CN1800812A - Mass spectrum method for identifying low-molecular-weight glutenin subunit of wheat - Google Patents
Mass spectrum method for identifying low-molecular-weight glutenin subunit of wheat Download PDFInfo
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Abstract
The invention discloses a precise mass spectrum method to identify wheat low-molecular-weight glutelin subunit, which comprises: (1) to prepare the sample, using 35-45% acetone to deposit protein extract and centrifuge, selecting supernatant, and using 75-85% acetone to deposit the glutelin subunit; (2) using dissolution liquid to dissolve the dissolution; wherein, the dissolution liquid comprises 15-25v% acetonitrile, 0.1v% TFA, and water.
Description
Technical field
The invention belongs to life science protein technique field, particularly the accurate evaluation of low-molecular-weight glutenin subunit of wheat molecular weight and based on the further sign of this technical method to glutenin subunit.
Background technology
Wheat is cultivated area maximum, output is the highest, geographic distribution is the widest important crops in the world, and its cultivated area and output account for 30% of bread crop, are widely used in food processing industry and cattle breeding industry.Contain the mucedin that is short of in other cereal crops in the wheat seed, can make multiple special foods such as bread, noodles, biscuit, cake and steamed bun.Therefore, the composition of seed albumen and content determine the quality of wheat processing quality to a great extent.
Osborne (1907) is divided into following four classes according to the difference of dissolving characteristics with wheat seed albumen: albumin and globulin, gliadin, glutenin.Studies show that the wheat gluten polymer is protein molecular (Wrigley, 1996 of nature maximum; Stevenson and Preston, 1996).Usually said wheat storage protein is meant alcohol soluble protein and glutenin, and wherein glutenin accounts for about 40% of storage protein.According to the mobility difference in SDS-PAGE under the reducing condition, glutenin can be divided into two classes again: high-molecular-weight glutelin subunit (HMW-GS) and low-molecular-weight glutenin subunit (LMW-GS).The composition of HMW-GS and content directly influence the baking quality of wheat flour, and LMW-GS is then closely related with dough elasticity.
Studies show that in a large number the allelic variation in LMW-GS site and quality of wheat difference have high correlation.Payne in 1984 etc. find that at first LMW-GS forms and the tetraploid quality has direct relation, the kind that for example has LMW-1 type electrophoresis pattern often has relatively poor processing quality, kind with LMW-2 type electrophoresis pattern is then on the contrary, and characteristic often has fine qualities.Discovery such as Nieto-Taladriz in 1997 constitutes the subunit of LMW-1, LMW-2 type and is controlled by the allele on Glu-3, the Glu-2 site.Luo etc. (2001) find when relatively common wheat HMW-GS and LMW-GS allelic variation are to the flour quality influence, the allelic variation in Glu-3 site has remarkable influence to the relevant technical parameter of wheat quality, as flour protein content, SDS precipitation number, seed hardness etc.Up to now, it is also fewer to the influence of wheat quality to study single LMW-GS.Sissons in 1998 etc. have separated some LMW-GS with RP-HPLC and have carried out quality research, find that the N-end can improve the mixed characteristic of flour for the LMW-GS of METSHIPGL-.Lee in 1999 etc. have carried out Study on Quality to 3 LMW-GS of bacterial expression, discovery recently can more effectively improve flour from the genomic LMW-16/10 of one grained wheat D from the genomic LMW-E2 of one grained wheat A and LMW-E4 and close the face characteristic, increases the ductility of dough.
Based on the difference of mobility in SDS-PAGE, LMW-GS can be divided into B, C and D type subunit.The relative molecular mass of Type B subunit is 40000-50000Da, and the relative molecular mass of C type subunit is 30000-40000Da, and D type subunit is a very small part, only occurs in the minority wheat breed.The gene loci of coding Type B subunit is in the Glu-3 site of the homology group of first the short arm of a chromosome.The gene loci of coding C type subunit may be in the Gli-1 site or the Gli-2 site on the 6th homoeologous group the short arm of a chromosome of the homology group of first the short arm of a chromosome.D type subunit is perhaps in the Gli-1 site.Studies show that the low-molecular-weight glutenin subunit gene loci closely links to each other with the gene loci of alcohol soluble protein, as the Gli-1 site, contain 3 kinds of protein genes: ω alcohol soluble protein gene, γ alcohol soluble protein gene and low-molecular-weight glutenin subunit gene.
The Type B subunit has similar structure with C type subunit.Central authorities all are made up of the repetitive sequence of proline and paddy ammonia phthalein amine the duplicate block, and these repetitive sequences mainly form βZhuan Jiao.The N end is a very short non repetitive sequence, and the C end also is made up of non repetitive sequence, and its secondary structure is mainly the α spiral, and comprises its all cysteine residues.The position of 8 cysteine residues of C end is a high conservative.6 cysteine residues in 1～No. 8 (1-5 and No. 7) are arranged in the Type B subunit, also have one 6 ' to hold in addition at C.Order-checking to the Type B low-molecular-weight glutenin subunit is found: some Type B low-molecular-weight glutenin subunits also contain the another one cysteine residues.Gene sequencing to coding Type B low-molecular-weight glutenin subunit shows, its N end should have a cysteine residues, but in the Type B low-molecular-weight glutenin subunit of most of wheat breeds, but do not find, and find to have a cysteine residues at the N of central duplicate block end.
C type low-molecular-weight glutenin subunit and α or γ alcohol soluble protein structural similarity.May be because sudden change has taken place alcohol soluble protein gene, make it can form intermolecular disulfide bond, so belong to the glutelin class.Alcohol soluble protein is a monomeric protein, can not form aggregation.C α type and C γ type contain 6 and 8 conserved cysteine residue respectively, yet the gene order of C α type subunit is presented at the N end cysteine residues should be arranged.N end in C γ type central authorities duplicate block has a cysteine residues.In addition, in C γ type subunit central authorities duplicate block, a cysteine residues has appearred in addition in some wheat breeds.
D type low molecular weight glutenin subunit is the same with C type subunit to have similar biochemical characteristic to alcohol soluble protein, even amino acid sequence also is like this.D type subunit is the same with ω type alcohol soluble protein to belong to poor sulfoprotein, is made up of very little N end, C end and central duplicate block.Glutamine is rich in the duplicate block in central authorities, and proline and phenylalanine are made up of the bracing βZhuan Jiao.It is generally acknowledged that D type low-molecular-weight glutenin subunit may be to cause a cysteine residues can form intermolecular disulfide bond by the sudden change of ω type alcohol soluble protein encoding gene, forms condensate and causes.
Compare with HMW-GS, the composition of low molecular weight glutenin subunit is more complicated, and a kind generally has 20-50 gene.Therefore, for a long time owing to lack effective separation method, compare with HMW-GS, research to LMW-GS both at home and abroad relatively lags behind, because conventional separation method SDS-PAGE resolution is not high, complicated operation, resulting molecular weight often differ bigger with the molecular weight of reality, therefore are badly in need of setting up new, more effective isolation identification technology.
Summary of the invention
The objective of the invention is to establish a kind of stable, mass spectrometry method fast, thereby can carry out accurate molecular weight determination low-molecular-weight glutenin subunit of wheat.By this proteomic techniques, can identify the molecular weight of low-molecular-weight glutenin subunit of wheat fast and accurately, analyze the low-molecular-weight subunit of wheat gluten and form, thus the wheat of evaluation and sign different cultivars.
The technical solution used in the present invention is:
A kind of mass spectrometry method of identifying low-molecular-weight glutenin subunit of wheat comprises the steps:
1. when the mass spectrum sample of preparation low-molecular-weight glutenin subunit of wheat, earlier with the acetone precipitation protein extract of 35-45%, get supernatant after centrifugal, use low-molecular-weight glutenin subunit in the acetone precipitation clear liquid of 75-85% again;
2. obtaining the low-molecular-weight glutenin subunit post precipitation, with lysate dissolving wheat low-molecular-weight glutenin subunit, the proportioning of lysate is: with the lysate volume is mete-wand, the volume of acetonitrile solution is the 15-25% of lysate volume in the lysate, the shared volume of TFA is 0.1% of a lysate volume, and all the other are water.
When the mass spectrum sample of preparation low-molecular-weight glutenin subunit of wheat, with the acetone precipitation protein extract of 35-45%, purpose is to remove earlier the high-molecular-weight glutelin subunit in the protein extract earlier.
The mensuration of low-molecular-weight glutenin subunit of wheat molecular weight mainly adopts the SDS-PAGE gel electrophoresis method at present.But the molecular weight of SDS-PAGE mensuration protein is according to the linear relationship between the logarithm of the mobility of protein and molecular weight it to be carried out the mensuration of molecular weight; but relative mobility and its molecular weight of some protein in the SDS system is not linear; the protein of or abnormal conformation unusual as electric charge; histone F1 itself has a large amount of positive charges; in conjunction with SDS be not enough to eliminate the influence of original positive charge; have the protein (as some glycoprotein) of big prothetic group and some structural proteins (for example collagen) and some contain the more protein of disulfide bond, the relative mobility of electrophoresis and the logarithm of molecular weight are not linear.So there is very mistake in traditional SDS-PAGE to the mensuration of molecular weight of albumen.Mass spectrum is the method for carrying out molecular weight determination by the mass-to-charge ratio of working sample ion, and its degree of accuracy is high.The present invention is a core with the mass-spectrometric technique, has set up the accurately method of mensuration low-molecular-weight glutenin subunit of wheat molecular weight of a cover.
Mass spectrum is the method for carrying out composition and structure analysis by the mass-to-charge ratio of working sample ion, and its development has benefited from the development (Zhang Guoan etc., 2003) of two kinds of soft ionization technology: electron spray ionisation and substance assistant laser desorpted ionized (MALDI).The electron spray ionisation method is to utilize high electric field to make liquid droplet charged that the capillary column of mass spectrum sample introduction end flows out under the effect of N air-flow, and the drop explosion also finally becomes spray form, and analyte ionsization enters mass analyzer in electrically charged mode.The ion that the MALDI ion gun produces mostly is single charge ion, and the mass number in the spectrum peak in the mass spectrogram and each component of sample has one-to-one relationship, and MALDI has strong tolerance to salt and surfactant, suitable high throughput analysis.Therefore the most suitable analysis polypeptide of MALDI-MS and protein mixture, this instrument are the highest mass spectrometers of sensitivity now, can analyze the denier sample.
Mass spectrum is mainly used in and solves two problem analyses: accurately measure biomacromolecule, as the molecular weight of protein, nucleotide, carbohydrate etc., and provide molecular structure information; To being present in the trace in the life complex system or trace small molecule bioactive material carries out qualitative or quantitative test (Wu Shirong etc., 2004).The mensuration of protein-based biomacromolecule molecular weight has crucial meaning, as the mensuration to the homogeneous prlmary structure of protein, should measure the molecular weight of protein, measures the molecular weight of subunit and oligomer again, and the molecular weight of hydrolysis, enzymatic fragment.Conventional molecular weight determination mainly contains osmometry, light scattering method, supercentrifugation, gel chromatography and polyacrylamide gel electrophoresis etc.These methods exist the sample consumption big, the shortcomings (Zhao Shankai etc., 1996) such as shape influence that degree of accuracy is low, be subject to protein.
The present invention utilizes substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF-MS), fast characterizing wheat LMW-GS, and a kind of new method of definite its accurate molecular weight, and this method has following feature:
(1) amount of samples is few, and general half granule seed (about 20mg) can be satisfied the demand.
(2) extracting method is simple, no toxic reagent, and usually contain toxic reagent in the SDS-PAGE extract, the specimen preparation time is long.
(3) used time weak point separates rapidly, is fit to high throughput analysis.SDS-PAGE separates LMW-GS and generally needs 1-2 hour (sulculus thin layer glue) or more than 10 hour (vat), through dyeing, decolouring, to the end electrophoresis pattern often need 1-2 days, and MALDI-TOF-MS analyzes a sample and only needs a few minutes.
(4) resolution height.The subunit that is difficult to distinguish in the SDS-PAGE collection of illustrative plates is easy to distinguish in mass spectrum.
(5) detect sensitive sensitivity.The general 10-100pmol protein sample that only needs can detect, and the applied sample amount of SDS-PAGE needs micro updating at least.
(6) detectable relative molecular mass scope wide (upper limit can reach hundreds of thousands).
(7) resolution (can reach 600m/ Δ m) and degree of accuracy height (internal standard method can reach 0.01%).
(8) result preserves and analyzes easily.Directly, only need do simple process and promptly can be used for analyzing relatively to scheme the sheet mode storage.
(9) can obtain accurate molecular weight information, precision is higher than the SDS-PAGE method far away.
It is extremely important to the structure and the function of further research protein to obtain accurate molecular weight information, particularly protein post-translational modification (Protein translational modifications, research PTMs).Therefore, mass-spectrometric technique probably becomes the microtechnique that a kind of high sensitivity is identified low-molecular-weight glutenin subunit of wheat, has broad application prospects.
Description of drawings
Fig. 1: hexaploid bread wheat kind " capital 411 " (Triticum aestivum L.) low-molecular-weight glutenin subunit SDS-PAGE (A) and mass spectrum are identified (B), among Figure 1A, 1 is whole glutenins, 2 is low molecular weight glutenin, and 3 are high molecular weight glutenin (containing the part low-molecular-weight glutenin);
Fig. 2: the mass spectrum of tetraploid wild emmer (Triticum dicoccoides) YS-5 and YS-13 low-molecular-weight glutenin subunit is identified;
Fig. 3: the mass spectrum of dliploid aegilops tauschii (Aegilops tauschii) low-molecular-weight glutenin subunit is identified;
Fig. 4: the mass spectrum of einkorn wheat (Triticum monococcum) TM-1 and TM-3 low-molecular-weight glutenin subunit is identified.
Embodiment
The SDS-PAGE of embodiment 1 low-molecular-weight glutenin subunit of wheat identifies
(1) vegetable material: wheat breed " capital 411 ".
(2) extraction of LMW-GS
The 20mg seed is pounded the powdered 1.5ml of putting into centrifuge tube, adds 70% ethanol, 150 μ l, vortex 30-60min, and the centrifugal 10min of 10000g removes supernatant.Add isopropyl alcohol 250 μ l, 65 ℃ of water-bath 30min, the centrifugal 10min of 10000g removes supernatant and blots with filter paper, repeats 3 times.Add 0.1ml 50% isopropyl alcohol (contain 80mM Tris-HCl[pH8.0]+1%DTT) vortex mixing, 65 ℃ of water-bath 30min.Add 0.1ml50% isopropyl alcohol (contain 80mM Tris-HCl[pH8.0]+1.4% 4-vinylpyridine) vortex mixing again, 65 ℃ of water-bath 30min, the centrifugal 15min of 12000g.Get supernatant with 40% acetone precipitation at room temperature 3 hours, the centrifugal 15min of 12000g collects supernatant; Supernatant is spent the night-20 ℃ of precipitations with 80% acetone, the centrifugal 15min of 13000g, collecting precipitation, precipitation is low-molecular-weight glutenin subunit of wheat.
(3) SDS-PAGE of LMW-GS separates and identifies
1. the preparation of SDS-PAGE electrophoresis sample adds sample buffer (2%SDS, 0.02% bromophenol blue, the 0.08M Tris-HCl of 100 μ l in precipitation, pH8.0,40% glycerine), vibration is 30 minutes in 65 ℃ of water-baths, centrifugal 10 minutes of 12000g makes SDS-PAGE and goes up sample usefulness.
2. the SDS-PAGE electrophoresis utilizes Boi-Rad Mini-PROTEAN 3 electrophoresis tanks, 5% to concentrate glue, 12% separation gel, Tris-glycine buffer liquid systems, 15mA electrophoresis 2 hours, 0.1% Coomassie brilliant blue R-250 dyeing, 10% ethanol and the decolouring of 10% acetate, electrophoresis pattern is shown in Figure 1A.
The MALDI-TOF-MS of embodiment 2LMW-GS identifies
(1) vegetable material
The hexaploid bread wheat (Triticum aestivum L., AABBDD): kind " capital 411 ".
The tetraploid wild emmer (Triticum dicoccoides, AABB): YS-5, YS-13
The dliploid aegilops tauschii (Aegilops tauschii, DD): TD121, TD128, TD132
Dliploid einkorn wheat (Triticum monococcum L., A
mA
m): TM1, TM3
(2) preparation of mass spectrophotometry sample
The 20mg seed is pounded the powdered 1.5ml of putting into centrifuge tube, adds 0.5M NaCl solution 100 μ l, vortex 30-60min, and the centrifugal 10min of 5000g removes supernatant.Add ddH
2O 200 μ l, vortex 30min, the centrifugal 10min of 5000g removes supernatant, repeats 3 times.Add 0.4ml 50% n-propanol vortex mixing, room temperature is placed 30min, and the centrifugal 5min of 12000g removes supernatant, and blots only with filter paper, and this step repeats 3 times.Adding 0.2ml 50% n-propanol (contain 80mM Tris-HCl[pH8.5]+1%DTT), vibration mixing, 65 ℃ of water-bath 30min.Get supernatant with 40% acetone precipitation at room temperature 3 hours, the centrifugal 15min of 13000g collects supernatant.Supernatant is spent the night-20 ℃ of precipitations with 80% acetone, the centrifugal 15min of 13000g, collecting precipitation, precipitation is low-molecular-weight glutenin subunit of wheat.Use the vacuum drying instrument drying precipitated then, add 50 μ l, 20% acetonitrile solutions (containing 0.1%TFA) dissolving.
(3)MALDI-TOF-MS
Use Tianjin, island AX1MA-CFRTMPlus type ground substance assistant laser desorption ionization flight mass spectrometer (be furnished with software and be used for system operation and data processing), full-automatic sample preparation, software control is accurately located, and has to postpone to extract cracking (PSD) function behind (DE), the source.
1. the cleaning of target plate (384 type) picks methyl alcohol with cotton balls, wipes the sample on the target plate, the ultrasonic 10-15min of 1% formic acid, ddH
2The O flushing adds the ultrasonic 10-15min of acetone, adds the ultrasonic 10-15min of methyl alcohol, adds the ultrasonic 10-15min of water, takes out target plate, uses washed with methanol, and is dry in the fuming cupboard.
2. choice of base is selected sinapic acid SA (3,5-dimethoxy-4 '-hydroxycinnamic acid, 3,5-Dimethexycinnamic acid) for use with preparation matrix, and 10mg matrix is dissolved among 1ml50% acetonitrile+0.05%TFA, keeps in Dark Place.
3. the preparation of standard specimen and the mensuration of selecting the LMW-GS molecular weight: Albumin-Aldrase (66431.08,33216.0,39212.88), the 0.1%TFA dissolving.
4. go up sample pre-treatment 1%TFA balance ZIPtip, filtered sample is aspirated 5-10 time repeatedly, and 1%TFA filters, 0.4 μ l Elution Buffer elution samples.
5. the mensuration that molecular weight is set of parameter adopts linear model, molecular weight ranges 10000-100000Da.
6. point sample (Sanming City therapy) matrix 0.5 μ l, sample 0.5 μ l, matrix 0.5 μ l.
7. mass spectroscopy is treated behind the sample drying target plate to be put into mass spectral sample chamber, carries out according to following program: the correction of the mensuration of the correction → standard specimen of target plate position, correction (selecting Average, Threshold-Apex for use) → sample, mensuration (selecting the instrument parameter consistent with standard specimen) → data are preserved.
8. Flame Image Process utilizes KratosAnalytical ltd.lanchpad V2.4.0 software to carry out Flame Image Process.According to result, can obtain the accurate molecular weight that different cultivars LMW-GS forms collection of illustrative plates and each subunit, shown in Figure 1B.
The application of embodiment 3 low-molecular-weight glutenin subunit mass spectrum authentication methods
(1) LMW-GS characterizes with wheat breed and identifies
Common wheat is allohexaploid kind (2n=6x=42), many abundant sibling species matter resources are arranged, comprise dliploid aegilops tauschii (DD), dliploid cultivation one (AA), tetraploid wild two and cultivation emmer wheat (AABB) etc., in these sibling specieses, the LMW-GS variation is very abundant, containing a large amount of high-quality candidate genes, is the important gene resource of bread wheat quality-improving.Therefore effectively identification of means is the key that makes full use of these resources.Simultaneously, the LMW-GS collection of illustrative plates can be used as the reliable mark of cultivar identification, and is significant to marker assisted selection in the breeding and the protection of kind rights and interests.Utilize the LMW-GS mass spectrum authentication method set up, the wheat of Different Ploidy is carried out the evaluation of low-molecular-weight glutenin subunit, obtained the LMW-GS collection of illustrative plates (result such as Fig. 1-shown in Figure 4) of high-resolution.According to the mass-spectrogram of these low-molecular-weight glutenin subunits, can characterize and identify different wheat breeds or germplasm.In view of the high sensitivity and the pinpoint accuracy of mass-spectrometric technique, the isolation identification effect of LMW-GS obviously is better than traditional SDS-PAGE method.
(2) accurate molecular weight of LMW-GS is determined
The MALDI-TOF-MS method can accurately be measured the molecular weight of low-molecular-weight glutenin subunit of wheat, for further its structure of research and function lay the foundation.The measurement result of the different sibling species low-molecular-weight glutenin subunit of wheat molecular weight is seen Fig. 2-4 figure.The quantity of the LMW-GS that mass spectrometry method detects is more than SDS-PAGE, and the molecular weight precision that records is also much higher, so this technology is accurately to measure the effective ways of low-molecular-weight glutenin subunit of wheat molecular weight.
(3) LMW-GS posttranslational modification (PTMs) is identified
The flower characters of wheat and the composition of glutenin subunit are closely related, and dissimilar glutenin subunits is for the contribution difference of gluten quality.Yet, there are some researches show that also even same subunit, the contribution to flower characters in different wheat breeds is also incomplete same.At this phenomenon, infer that may there be the posttranslational modification phenomenon in the wheat glutenin subunit, modification in various degree might cause in the different cultivars same subunit different to the contribution of flower characters as glycosylation and phosphorylation etc.Yet, in research, obtained different results of study in recent years to the posttranslational modification of wheat glutenin subunit.Someone thinks that there is the phenomenon of glycosylation and phosphorylation modification in the wheat glutenin subunit, and the degree that generation is modified is higher, has the people to think that then there is not posttranslational modification in glutenin subunit.The reason that produces this result is because the difference of research method causes to a great extent.The foundation of MALDI-TOF-MS accurate molecular weight assay method will further promote the further investigation of low-molecular-weight glutenin subunit posttranslational modification.
Molecular weight size by the gluten subunit relatively measured with mass spectrometry method and with the derive difference of the gluten subunit molecular weight size that obtains of gene order can tentatively be judged the modification situation after the wheat glutenin subunits translation.When molecular weight difference surpasses 0.1% (mass spectrometer permissible error) of gene order derivation, can infer tentatively that the posttranslational modification of certain form may take place gluten subunit.
According to above imagination, we adopt pair of alleles specific PCR primer to einkorn wheat (Triticum monococcum L., A
mA
m, 2n=2x=14) genomic DNA of TM-1 and TM-3 increases, and obtains 2 different full gene coded sequences, comprises upstream promoter, open reading frame and downstream sequence, intronless.According to the amino acid sequence of deriving, 2 gene LMW-M1 and LMW-M3 are typical LMW-i type subunit, and molecular weight is respectively 38708.7Da and 38720.8Da.Extract the LMW gluten subunit of TM-1 and TM-3 then, utilize MALDI-TOF-MS to analyze the accurate molecular weight that has obtained above-mentioned 2 subunits, be respectively 38789.6Da and 39026.9Da (the results are shown in Figure 4).Through comparing, the molecular weight of finding the LMW-M1 subunit and gene derivation very near (less than 0.1%), and the molecular weight difference of LMW-M3 subunit is greater than permissible error, infer that thus posttranslational modification does not take place the LMW-M1 subunit, then there is the posttranslational modification of certain form in the LMW-M3 subunit, as glycosylation and phosphorylation modification etc.Can further carry out LMW-GS posttranslational modification type, decorating site and functional study thereof on this basis.
Claims (3)
1. a mass spectrometry method of identifying low-molecular-weight glutenin subunit of wheat comprises the steps:
(1) when the mass spectrum sample of preparation low-molecular-weight glutenin subunit of wheat, earlier with the acetone precipitation protein extract of 35-45%, get supernatant after centrifugal, use low-molecular-weight glutenin subunit in the acetone precipitation clear liquid of 75-85% again;
(2) obtaining the low-molecular-weight glutenin subunit post precipitation, with lysate dissolving wheat low-molecular-weight glutenin subunit, the proportioning of lysate is: with the lysate volume is mete-wand, the volume of acetonitrile solution is the 15-25% of lysate volume in the lysate, the shared volume of TFA is 0.1% of a lysate volume, and all the other are water.
2. to play the mass spectrometry method of 1 described evaluation low-molecular-weight glutenin subunit of wheat as right, it is characterized in that when the mass spectrum sample of preparation low-molecular-weight glutenin subunit of wheat, earlier with 40% acetone precipitation protein extract, get supernatant after centrifugal, use the low-molecular-weight glutenin subunit in 80% the acetone precipitation clear liquid then.
3. to play the mass spectrometry method of 1 described evaluation low-molecular-weight glutenin subunit of wheat as right, it is characterized in that with lysate dissolving wheat low-molecular-weight glutenin subunit, the optimum ratio of lysate is: with the lysate volume is mete-wand, the volume of acetonitrile solution is 20% of a lysate volume in the lysate, the shared volume of TFA is 0.1% of a lysate volume, and all the other are water.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101201337B (en) * | 2006-12-15 | 2012-06-13 | 首都师范大学 | Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit |
| CN103278453A (en) * | 2013-03-09 | 2013-09-04 | 青海省农林科学院 | Method for obtaining wheat root related drought resistant protein through utilizing dimensional electrophoresis and MALDI-TOF-MS technology |
| CN108828049A (en) * | 2018-08-27 | 2018-11-16 | 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） | A kind of high low molecular weight glutenin subunit SDS-PAGE separation method of wheat |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100337113C (en) * | 2004-04-30 | 2007-09-12 | 首都师范大学 | Acidic capillary electrophoresis identification method for high molecular weight glutelin subunit of wheat |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101201337B (en) * | 2006-12-15 | 2012-06-13 | 首都师范大学 | Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit |
| CN103278453A (en) * | 2013-03-09 | 2013-09-04 | 青海省农林科学院 | Method for obtaining wheat root related drought resistant protein through utilizing dimensional electrophoresis and MALDI-TOF-MS technology |
| CN108828049A (en) * | 2018-08-27 | 2018-11-16 | 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） | A kind of high low molecular weight glutenin subunit SDS-PAGE separation method of wheat |
| CN108828049B (en) * | 2018-08-27 | 2020-05-12 | 河北省农林科学院遗传生理研究所（河北省农林科学院农产品质量安全研究中心） | SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separation method for high and low molecular weight glutenin subunits of wheat |
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