Method for identifying quantity of cysteine in protein and application thereof
A technology of cysteine residues and proteins, which is applied in the field of identifying the number of cysteine in proteins to achieve the effect of improving efficiency
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Embodiment 1
[0026] Example 1, identification of the number of cysteine residues in wheat high molecular weight glutenin subunits
[0027] 1. Extraction and sample preparation of wheat high molecular weight glutenin subunits
[0028] 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...
Embodiment 2
[0035] Embodiment 2, utilize the wheat variety that 17 known subunits are composed to verify the method of the present invention
[0036] 1. Extraction and sample preparation of wheat high molecular weight glutenin subunits
[0037] 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.
[0038] The mass spectrometry results of wheat variety Shan229 are shown in Figure 2. in, Figure 2A Mass spectrum of high molecular weight glutenin subunits in common wheat variety Shan229 treated with 4-vinylpyridine, Figure 2B It is the mass spectrum of high molecular weight glutenin subunits in common wheat variety Shan229 without 4-vinylpyridine treatment.
[0039] The mass spectrometry results of 17 different wheat varieties are shown in Table 1 and T...
Embodiment 3
[0046] 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
[0047] 1. Sample extraction and preparation
[0048] 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 ...
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