CN101201337B - Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit - Google Patents
Method for identifying biomass spectrum of wheat macromolecule weight glutelin subunit Download PDFInfo
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Abstract
The invention discloses a mass spectroscopy to examine the high molecular weight glutenin subunit of wheat, and belongs to the technical field of Proteome of life sciences, which in particular relates to accurate examination of molecular weight of the high molecular weight glutelin subunit of wheat and further characterization of the glutenin subunit based on the technology. The invention comprises the following steps: (1) preparation of biologic mass spectrometric analysis specimens of the high molecular weight glutenin subunit of wheat; (2) dissolving the high molecular weight glutenin subunit with dissolving solution; (3) making use of matrix assisted laser desorption-ionization time of flight mass sepectrometry for examination of the high molecular weight glutenin subunit of wheat. The method has the characteristics of use of few specimens, simple method of extraction, no toxic reagent, quick separation, high distinguishability, high detection sensitivity and so on.
Description
Technical field
The invention belongs to the phytoprotein group field that learns a skill, particularly the biological mass spectrometry method accurately identified of wheat high-molecular-weight glutelin subunit and molecular weight thereof and based on the further sign of the method to glutenin subunit.
Background technology
Wheat is the widest important cereal crops that distribute in the world, and its endosperm contains the mucedin that is short of in other cereal crops, therefore can make multiple special foods such as bread, noodles, biscuit, cake and steamed bun.Research shows that the composition of seed albumen and content determine the quality of flour processing quality to a great extent.Wheat seed albumen can be divided into albumin, globulin, alcohol soluble protein and glutenin according to the difference of dissolving characteristics, and they form the glutelin polymer through disulfide bond, thus the viscoelasticity of decision dough.Have research to show, the wheat gluten polymer is the maximum protein molecular of nature, and its molecular weight can reach millions of and even up to ten million (Wrigley, 1996; Stevenson and Preston, 1996).Usually said wheat storage protein mainly refers to alcohol soluble protein and glutelin, and wherein glutenin accounts for about 40% of storage protein.Different according to the mobility in SDS-PAGE under the reducing condition, can glutenin be divided into two types of high-molecular-weight glutelin subunit (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) again.HMW-GS only accounts for 10% of wheat storage protein, because of it plays very important effect (giving flour elasticity) in forming high molecular polymer, so its composition and function directly influence the baking quality of wheat flour.
The correlative study of HMW-GS and baking properties is carried out early.Payne (1987) etc. marks to the subunit baking properties according to precipitation number difference between subunit, for closely linked site, then with subunit to being unit attending to judge.What grading system was the highest is that the 1Dx5+1Dy10 subunit is right, is thereafter 1Ax1,1Ax2
*, 1Bx17+1By18,1Bx7+1By8, and the subunit inferior quality of 1Dx2+1Dy12, the grading system of 1Bx6+1By8 is minimum, and infers that 1Bx7 is better than 1Bx6 in B group subunit, 1By8 is better than 1By9.Seilmeier etc. (1991) then choose single subunit 1Dx5,1By9,1Dy10 according to the mensuration of anti-stretching force has quality preferably, and 1By8 and 1Dy12 are relatively poor relatively.Payne etc. (1983) have reported that also the subunit 1Dx2.2 that originates from Japan is a subunit relevant with high-quality.Result of study finds that also 1Bx13+1By16,1Bx4+1By12 also have vital role to flavor evaluation, and wherein the scoring of 1Bx4+1By12 is only second to 1Dx5+1Dy10.In addition, 7+9 and 14+15 also have forward effect (Liu Li etc., 2003) to baking properties.
The amino acid sequence of ripe HMW-GS can be divided into three structural regions: the duplicate block at conservative N-petiolarea, non-repetitive C-petiolarea and middle part.N-end and C-end have very high conservative property in the HMW glutelin; Wherein N-holds non-duplicate block to contain 81-104 amino acid residue; All obtain preceding 5 amino acid sequences of HMW-GS identical (EGEAS) of sequential analysis, and N-holds and also contains 3-5 halfcystine in the non-duplicate block; The C-end has 42 amino acid residues, includes a halfcystine.The duplicate block, middle part contains 490-700 amino acid residue; Repeat to occur constituting by polypeptide unit's six peptides, nonapeptide and tripeptides; Wherein six peptides appear in all x-types and the y-type subunit with the conservative form of GYYPTSLQQ with PGQGQQ, nonapeptide; And tripeptides only appears in the x-type subunit with the conservative form of GQQ, and indivedual subunits contain a halfcystine.The difference of size mainly is because due to the variation in middle part repetitive structure zone between the different subunits of HMW, especially different the causing of quantity (Shwery and Tatham, 1997) of six peptides and tripeptides.The design feature that repeats the peptide section has not only determined HMW-GS to have the lysine of the glutamine of high-load, proline and glycocoll and low content, has also formed the hydrophobic property (Shewry etc., 1989) of duplicate block, middle part.
Secondary structure to HMW-GS is analyzed discovery, and the duplicate block, center is by the β-folded inverted helical structure of forming that repeats, and the non-duplicate block at two ends is to constitute two chondritics by alpha-helix.The halfcystine of non-duplicate block, two ends can make HMW-GS form intermolecular disulfide bond, reaches up to ten million daltonian glutelin condensates (MacRitchie, 1992) thereby aggregate into.The glutamine of duplicate block high-load can form in the molecule and intermolecular hydrogen bond, and the part hydrogen bond rupture forms the sequence of loose region and bonding, and loose region can be stretched draws also and can make glutelin have elasticity (Belton etc., 1994 in external force disappearance back recovery; 1999); The crosslinking degree of two ends disulfide bond then determines group's elasticity (Shewry etc., 1992) of dough.
SDS-PAGE is the conventional method of present glutelin isolation identification.Experiment shows that as long as deposition condition is consistent, the bar number and the relative mobility of HMW-GS electrophoretic band (subunit) are stable, do not receive the influence (Bietz, 1972) of environmental baseline.Be utilized in the SDS-PAGE collection of illustrative plates that the contrast of basis and standard banding pattern under the SDS-PAGE deposition condition is set up, Wheat HMW-GS is carried out compartment analysis, be one of important means of the assignment of genes gene mapping of research Wheat HMW-GS, attributional analysis always.Though SDS-PAGE is a kind of not only fast but also economic method; But also exist the not identical phenomenon of HMW-GS mobility and molecular weight actual size (D ' Ovidio etc.; 1994); HMW-GS molecular weight by SDS-PAGE estimates is often higher with the protein molecular weight that its coding gene sequence is derived, and in practical application, generally can only carry out just estimation slightly to the molecular weight of different subunits with the SDS-PAGE method, also lacks a kind of method of confirming the HMW-GS accurate molecular weight fast.
With respect to " a kind of mass spectrometry method of identifying low-molecular-weight glutenin subunit of wheat " (CN200510135316.2) patented technology; The present invention is a biological mass spectrometry method of identifying the hmw glutenin subunit (HMW-GS) in the wheat storage protein, and " a kind of mass spectrometry method of identifying low-molecular-weight glutenin subunit of wheat " is the biological mass spectrometry method of identifying the low-molecular-weight glutenin subunit (LMW-GS) in the wheat storage protein (CN200510135316.2).
Summary of the invention
The objective of the invention is to set up a kind of stable biological mass spectrometry method of identifying wheat high-molecular-weight glutelin subunit fast and accurately measuring its molecular weight.Through this proteomic techniques, can identify fast and accurately that the high-molecular-weight glutelin subunit of Wheat Cultivars is formed and accurate molecular weight, for further research storage protein molecular structure and function provide foundation.
The present invention identifies the biological mass spectrometry method of wheat high-molecular-weight glutelin subunit, comprises the steps:
(1) when the biological mass spectrometry analytic sample of preparation wheat high-molecular-weight glutelin subunit, earlier with the acetone precipitation protein extract of 35-45% 2-4 hour, get deposition after centrifugal, be high-molecular-weight glutelin subunit;
(2) obtaining the high-molecular-weight glutelin subunit post precipitation; Dissolve high-molecular-weight glutelin subunit with lysate; The proportioning of lysate is: with the lysate volume is mete-wand, and the volume of acetonitrile solution is the 25-35% of lysate volume in the lysate, and the shared volume of trifluoroacetic acid (TFA) is 0.4% of a lysate volume; All the other are high purity water, and using the dissolution time of lysate dissolving high-molecular-weight glutelin subunit is 1 hour;
When (3) utilizing substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF-MS) to identify wheat high-molecular-weight glutelin subunit, matrix select for use sinapic acid SA (3,5-Dimethexycinnamic acid; 3,5-dimethoxy-4 '-hydroxycinnamic acid), be dissolved among 1ml50% acetonitrile+0.05%TFA with 10mg matrix; Standard specimen is selected Albumin (66431.08Da) for use, the 0.1%TFA dissolving, and the mensuration of molecular weight adopts linear model; Molecular weight ranges 10000-100000; Point sample adopts to mix goes up appearance method, matrix 1 μ l, sample 0.1 μ l.
The biological mass spectrometry method of above-mentioned evaluation wheat high-molecular-weight glutelin subunit; Preferred scheme is: when the mass spectrum sample of preparation high-molecular-weight glutelin subunit;, precipitate after 3 hours earlier, be precipitated as high-molecular-weight glutelin subunit after centrifugal with 40% acetone precipitation protein extract; Be to dissolve high-molecular-weight glutelin subunit with lysate; The optimal proportion of lysate is: with the lysate volume is mete-wand; The volume of acetonitrile solution is 30% of a lysate volume in the lysate; The shared volume of TFA is 0.4% of a lysate volume, and all the other are high purity water, and dissolution time is 1 hour.
When technical scheme of the present invention is sent out in development: at first, establish the method for distilling of wheat high-molecular-weight glutelin subunit; Secondly, the dissolution conditions before the mass spectrum on the protein sample is groped, comprised following three aspects: determination of ratio between each composition of lysate, confirming of the righttest dissolution time and confirming that volume of dissolution is big or small.At last, select suitable dissolving method and mass spectrum parameter to accomplish the accurate mensuration of wheat high-molecular-weight glutelin subunit molecular weight.
HMW-GS has important role in Wheat Quality Improvement, traditional research method mainly adopts SDS-PAGE with RP-HPLC it to be separated, but all there is certain deficiency in these methods.Utilize MS that the mensuration that biomacromolecule carries out accurate molecular weight is considered to the megatrend that mass spectrum develops, its maximum characteristics are that detectable relative molecular mass scope is wide, highly sensitive, and degree of accuracy is high, and are easy and simple to handle rapid.After this test is carried out roughing out to protein sample; Adopt appearance on the potpourri, simplified experimental procedure greatly, thereby avoided in the sample preparation process infringement to greatest extent albumen; Through revision test repeatedly; Confirmed the optimum test condition that accurate molecular weight is measured, the result shows that mass spectrometry method can carry out molecular weight to biomacromolecule and accurately measure, and kind evaluation to HMW-GS is feasible on the employing potpourri.
Technical characterictic
Biological mass spectrometry is the analytical approach of carrying out composition and structure evaluation through 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 ionization techniques: 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 drop explosion and final becomes spray form, and analyte ionsization gets into mass analyzer with electrically charged mode.Mostly the ion that the MALDI ion gun produces is single charge ion; Mass number in 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, is fit to high throughput analysis, so the most suitable analysis polypeptide of MALDI-MS and protein mixture; This instrument is the highest mass spectrometer of sensitivity now, can analyze the denier sample.
The biological mass spectrometry technology is mainly used in and solves two major issues: accurately measure biomacromolecule, like the molecular weight of protein, nucleotide, carbohydrate etc., and molecular structure information is provided; 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, like 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.; But these methods all exist, and the sample consumption is big, degree of accuracy is low, be subject to shortcomings (Zhao Shankai etc., 1996) such as protein form influence.
The present invention utilizes substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF-MS) technology, Rapid identification Wheat HMW-GS, and a kind of new method of definite its accurate molecular weight, and this method has following characteristic:
(1) amount of samples is few, and general half granule seed (about 10-20mg) can be satisfied the demand.
(2) method for distilling is simple, no toxic reagent, and usually contain noxious material in the SDS-PAGE extract, and the specimen preparation time is long.
(3) used time weak point separates rapidly, is fit to high throughput analysis.SDS-PAGE separates HMW-GS and generally needs 3-4 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 time a few minutes.
(4) resolution is high.The subunit that in the SDS-PAGE collection of illustrative plates, is difficult to distinguish is easy to distinguish in mass spectrum.
(5) detection sensitivity is high.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 high (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 the research of protein post-translational modification (PTMs).Therefore, the biological mass spectrometry technology probably becomes the trace detection technology that a kind of high sensitivity is identified wheat high-molecular-weight glutelin subunit, has broad application prospects.
Description of drawings:
Fig. 1. the different dissolution times of wheat breed capital 411 high-molecular-weight glutelin subunits biological mass spectrometry identify;
The biological mass spectrometry of the different dissolution times of Fig. 2 wheat breed capital 411 high-molecular-weight glutelin subunits is identified;
Fig. 3. the biological mass spectrometry of the different volume of dissolution of wheat breed capital 411 high-molecular-weight glutelin subunits is identified;
Fig. 4. the biological mass spectrometry of Wheat Cultivars high-molecular-weight glutelin subunit is identified;
Fig. 5. the biological mass spectrometry with wheat breed of identical HGMW-GS composition is identified.
Embodiment:
The SDS-PAGE of embodiment 1 wheat high-molecular-weight glutelin subunit identifies
(1) vegetable material: wheat breed " capital 411 ".
(2) extraction of HMW-GS:
The 10-20mg 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 and (contain 80mM Tris-HCl [pH8.0]+1%DTT) vortex mixing, 65 ℃ of water-bath 30min.Add 0.1ml50% isopropyl alcohol (4-vinylpyridine that contains 80mM Tris-HCl [pH8.0]+1.4%) 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 13000g, collecting precipitation is wheat high-molecular-weight glutelin subunit.
(3) SDS-PAGE of HMW-GS identifies
1. the sample buffer (2%SDS, 0.02% bromophenol blue, 0.08M Tris-HCl, pH8.0,40% glycerine) that is prepared in adding 100 μ l in the deposition of electrophoresis sample vibrated 30 minutes in 65 ℃ of water-baths, and centrifugal 10 minutes of 12000g makes SDS-PAGE and goes up appearance usefulness.
2. electrophoresis utilizes Bio-Rad Mini-PROTEAN 3 electrophoresis tanks, 5% to concentrate glue, 12% separation gel; Tris-glycine buffer liquid systems; 15mA electrophoresis 1.5 hours; 0.1% Coomassie brilliant blue R-250 dyeing, 10% ethanol and 10% acetate decolour, and confirm the preparation effect of HMW-GS according to electrophoresis pattern.
Embodiment 2HMW-GS biological mass spectrometry authentication method
(1) vegetable material: wheat breed " capital 411 ".
(2) preparation of mass spectrophotometry sample
The 10-20mg seed is pounded the powdered 1.5ml of putting into centrifuge tube, adds 0.5M NaCl solution 100 μ l, vortex 1/2 to 1 hour, and the centrifugal 10min of 5000g removes supernatant.Add ddH
2O 200 μ l, vortex 1/2 hour, 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.Add 0.2ml 50% n-propanol and (contain 80mM Tris-HCl [pH8.5]+1%DTT), vibration mixing, 65 ℃ of water-bath 30min.Get supernatant, add acetone to final concentration and reach 40%, precipitation at room temperature 3 hours, the centrifugal 15min of 13000g, collecting precipitation, with the proper amount of acetone washing, the vacuum drying deposition is wheat high-molecular-weight glutelin subunit.
(3) optimization of mass spectrum sample dissolution conditions
1. lysate (acetonitrile: TFA: H
2O) component ratio really normal root select suitable lysate component ratio according to known protein lysate composition and ratio design lysate ratio gradient according to the mass spectrum qualification result.Each component ratio gradient of lysate is seen table 1, and Mass Spectrometer Method result sees Fig. 1, and optimal proportion is acetonitrile: TFA: H
2O=30%: 0.4%: 69.6%.
Table 1 wheat high-molecular-weight glutelin subunit lysate (acetonitrile: TFA: H
2O) the component ratio gradient detects
| Acetonitrile TFA H 2O | Acetonitrile TFA H 2O | Acetonitrile TFA H 2O | Acetonitrile TFA H 2O | Acetonitrile TFA H 2O | Acetonitrile TFA H 2O | Acetonitrile TFA H 2O | Acetonitrile TFA H 2O |
| 0\0\100 | 0\0.1\100 | 0\0.2\100 | 0\0.3\100 | 0\0.4\100 | 0\0.5\100 | 0\0.75\100 | 0\1\100 |
| 10\0\90 | 10\0.1\90 | 10\0.2\90 | 10\0.3\90 | 10\0.4\90 | 10\0.5\90 | 10\0.75\90 | 10\1\90 |
| 20\0\80 | 20\0.1\80 | 20\0.2\80 | 20\0.3\80 | 20\0.4\80 | 20\0.5\80 | 20\0.75\80 | 20\1\80 |
| 30\0\70 | 30\0.1\70 | 30\0.2\70 | 30\0.3\70 | 30\0.4\70 | 30\0.5\70 | 30\0.75\70 | 30\1\70 |
| 40\0\60 | 40\0.1\60 | 40\0.2\60 | 40\0.3\60 | 40\0.4\60 | 40\0.5\60 | 40\0.75\60 | 40\1\60 |
| 50\0\50 | 50\0.1\50 | 50\0.2\50 | 50\0.3\50 | 50\0.4\50 | 50\0.5\50 | 50\0.75\50 | 50\1\50 |
2. dissolution time really normal root select Mass Spectrometer Method effect lysate ratio preferably according to the experimental result of the lysate ratio that tentatively obtains, strict control dissolution time is done the gradient dissolving.Consider that the too short protein dissolution of dissolution time is insufficient, and overlong time can cause the part degraded of albumen, so the initial setting dissolution time is 0.5-2 hour.If dissolution time length is to the protein dissolution effect, promptly the Mass Spectrometer Method effect does not have too big influence, and then dissolution time is short more good more.Mass Spectrometer Method result sees Fig. 2, and the optimal dissolution time is 1 hour.
3. volume of dissolution fixes on the optimal proportions of confirming each composition of lysate and optimal dissolution after the time really, and design lysate volume gradient is carried out groping of volume of dissolution size.Dissolving experience and mass spectral high sensitivity characteristic according to SDS-PAGE; Design lysate gradient is 5 μ l, 25 μ l and 50 μ l, and in order to the deposition of the wheat high-molecular-weight glutelin subunit that dissolves 15 μ l, Mass Spectrometer Method result sees Fig. 3 respectively; The resolution that can find out three kinds of volume of dissolution is similar; Volume of dissolution is described, promptly protein concentration is little to the influence that biological mass spectrometry detects, and explains that also the sensitivity of this technology for detection is very high.Consider the ease for operation of experiment, with the volume of dissolution of 50 μ l for well.
(4)MALDI-TOF-MS
Use the substance assistant laser desorpted ionized flight mass spectrometer of Tianjin, island AX1MA-CFRTMPlus type (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 functions such as (PSD) behind (DE) and the source.Main operational steps and technical essential are following:
1. the cleaning of target plate (384 type): pick methyl alcohol with cotton balls, wipe 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 and preparation: sinapic acid SA (3,5-Dimethexycinnamic acid, 3,5-dimethoxy-4 '-hydroxycinnamic acid), 10mg matrix is dissolved among 1ml50% acetonitrile+0.05%TFA, keeps in Dark Place.
3. the preparation of standard specimen and selection: the mensuration of HMW-GS molecular weight: Albumin (66431.08), the 0.1%TFA dissolving.
4. the setting of parameter: the mensuration of molecular weight adopts linear model, molecular weight ranges 10000-100000.
5. point sample (in the mixing appearance method): matrix 1 μ l, sample 0.1 μ l.
6. detect: treat behind the sample drying target plate to be put into mass spectral sample chamber, undertaken by 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
7. Flame Image Process: utilize KratosAnalytical ltd.lanchpad V2.4.0 software to carry out Flame Image Process and interpretation of result.
The application of embodiment 3 high-molecular-weight glutelin subunit biological mass spectrometry authentication methods
(1) accurate molecular weight is confirmed
The important step of confirming further to study its hereditary variation, molecular structure and function of wheat high-molecular-weight glutelin subunit accurate molecular weight.Accurate evaluation through molecular weight not only can accurately be distinguished the HMW-GS of difference in functionality, but also can understand some structural informations of protein protomer, for functional study lays the foundation.The mensuration of wheat high-molecular-weight glutelin subunit molecular weight mainly adopts the SDS-PAGE gel electrophoresis method at present; But relative mobility and its molecular weight of some albumen in the SDS system is not linear, so there is very mistake in traditional SDS-PAGE to the mensuration of molecular weight of albumen.The glutenin subunit molecular weight that utilizes SDS-PAGE to measure is compared often higher with the molecular weight that encoding gene is derived.The biological mass spectrometry technology just in time can remedy the deficiency of SDS-PAGE method.We use the method for being set up; The HMW-GS of aegilops tauschii TD38, bread wheat kind Kontrast and this inferior you special wheat Spelt130 has been carried out accurate molecular weight to be confirmed; Obtained the mass spectrogram of steady and audible high-molecular-weight glutelin subunit; Measured the accurate molecular weight of the 1Dx4,1By18,1Bx13 and the 1By16 subunit that are contained in these wheats, the result is as shown in Figure 4.The coding gene sequence of these subunits is not also cloned at present, therefore, utilizes the accurate molecular weight information of the glutenin subunit of MALDI-TOF-MS acquisition to lay a good foundation for separation, molecular structure and the functional study of these subunit genes.
The evaluation of (2) gluten subunit posttranslational modification (PTMs)
The flower characters of wheat and the composition of gluten subunit are closely related, and dissimilar glutenin subunits is different to the contribution of flower characters.Yet some research report is pointed out, even same subunit is in different wheat breeds, also incomplete same to the contribution of flower characters.To this phenomenon, can infer that possibly there is the posttranslational modification phenomenon in glutenin subunit, in various degree be modified with the difference that possibly cause flower characters.Yet, whether exist posttranslational modification to have dispute for the wheat glutenin subunit at present always.Have research to think that gluten subunit exists glycosylation and phosphorylation modification phenomenon, and the modification level is very high, has the people to think that then there are not modifications such as glycosylation in glutenin subunit.Confirm that this key of problem depends on effective detection method, and high-molecular-weight glutelin subunit MALDI-TOF-MS authentication method be established as address the above problem brought maybe.
The difference of the molecular weight size that the molecular weight size through the gluten subunit relatively measured with the biological mass spectrometry method and its coding gene sequence are derived can tentatively be inferred the modification situation of glutenin subunit.When molecular weight difference exceeded 0.1% (the mass spectrometer permissible error) that gene order derives, posttranslational modification had taken place can to infer tentatively that gluten subunit has.
Identifying with MALDI-TOF-MS in the process of Wheat HMW-GS; Find that there is notable difference in some subunits molecular weight in different wheat breeds; The molecular weight that the HMW-GS molecular weight of some kind is obviously derived greater than gene order, so modify phenomenon after might having protein translation.For example, identical HMW-GS composition is contained with China spring in common wheat (TriticumaestivumL.AABBDD) kind capital 411: 1Dx2,1Bx7,1By8 and 1Dy12, MALDI-TOF-MS identifies the not quite identical (see figure 5) of molecular weight of finding them.Calculate according to the mass spectrum permissible error, the molecular weight of these three kinds of subunits of 1Dx2,1By8 and 1Dy12 is little in these two interracial differences.But; The obvious difference of 1Bx7 subunit has surpassed permissible error; Further compare the molecular weight that its encoding gene is derived again, find that the molecular weight of the 1Bx7 subunit in capital 411 conforms to the molecular weight of deriving, and the molecular weight of the 1Bx7 subunit of China spring is obviously greater than the gene derived value.Therefore, infer that posttranslational modification possibly take place the 1Bx7 subunit.Therefore can judge tentatively that through this method the possibility of posttranslational modification takes place HMW-GS, for further analyzing posttranslational modification type, glycosylation or phosphorylation modification site and the influence of glutelin polymer structure and function being laid a good foundation.
(3) HMW-GS forms evaluation and attributional analysis
The method of identifying Wheat HMW-GS adopts the SDS-PAGE method more, compares the back with the HMW-GS standard diagram that contrasts and confirms the subunit composition.Experiment shows that as long as deposition condition is consistent, the bar number and the relative mobility of HMW-GS electrophoretic band (subunit) are stable, do not receive the influence of environmental baseline.Yet the control not a duck soup of experiment condition has some non-artificial factors to tend to influence the consistance of experiment condition.In some cases, micro displacement can take place on the SDS-PAGE collection of illustrative plates band of some subunits changes, if these variations occur on the close subunit of some mobility speeds, just is easy to they are carried out wrong evaluation.In addition, the improper mistake that also can cause subunit to judge of the control of electrophoresis time.Therefore, HMW-GS is to some subunits, like 1Ax2
*Distinguish with 1Dx2 etc. is difficult, the error of qualification result often between the different experiments chamber causes the quality evaluation mistake.
The MALDI-TOF-MS that the present invention set up can accurately identify the wheat high-molecular-weight glutelin subunit composition, is attributional analysis and correct estimate provide safeguard (Fig. 4-5).This method is comparatively directly perceived, and sensitivity and degree of accuracy obviously be superior to SDS-PAGE, operates also easier, rapid.In kind and Germplasm Identification, particularly advantage is remarkable in fine quality Rapid identification and the screening.Therefore, biological mass spectrometry might become the trace detection technology that a kind of high sensitivity is identified wheat high-molecular-weight glutelin subunit, has a extensive future.
Claims (1)
1. a biological mass spectrometry method of identifying wheat high-molecular-weight glutelin subunit comprises the steps:
(1) when the biological mass spectrometry analytic sample of preparation wheat high-molecular-weight glutelin subunit, earlier with 40% acetone precipitation protein extract 3 hours, get deposition after centrifugal, be high-molecular-weight glutelin subunit;
(2) obtaining the high-molecular-weight glutelin subunit post precipitation; Dissolve high-molecular-weight glutelin subunit with lysate; The proportioning of lysate is: with the lysate volume is mete-wand; The volume of acetonitrile solution is 30% of a lysate volume in the lysate, and the shared volume of trifluoroacetic acid (TFA) is 0.4% of a lysate volume, and all the other are high purity water; Using the dissolution time of lysate dissolving high-molecular-weight glutelin subunit is 1 hour;
When (3) utilizing substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF-MS) to identify wheat high-molecular-weight glutelin subunit; Matrix is selected sinapic acid SA (3 for use; 5-Dimethexycinnamic acid; 3,5-dimethoxy-4 '-hydroxycinnamic acid), be dissolved among 1ml50% acetonitrile+0.05%TFA with 10mg matrix; Standard specimen is selected the Albumin of 66431.08Da for use, the 0.1%TFA dissolving; The mensuration of molecular weight adopts linear model, molecular weight ranges 10000-100000; Point sample adopts to mix goes up appearance method, matrix 1 μ l, sample 0.1 μ l.
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| CN105203677A (en) * | 2015-10-16 | 2015-12-30 | 江苏大学 | Method for quickly detecting proteins in bombyx batryticatus according to biological mass spectrometry technology |
| CN107991491A (en) * | 2017-10-31 | 2018-05-04 | 北京毅新博创生物科技有限公司 | Correct the method and product of the accuracy rate of Mass Spectrometer Method protein sample |
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| Title |
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| Hsam, SLK |
| Yan, Y |
| Yan, Y;Hsam, SLK;Yu, JZ, et al..HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat (Triticum spelta L.) progenitors.《THEORETICAL AND APPLIED GENETICS》.2003,第107卷(第7期),1321-1330. * |
| Yu, JZ, et al..HMW and LMW glutenin alleles among putative tetraploid and hexaploid European spelt wheat (Triticum spelta L.) progenitors.《THEORETICAL AND APPLIED GENETICS》.2003,第107卷(第7期),1321-1330. |
| 司晓敏.利用蛋白质组技术鉴定小麦高分子量谷蛋白亚基的研究.《中国优秀硕士学位论文全文数据库》.2005,(第4期),25-28. * |
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