which leads to larger aggregates and greatly improves the quality of flour. Therefore, the number of cysteine residues has a very important impact on the quality of wheat. Detecting the number of cysteine residues plays a very important role in the discovery of high-quality subunits and the improvement of wheat quality, but there is still a lack of rapid and efficient methods for identifying the number of cysteine in wheat high molecular weight glutenin subunits at home and abroad.

Contents of the invention

In recent years, with the development of biological mass spectrometry, different mass spectrometry techniques have become a powerful tool for the identification of wheat glutenin subunits (Zhang Y, 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 genes from Aegilops tauschii and implications for the evolution of Glu-D1-1 alleles. Genetics, 2008, 178: 23-33; Qian Zhang, Yanmin Dong, 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-MSALDI-TOF ).Journal of Cereal 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 of Cereal Science, 2009, 50: 295-301). Compared with previous traditional methods, mass spectrometry is more accurate, more sensitive and faster.

The purpose of the present invention is to establish a stable, rapid mass spectrometry method, thereby the number of cysteine residues in the protein to be tested, especially the cysteine residues in the wheat high molecular weight glutenin subunit method of identification. Through the method, the composition of wheat high molecular weight glutenin subunits and alleles can be quickly and accurately identified, and the number of cysteine residues contained in each high molecular weight glutenin subunit can be determined.

The above-mentioned proteins to be tested are from wheat or lupine.

Principle of the method of the present invention: 4-vinylpyridine can combine with cysteine to prevent cysteine from forming a disulfide bond, therefore, one 4-vinylpyridine can be combined with one cysteine. During the extraction of glutenin subunits, one set of samples was spiked with 4-vinylpyridine and the other without 4-vinylpyridine, followed by mass spectrometric identification. The increased molecular weight of the glutenin subunit is related to the number of cysteine contained in the subunit, that is, for every cysteine residue contained in the high molecular weight glutenin subunit, the molecular weight will increase by about 105.14Da (ie one cysteine residue). Molecular weight of 4-vinylpyridine).

Therefore, it is first necessary to obtain the molecular weight of the same protein under the two treatments (with 4-vinylpyridine treatment and without 4-vinylpyridine treatment) by mass spectrometry, and then the molecular weight of the same protein treated with 4-vinylpyridine Subtract the molecular weight not treated with 4-vinylpyridine, and finally divide by 105, the result obtained is the number of cysteine residues contained in the protein to be tested.

When the protein to be tested is from wheat, the method of the present invention includes sample preparation, mass spectrometry identification, data processing analysis and cysteine number determination, specifically including the following steps:

(1) Sample preparation

Take two portions of 15 mg of wheat Bumper flour, put them into 1.5 ml centrifuge tubes respectively; add 1 ml of 70% ethanol by volume, vortex for 30 min, centrifuge at 12000 rpm for 10 min, and remove the supernatant. Then add 1 ml of isopropanol with a volume percentage of 55% respectively, bathe in water at 65° C. for 30 min, centrifuge at 12000 g for 10 min, and remove the supernatant. Then the remaining supernatant was blotted dry with filter paper, and the above steps were repeated 3 times. Then add 0.1ml of solution A containing DTT (the mass percentage of DTT and solution A is 1:100), wherein the composition of solution A is: isopropanol: 1M Tris-HCl (pH8): double distilled water=50:8:42 (volume ratio); vortex to mix, 65 ° C water bath for 30 min.

Then, centrifuge at 12000g for 10min, take the supernatant from the first sample, and precipitate overnight at room temperature with acetone of 40% by volume; add 0.1ml of solution A (4-vinylpyridine and The volume percentage of solution A is 1.4:98.6), vortex mixing, 65 ° C water bath for 30 min; 12000 rpm centrifugation for 10 min; take the supernatant and use 40% volume percentage of acetone to precipitate overnight at room temperature; after overnight precipitation, both samples are centrifuged at 12000 rpm 10min, discard the supernatant, add 0.1ml of solution A containing DTT (the mass percentage of DTT and solution A is 1:100), bathe in water at 65°C for 30min; take the supernatant of the first sample and precipitate it with 40% by volume acetone at room temperature Overnight; add 0.1ml of solution A containing 4-vinylpyridine to the second sample (volume percentage of 4-vinylpyridine and solution A is 1.4:98.6), vortex and mix well, 30min in 65°C water bath; centrifuge at 12000rpm for 10min Get the supernatant and precipitate overnight at room temperature with 40% volume percent acetone; after the overnight precipitation, both samples are centrifuged at 12000rpm for 10min, discard the supernatant, dry the precipitation for 10min, and add 70 μl of chromatographic grade acetonitrile solution (which contains a final concentration of 0.1% TFA) was dissolved for more than 4 hours, and the resulting supernatant was analyzed by mass spectrometry using a mixed sample loading method.

(2) Identification by mass spectrometry

The mass spectrometry instrument 4800 produced by Applied Biosystems was adopted. The mass spectrometer parameters were laser intensity 2,500, mass range 65-95kDa, acceleration voltage 25kV, plate voltage 92%, scale 0.3%, and delay time 1,000ns. The obtained protein mass spectra were processed and analyzed by software.

Calculation of the number of cysteine residues: The number of cysteine residues in the high molecular weight glutenin subunits was calculated using the change in the molecular weight of the 4-vinylpyridine-treated and untreated protein during sample preparation.

The application value of the inventive method:

(1) Not only can the number of cysteine residues contained in the protein be obtained, but also the molecular weight of the protein can be obtained, so as to identify the allelic composition and wheat varieties and realize the rapid screening and selection of high-quality subunits in the early hybrid generation , greatly improving the efficiency of wheat quality improvement.

(2) The number of cysteine residues can be detected for the high-molecular-weight glutenin subunits of common wheat and its related species, so as to provide help for further research on the function of disulfide bonds.

(3) It can be used to detect the number of cysteine residues in glutenin subunits in wheat and its close relatives on a large scale, thereby rapidly screening new high-quality glutenin candidate subunits, and the method of the present invention is faster than gene identification ,more acurrate.

The present invention utilizes the accuracy of mass spectrometry to invent a method for identifying the amount of cysteine in wheat high molecular weight glutenin subunits using mass spectrometry technology, which can be applied to wheat quality improvement and high molecular weight glutenin subunits Rapid detection of the number of cysteine, so as to discover and screen high-quality subunits, and improve the efficiency of quality improvement. In addition, the method of the present invention can also identify the number of cysteine residues contained in the salt-soluble protein and water-soluble protein in lupine, and then form a complete technical system for identifying the number of cysteine residues.

Description of drawings

Fig. 1 is the mass spectrogram comparison pattern of high molecular weight glutenin subunits treated with 4-vinylpyridine and untreated in common wheat variety Bumper;

Fig. 2 is the comparison spectrum of mass spectrometry of high molecular weight glutenin subunits treated with 4-vinylpyridine and untreated in common wheat variety Shan229;

Fig. 3 is the identification pattern of the number of cysteine residues in lupine treated with 4-vinylpyridine and untreated salt-soluble protein;

Fig. 4 is a chart for identifying the number of cysteine residues in lupine water-soluble proteins treated with 4-vinylpyridine and untreated.

Detailed ways

The experimental materials involved in the present invention are 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 Soybean (Wang Ke, Molecular cloning and phylogenetic research on glutenin coding genes of common wheat and its close relatives, doctoral dissertation of Capital Normal University, 2011), the above biological materials were bred and preserved by the Genetic and Bioengineering Laboratory of Capital Normal University .

Example 1, identification of the number of cysteine residues in wheat high molecular weight glutenin subunits

1. Extraction and sample preparation of wheat high molecular weight glutenin subunits

Take two portions of 15 mg of wheat Bumper flour, put them into 1.5 ml centrifuge tubes respectively; add 1 ml of 70% ethanol by volume, vortex for 30 min, centrifuge at 12000 rpm for 10 min, and remove the supernatant. Then add 1 ml of isopropanol with a volume percentage of 55% respectively, bathe in water at 65° C. for 30 min, centrifuge at 12000 g for 10 min, and remove the supernatant. Then the remaining supernatant was blotted dry with filter paper, and the above steps were repeated 3 times. Then add 0.1ml of DTT-containing solution A (the mass percentage of DTT and solution A is 1:100), wherein the composition of solution A is, isopropanol: 1M Tris-HCl (pH8): double distilled water=50:8: 42 (volume ratio); vortex to mix, 65 ° C water bath for 30 min.

Then, centrifuge at 12000g for 10min, take the supernatant from the first sample, and precipitate overnight at room temperature with acetone of 40% by volume; add 0.1ml of solution A (4-vinylpyridine and The volume percentage of solution A is 1.4:98.6), vortex mixing, 65 ° C water bath for 30 min; 12000 rpm centrifugation for 10 min; take the supernatant and use 40% volume percentage of acetone to precipitate overnight at room temperature; after overnight precipitation, both samples are centrifuged at 12000 rpm 10min, discard the supernatant, add 0.1ml of solution A containing DTT (the mass percentage of DTT and solution A is 1:100), bathe in water at 65°C for 30min; take the supernatant of the first sample and precipitate it with 40% by volume acetone at room temperature Overnight; add 0.1ml of solution A containing 4-vinylpyridine to the second sample (volume percentage of 4-vinylpyridine and solution A is 1.4:98.6), vortex and mix well, 30min in 65°C water bath; centrifuge at 12000rpm for 10min Get the supernatant and precipitate overnight at room temperature with 40% volume percent acetone; after the overnight precipitation, both samples are centrifuged at 12000rpm for 10min, discard the supernatant, dry the precipitation for 10min, and add 70 μl of chromatographic grade acetonitrile solution (which contains a final concentration of 0.1% TFA) was dissolved for more than 4 hours, and the resulting supernatant was subjected to mass spectrometry analysis by the mixed loading method.

The mass spectrometry results are shown in Figure 1. 1A is the mass spectrum of high molecular weight glutenin subunits in common wheat variety Bumper treated with 4-vinylpyridine, and FIG. 1B is the mass spectrum of high molecular weight glutenin subunits in common wheat variety Bumper not treated with 4-vinylpyridine.

2. Identification of the number of cysteine residues in glutenin subunits

(1) Instruments and equipment: a mass spectrometer 4800 produced by Applied Biosystems was used.

(2) Mass spectrometer parameters: laser intensity 2,500, mass range 65-95kDa, acceleration voltage 25kV, plate voltage 92%, scale 0.3%, delay time 1,000ns.

(3) Data processing and determination of the number of cysteine residues: use software for data processing and analysis. Calculation of the number of cysteine residues: As can be seen from Figure 1, the molecular weight of the 1Dx5 subunit in wheat Bumper increased by 517.67Da after adding 4-vinylpyridine; while other x- type and y-type HMW-GS increased by about 400Da and 700Da, respectively, than those without 4-vinylpyridine treatment. These results indicate that the 1Dx5 subunit contains 5 cysteine residues, and the other x-type and y The numbers of cysteine residues in -type HMW-GS were 4 and 7, respectively. These results are consistent with known results, illustrating the reliability of the method.

Embodiment 2, utilize the wheat variety that 17 known subunits are composed to verify the method of the present invention

1. Extraction and sample preparation of wheat high molecular weight glutenin subunits

Take Ajana, Banks, Bullaring, Bumper, Calingiri, Chara, EGA Blanco, Endure, IGW2944, IGW3240, Pugsley, M12, M25, M26, Shan229, Wanmai33, Halberd and other 17 different varieties of wheat seeds, sampling and sample preparation and mass spectrometry Identification is equivalent to Example 1.

The mass spectrometry results of wheat variety Shan229 are shown in Figure 2. 2A is the mass spectrum of high molecular weight glutenin subunits in common wheat variety Shan229 treated with 4-vinylpyridine, and FIG. 2B is the mass spectrum of high molecular weight glutenin subunits in common wheat variety Shan229 not treated with 4-vinylpyridine.

The mass spectrometry results of 17 different wheat varieties are shown in Table 1 and Table 2.

Table 117 Detection results of the number of cysteine residues in high molecular weight glutenins encoded by Glu-A1 and Glu-B1 sites in wheat varieties with known high molecular weight glutenin subunit composition

Table 217 Detection results of the number of cysteine residues in the high molecular weight glutenin encoded by the Glu-D1 site in wheat varieties with known high molecular weight glutenin subunit composition

2. Data analysis

As can be seen from Table 1, both the special 1Bx20 subunits and 1Dx5 subunits containing 2 and 5 cysteine residues, and all other conventional high molecular weight subunits containing 4 cysteine residues Glutenin subunits, the increased molecular weight of the high molecular weight glutenin subunits treated with 4-vinylpyridine corresponds to the number of cysteine residues they contain, that is, each high molecular weight glutenin subunit contains one and a half The molecular weight of the cystine residue will increase by about 105.14Da accordingly.

Example 3, using the number of cysteine residues in salt-soluble protein and water-soluble protein in lupine to verify the method of the present invention

1. Sample extraction and preparation

Salt-soluble protein: Take two 15mg lupine powders, add 0.2ml of 0.5M NaCl solution to one of the samples, vortex for 30 minutes, and then add 0.2ml of 0.5M containing 4-vinylpyridine to the other sample M NaCl solution (volume percent of 4-vinylpyridine and 0.5M NaCl solution is 1.4:98.6), vortexed, 30min in a water bath at 65°C, and centrifuged at 12000rpm for 10min; Acetone precipitated overnight at room temperature; after overnight precipitation, both samples were centrifuged at 12000rpm for 10min, the supernatant was discarded, the precipitate was dried for 10min, and 70 μl of chromatographic grade acetonitrile solution (which contained TFA with a final concentration of 0.1%) was added, dissolved for more than 4 hours, and the obtained The supernatant was analyzed by mass spectrometry using the mixed loading method.

Water-soluble protein: Take two 15mg lupine powders, add 0.2ml of distilled water to one of the samples, vortex for 30 minutes, then add 0.2ml of 1.4% 4-vinylpyridine aqueous solution to the other sample , vortexed and mixed, 65 ℃ water bath for 30min, 12000rpm centrifugation for 10min; take the supernatant and precipitate overnight at room temperature with 80% volume percent acetone; after overnight precipitation, both samples were centrifuged at 12000rpm for 10min, discard the supernatant, and dry the precipitate for 10min. 70 μl of chromatographic grade acetonitrile solution (containing TFA with a final concentration of 0.1%) was added, dissolved for more than 4 hours, and the obtained supernatant was subjected to mass spectrometry analysis by the mixed loading method.

2. Spectrum analysis

The number of cysteine residues per subunit was calculated from the difference in molecular weight between 4-vinylpyridine-treated and untreated proteins. The results are shown in Figure 3 and Figure 4. Wherein, Fig. 3A is the mass spectrum of the salt-soluble protein of lupine treated with 4-vinylpyridine, and Fig. 3B is the mass spectrum of the salt-soluble protein of lupine not treated with 4-vinylpyridine. Fig. 4A is the mass spectrum of water-soluble protein of lupine treated with 4-vinylpyridine, and Fig. 4B is the spectrum of water-soluble protein of lupine not treated with 4-vinylpyridine.

Between Figure 3A and 3B, only the molecular weight difference of the subunit marked with a box is about 100 Da, while the molecular weight of the other subunits changes very little within the error range, so only the first subunit contains 1 cysteine acid residues, the other two subunits do not contain cysteine residues.

The molecular weight difference between Figures 4A and 4B for all subunits is about 100 Da, therefore, all subunits contain 1 cysteine residue.

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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