CN109651487A - A kind of extracting method of the endogenous peptide of plant - Google Patents

A kind of extracting method of the endogenous peptide of plant Download PDF

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CN109651487A
CN109651487A CN201910167031.9A CN201910167031A CN109651487A CN 109651487 A CN109651487 A CN 109651487A CN 201910167031 A CN201910167031 A CN 201910167031A CN 109651487 A CN109651487 A CN 109651487A
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plant
peptide
powder
mixed solution
extracting method
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吴刘记
陈雪艳
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Henan Agricultural University
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Henan Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

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  • Medicinal Chemistry (AREA)
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Abstract

The invention belongs to plant peptide extractive technique field, in particular to the extracting method of a kind of endogenous peptide of plant includes the following steps: to freeze plant first with liquid nitrogen, is then ground to powder, and powder is placed in centrifuge tube；It is placed in water-bath and heats again；Add the mixed solution of TCA and acetone, after precipitating, sediment is collected in centrifugation；Washing precipitate redissolves sediment in the TFA aqueous solution dissolved with protease inhibitors to after colourless；Then it is incubated for；Under low temperature after ultrasound cracking, centrifugation obtains supernatant；Supernatant is crossed into desalting processing after super filter tube again, vacuum drying obtains peptide fragment.The method of the present invention is easy to operate, and effectively retains natural long chain peptide in plant, therefore extracting method yield of the invention is high, and the peptide fragment number of acquisition is more, and peptide segment length is long.

Description

A kind of extracting method of the endogenous peptide of plant
Technical field
The invention belongs to plant peptide extractive technique field, in particular to the extracting method of a kind of endogenous peptide of plant.
Background technique
Peptide is made of and arrangement mode is constituted slave dipeptides to complicated linear, ring structure natural amino acid with different Different peptides general name, be usually made of 2 to 100 amino acid.Peptide is by the irreversible hydrolysis of peptide bond from biggish (egg White matter) generate in precursor.Compared with protein, peptide not only is easier to digest and assimilate than protein, also has and promotes immune, adjusting The physiological functions such as hormone, antibacterial, antiviral, blood pressure lowering and reducing blood lipid.Land plant is many kinds of, makes full use of cheap plant Peptide development of resources has the peptide product of high added value, can produce huge economic benefit and social benefit.
Peptide group is a branch of proteomics developed in recent years, the main neuropeptide and egg for studying animal The degradation of white enzyme, but peptide group, in plant science or a relatively new field, in the coming years, it may be helped In elaboration polypeptide group.Plant peptide is less than the protein molecule of 10kDa, can substantially be divided into two classes: one kind be by peptase compared with The biologically active peptide generated in selectively acting on larger precursor albumen, another kind of is proteolytic enzyme in albumen circular flow The degradation peptide of generation.Great diversity in view of polypeptide in biosystem and its participation during key regulatory improve more The demand of peptide discovery has already appeared, and therefore, develops for sample preparation, mass spectral analysis detection, sequencing and by comparing The peptide spectrum of different experiments device carries out the specific peptide fragment technology of discriminatory analysis.
Polypeptide classes are various, almost participate in the links such as the growth, development, immune, metabolism of organism.In body Existing endogenous peptide have the function of it is important, such as the peptide that is found in humans and animals have promote it is immune, adjust hormone, be anti- The biological functions such as bacterium, antiviral, blood pressure lowering and reducing blood lipid.Peptide is less in the research of plant, and presently found polypeptide is in plant Have the function of in terms of growth and development, reproduction, symbiosis interaction and pressure response important.On the one hand, peptide can pass through its antibacterial spy Property directly and pathogenic fungi interaction；On the other hand, peptide can be using interference signal molecule or as signaling molecule between plant cell It plays an important role in signal transmitting.Although the peptide found in plant is more and more, their function has also obtained preliminary solution Analysis, but peptide is often ignored in predictive genes and mass spectral analysis.
In addition, the acquisition of plant endogenous polypeptide is the basis of identification and research endogenous polypeptide, it is endogenous that plant is extracted at present There are two types of the main methods of peptide: one is acid extraction methods；Another kind is the mixed of sour Extraction buffer and plant protease inhibitor Close object.But the flux of both methods is not high, and Recent study is found, protease inhibitors is added can not be fine Inhibit plant in protease activity.Therefore, a kind of mentioning for polypeptide in efficient, quick, reproducible plant is probed into Take method very necessary.
Summary of the invention
It is an object of the present invention to extract the existing above problem for polypeptide in existing plant, a kind of plant is provided The extracting method of internal source peptide.
To achieve the goals above, the technical solution that the application uses are as follows:
A kind of extracting method of the endogenous peptide of plant, includes the following steps:
After the plant tissue being placed in mortar is freezed using liquid nitrogen, grind into powder, and powder is packed into centrifuge tube In, for use；
Centrifuge tube equipped with powder is placed in 94.5 DEG C~95.5 DEG C of water-bath after heating 4min~5min, for use；
Be added the mixed solution of TCA and acetone into powder again, the ratio of the powder and mixed solution be 1g:5mL~ 7mL；After precipitating 4.5h~5.5h, 18min~22min is centrifuged under 3 DEG C~5 DEG C and 9000r/min~11000r/min, it is heavy to collect Starch, for use；
By the acetone washing of sediment pre-cooling to after colourless, sediment is redissolved in dissolved with protease inhibitors TFA aqueous solution, and the volume fraction of TFA is 1% in the TFA aqueous solution；The ratio of the sediment and TFA aqueous solution is The ratio of 1g:4mL~6mL, the TFA aqueous solution and protease inhibitors is the 1mL:4 μ μ of L~6 L；Then at 3 DEG C~5 DEG C It is incubated for 0.8h~1.2h, obtains the first mixed solution, for use；
By the first mixed solution, ultrasound cracks 4min~5min at low temperature again, obtains the second mixed solution, for use, wherein Ultrasonic power is 40W, and every ultrasound 5.5s~6.5s stops ultrasound 7.5s~8.5s, and cracking temperature is 4 DEG C~10 DEG C；
Second mixed solution is centrifuged under 3 DEG C~5 DEG C and 9000r/min~11000r/min again 18min~ 22min obtains supernatant, for use；
The supernatant is crossed to the super filter tube of 10kD again, obtained ultrafiltrate is the extraction containing the endogenous peptide of plant Liquid；
After the extracting solution desalting processing containing the endogenous peptide of plant source, vacuum drying obtains peptide fragment.
Further, the plant tissue is plant leaf blade.
Further, the plant leaf blade is fresh maize leaf.
Further, the mixed liquor of the TCA and acetone, wherein the ratio of acetone and TCA are 10mL:1g.
Further, the temperature of the acetone of the pre-cooling is 4 DEG C~10 DEG C.
Compared with prior art, the beneficial effects of the present invention are: the method for the invention progress water-bath before peptide separation adds Heat and precipitating, wherein heating water bath can make vegetable protein deactivation, inhibit protein degradation, keep more under physiological status Peptide promotes polypeptide release dissolution；And TCA/ acetone precipitation can promote the dissolution of a large amount of secondary compounds, while protein precipitation Matter, and remove interfering substance；And the method for the present invention is easy to operate, and effectively retains natural long chain peptide in plant, because This extracting method yield original text of the invention, the peptide fragment number of acquisition is more, and peptide segment length is long.
Detailed description of the invention
Fig. 1 is the peptide fragment score distribution map of the embodiment of the present invention 1；
Fig. 2 is the peptide fragment staple diagram of the embodiment of the present invention 1；
Fig. 3 is the peptide fragment score distribution map of comparative example 1 of the present invention；
Fig. 4 is the peptide fragment staple diagram of comparative example 1 of the present invention.
Specific embodiment
Technological means of the invention, creation characteristic, achieving the goal is easy to understand with effect in order to make, below in conjunction with Specific embodiments of the present invention and attached drawing, are clearly and completely described technical solution of the present invention.
Label-free polypeptide group becomes important mass spectrum quantitative approach in recent years, according to its principle point, mainly there is two Kind.The wherein label-free method of spectrum counts class, development is more early, also forms many algorithms and is quantified, But main principle is all according to MS2Qualification result as quantitative basis, various methods the difference is that later period algorithm Amendment on large-scale data.The principle of second of label-free method is with MS1Based on, calculate each peptide segment signal Integral in LC-MS chromatography, this is also the principle of Maxquant algorithm employed in the following embodiment of the application.
Maxquant is leading proteomics/polypeptide group qualitative, quantitative algorithm, becomes albumen gradually now One of standard solution in matter group/polypeptide group field.Using the Labelfree algorithm in Maxquant to protein Group/polypeptide group data carry out label-free calculating, also become the important application of this algorithm.Maxquant both can be right Proteomics/polypeptide group data of SILAC label are analyzed, while can also be to the data of unstable isotope labelling It is analyzed.Label freequantification (LFQ) algorithm therein, it is simple for, be to each LCMS first Peptide segment signal in data is identified and is quantified, then using built-in Andromeda algorithm to all peptide segment signal MS2Into Row database retrieval, complete qualitative work, finally integrate all qualitative, quantitative data, complete entire quantitative proteomics/ Polypeptide group datamation.
The development of modern biotechnology mass-spectrometric technique is almost synchronous with the raising of resolution ratio, wherein the mass spectrum of orbitrap class Technology has become the Typical Representative of high resolution mass spec now.Maxquant algorithm can only be with the protein of high resolution mass spec Group/polypeptide group data are used cooperatively, orbitrap mass spectrum (Q-exactive) acquisition for the latest generation that this experiment uses LCMS/MS data.Q-exactive can reach perfect combination qualitatively and quantitatively, MS1And MS2Acquisition be all it is high-resolution, Wherein MS1Resolution ratio can achieve 140,000 (routine work 70,000), it more difficult to be MS2Routine work resolution ratio Also 17,500 be can achieve.MS2The HCD mode of use, without so-called " one third " rule, can perfection show MS2Whole Information.
The equal energy such as the chemical reagent used in following embodiment, such as TCA (trichloroacetic acid), acetone, TFA (trifluoroacetic acid) Enough pass through is commercially available.Wherein TCA and TFA is purchased from Shanghai Aladdin biochemical technology Co., Ltd.Acetone etc. is purchased from traditional Chinese medicines collection Group is chromatographic grade.
Embodiment 1
A kind of extracting method of the endogenous peptide of plant, includes the following steps:
Using liquid nitrogen by after the plant tissue being placed in mortar frost, grind into powder grinds 2min, and powder is filled Enter in the centrifuge tube of 2mL, for use；Plant tissue is fresh maize leaf；
Centrifuge tube equipped with powder is placed in 95 DEG C of water-bath after heating 5min, for use；
The mixed solution of TCA and acetone is added into powder again, the ratio of powder and mixed solution is 1g:6mL；Precipitate 5h Afterwards, it is centrifuged 20min under 4 DEG C and 10000r/min, collects sediment, for use；The wherein TCA and total 10mL of acetone mixture, and it is every 1g TCA is dissolved in the acetone of 10mL.
By the acetone washing of sediment pre-cooling to after colourless, sediment is redissolved in dissolved with protease inhibitors TFA aqueous solution, and the volume fraction of TFA is 1% in TFA aqueous solution；The ratio of sediment and TFA aqueous solution is 1g:5mL, institute The ratio for stating TFA aqueous solution and protease inhibitors is 1mL:5 μ L；Then it is incubated for 1h at 4 DEG C, obtains the first mixed solution, For use；Wherein protease inhibitors used is the protease inhibitors for originating from Missouri, USA Sigma-Aldrich；
By the first mixed solution, ultrasound cracks 5min at low temperature again, obtains the second mixed solution, for use, wherein ultrasonic function Rate is 40W, and every ultrasound 6s stops ultrasound 8s, and cracking temperature is 8 DEG C；
Second mixed solution is centrifuged 20min under 4 DEG C and 10000r/min again, obtains supernatant, for use；
The supernatant is crossed to the super filter tube of 10kD again, obtained ultrafiltrate is the extraction containing the endogenous peptide of plant Liquid；
After the extracting solution desalting processing containing the endogenous peptide of plant source, vacuum drying obtains peptide fragment.
Using the principle of Solid Phase Extraction, desalination is carried out using the method for needle column.Product used is the western lattice of Missouri, USA Product needle the column C18, internal diameter 7mm, capacity 3ml of Ma aldrich company.
Said extracted method in triplicate, is recorded as the 1st group, the 2nd group and the 3rd group, and respectively to this three groups of obtained peptides Duan Jinhang detection.
It is the detection method of the endogenous peptide of plant extracted to the extracting method of the endogenous peptide of above-mentioned plant below.
Specific detection method are as follows:
The peptide fragment of 2 μ g is redissolved in the TFA aqueous solution that volume fraction is 0.1%2mL, sample to be tested is obtained；
Sample to be tested is subjected to LC-MS/MS analysis again.
It is separated using a nanoliter flow velocity HPLC liquid phase systems.Liquid phase A liquid is that (acetonitrile is 0.1% formic acid acetonitrile solution 2%), B liquid is 0.1% formic acid acetonitrile solution (acetonitrile 84%).Chromatographic column (150 μ of ThermoEASY column SC200 M*100mm (RP-C18)) it is balanced with 100% A liquid.Sample is loaded to Thermo EASY column by autosampler 150 μm of * 20mm (RP-C18) of SC001traps, then through chromatography post separation, flow velocity 300nL/min.Related fluid phase gradient is such as Under: 0min~115min, B linear gradient are from 0%~45%；115min~117min, B linear gradient from 45%~ 100%；117min~120min, B liquid maintain 100%.Enzymolysis product uses Q- after capillary high performance liquid chromatography separates Exactive mass spectrograph is analyzed by mass spectrometry.Duration: 120min is analyzed, detection mode: cation, precursor scans range: The mass-charge ratio of the fragment of 300m/z~1800m/z, polypeptide and polypeptide acquires in following manner: MS1In the M/Z200 time-division Resolution is 70,000, AGC target:3e6, level-one Maximum IT:10ms, Number of scanranges:1, Dynamic exclusion:40.0s；20 fragment patterns storeds are acquired after each full scan, second level resolution ratio in M/Z 200 is 17,500, MS/MSActivation modes: HCD, Isolation window:2m/z, Microscans:1, second level MaximumIT:60ms, Normalized collision energy:30eV, Underfill ratio:0.1%.
The result such as table 1 that above-mentioned 3 extracting solutions are detected using LC-MS/MS method, from table 1 it can be seen that three It is secondary to identify 2607,2606,2594 peptide fragments respectively.Total identification peptide fragment 2620, corresponding 527, albumen.To these peptide fragments Score is counted, and the peptide fragment score extracted is higher, and the peptide fragment quality for illustrating that this method is extracted is preferable, as shown in Figure 1.It is right These peptide segment length are counted, and the polypeptide length extracted is as shown in Figure 2 in normal distribution within the scope of 8-25, and substantially.
1 Mass Spectrometric Identification of table is counted to peptide segment number and protein quantity
Comparative example 1
A kind of extracting method of the endogenous peptide of plant, includes the following steps:
Using liquid nitrogen by after the plant tissue being placed in mortar frost, grind into powder grinds 2min, and powder is filled Enter in the centrifuge tube of 2mL, for use；Plant tissue is fresh maize leaf；
In the powder be added be dissolved with protease inhibitors TFA aqueous solution, and in TFA aqueous solution TFA volume fraction It is 1%；The ratio of sediment and TFA aqueous solution is 1g:5mL, and the ratio of TFA aqueous solution and protease inhibitors is 1mL:5 μ L； Then it is incubated for 1h at 4 DEG C, obtains the first mixed solution, for use；
By the first mixed solution, ultrasound cracks 5min at low temperature again, the second mixed solution is obtained, for use, wherein surpassing Acoustical power is 40W, and every ultrasound 6s stops ultrasound 8s；
It obtains the second mixed solution by described again under 4 DEG C and 10000r/min and is centrifuged 20min, obtain supernatant, for use；
The supernatant is crossed to the super filter tube of 10kD again, obtained ultrafiltrate is the extraction containing the endogenous peptide of plant Liquid；
After the extracting solution desalting processing containing the endogenous peptide of plant source, vacuum drying obtains peptide fragment.
Said extracted method in triplicate, is recorded as 1' group, 2' group and 3' group, and this three groups are obtained respectively Peptide fragment is detected.
It is the detection method of the endogenous peptide of plant extracted to the extracting method of the endogenous peptide of above-mentioned plant below.
Specific detection method is identical as the detection method of embodiment 1, testing result are as follows:
The result such as table 2 that above-mentioned 3 groups of peptide fragment is detected using LC-MS/MS method, from table 2 it can be seen that three times 1125,969,1089 peptide fragments are identified respectively.Total identification peptide fragment 1400, corresponding 382, albumen.To these peptide fragment scores It is counted, the peptide fragment score extracted is lower, and the peptide fragment for illustrating that this method is extracted is second-rate, as shown in Figure 3.To these Peptide segment length is counted, and the polypeptide length extracted is as shown in Figure 4 in normal distribution within the scope of 8-25, and substantially.
The three groups of peptide segment numbers and protein quantity of 2 Mass Spectrometric Identification comparative example 1 of table count
By the way that embodiment 1 and the result of comparative example 1 compare, discovery the method for the present invention is easy to operate, and effective Retain natural long chain peptide in plant, therefore extracting method yield of the invention is high, the peptide fragment number of acquisition is more, and peptide segment length It is long.
Embodiment 2
A kind of extracting method of the endogenous peptide of plant, includes the following steps:
Using liquid nitrogen by after the plant tissue being placed in mortar frost, grind into powder grinds 2min, and powder is filled Enter in the centrifuge tube of 2mL, for use；Plant tissue is fresh maize leaf；
Centrifuge tube equipped with powder is placed in 94.5 DEG C of water-bath after heating 4min, for use；
The mixed solution of TCA and acetone is added into powder again, the ratio of powder and mixed solution is 1g:5mL；Precipitating After 4.5h, it is centrifuged 18min under 3 DEG C and 9000r/min, collects sediment, for use；The wherein TCA and total 10mL of acetone mixture, and 1g TCA is dissolved in the acetone of every 10mL.
By the acetone washing of sediment pre-cooling to after colourless, sediment is redissolved in dissolved with protease inhibitors TFA aqueous solution, and the volume fraction of TFA is 1% in TFA aqueous solution；The ratio of sediment and TFA aqueous solution is 1g:4mL, TFA The ratio of aqueous solution and protease inhibitors is 1mL:4 μ L；Then it is incubated for 0.8h at 3 DEG C, obtains the first mixed solution, to With；Wherein protease inhibitors used is the protease inhibitors for originating from Missouri, USA Sigma-Aldrich；
By the first mixed solution, ultrasound cracks 4min at low temperature again, obtains the second mixed solution, for use, wherein ultrasonic function Rate is 40W, and every ultrasound 5.5s stops ultrasound 7.5s, and cracking temperature is 4 DEG C；
Second mixed solution is centrifuged 18min under 3 DEG C and 9000r/min again, obtains supernatant, for use；
The supernatant is crossed to the super filter tube of 10kD again, obtained ultrafiltrate is the extraction containing the endogenous peptide of plant Liquid；
After the extracting solution desalting processing containing the endogenous peptide of plant source, vacuum drying obtains peptide fragment.
Embodiment 3
A kind of extracting method of the endogenous peptide of plant, includes the following steps:
Using liquid nitrogen by after the plant tissue being placed in mortar frost, grind into powder grinds 2min, and powder is filled Enter in the centrifuge tube of 2mL, for use；Plant tissue is fresh maize leaf；
Centrifuge tube equipped with powder is placed in 95.5 DEG C of water-bath after heating 5min, for use；
The mixed solution of TCA and acetone is added into powder again, the ratio of powder and mixed solution is 1g:7mL；Precipitating After 5.5h, it is centrifuged 22min under 5 DEG C and 11000r/min, collects sediment, for use；The wherein TCA and total 10mL of acetone mixture, And 1g TCA is dissolved in the acetone of every 10mL.
By the acetone washing of sediment pre-cooling to after colourless, sediment is redissolved in dissolved with protease inhibitors TFA aqueous solution, and the volume fraction of TFA is 1% in the TFA aqueous solution；The ratio of sediment and institute's TFA aqueous solution is 1g: The ratio of 6mL, TFA aqueous solution and protease inhibitors is 1mL:6 μ L；Then it is incubated for 1.2h at 5 DEG C, it is molten obtains the first mixing Liquid, for use；Wherein protease inhibitors used is the protease inhibition for originating from Missouri, USA Sigma-Aldrich Agent；
By the first mixed solution, ultrasound cracks 5min at low temperature again, obtains the second mixed solution, for use, wherein ultrasonic function Rate is 40W, and every ultrasound 6.5s stops ultrasound 8.5s, and cracking temperature is 10 DEG C；
Second mixed solution is centrifuged 22min under 5 DEG C and 11000r/min again, obtains supernatant, for use；
The supernatant is crossed to the super filter tube of 10kD again, obtained ultrafiltrate is the extraction containing the endogenous peptide of plant Liquid；
After the extracting solution desalting processing containing the endogenous peptide of plant source, vacuum drying obtains peptide fragment.
Disclosed above is only presently preferred embodiments of the present invention, and still, the embodiment of the present invention is not limited to this, Ren Heben What the technical staff in field can think variation should all fall into protection scope of the present invention.

Claims (5)

1. a kind of extracting method of the endogenous peptide of plant, which comprises the steps of:
After plant tissue is freezed using liquid nitrogen, grind into powder, and powder is fitted into centrifuge tube, for use；
Centrifuge tube equipped with powder is placed in 94.5 DEG C~95.5 DEG C of water-bath after heating 4min~5min, for use；
The mixed solution of TCA and acetone is added into powder again, the ratio of the powder and mixed solution is 1g:5mL~7mL； After precipitating 4.5h~5.5h, it is centrifuged 18min~22min under 3 DEG C~5 DEG C and 9000r/min~11000r/min, collects precipitating Object, for use；
By the acetone washing of sediment pre-cooling to after colourless, then sediment redissolved in the TFA for being dissolved with protease inhibitors Aqueous solution, and the volume fraction of TFA is 1% in the TFA aqueous solution；The ratio of the sediment and the TFA aqueous solution is The ratio of 1g:4mL~6mL, the TFA aqueous solution and protease inhibitors is the 1mL:4 μ μ of L~6 L；Then at 3 DEG C~5 DEG C It is incubated for 0.8h~1.2h, obtains the first mixed solution, for use；
By the first mixed solution, ultrasound cracks 4min~5min at low temperature again, obtains the second mixed solution, for use, wherein ultrasound Power is 40W, and every ultrasound 5.5s~6.5s stops ultrasound 7.5s~8.5s, and cracking temperature is 4 DEG C~10 DEG C；
Second mixed solution is centrifuged to 18min~22min under 3 DEG C~5 DEG C and 9000r/min~11000r/min again, Supernatant is obtained, for use；
The supernatant is crossed to the super filter tube of 10kD, obtained ultrafiltrate, the as extracting solution containing the endogenous peptide of plant again；
After the extracting solution desalting processing containing the endogenous peptide of plant source, vacuum drying obtains peptide fragment.
2. the extracting method of the endogenous peptide of plant as described in claim 1, which is characterized in that the plant tissue is leaves of plants Piece.
3. the extracting method of the endogenous peptide of plant as claimed in claim 2, which is characterized in that the plant leaf blade is fresh Maize leaf.
4. the extracting method of the endogenous peptide of plant as described in claim 1, which is characterized in that the mixing of the TCA and acetone Liquid, wherein the ratio of acetone and TCA are 10mL:1g.
5. the extracting method of the endogenous peptide of plant as described in claim 1, which is characterized in that the temperature of the acetone of the pre-cooling It is 4 DEG C~10 DEG C.
CN201910167031.9A 2019-03-06 2019-03-06 A kind of extracting method of the endogenous peptide of plant Withdrawn CN109651487A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110346479A (en) * 2019-08-06 2019-10-18 中国海洋大学 The extraction of the endogenous peptide of one seed oyster and identification method
CN112868881A (en) * 2021-02-15 2021-06-01 巨野恒丰果蔬有限公司 Extraction and preparation method of wheat polypeptide

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110346479A (en) * 2019-08-06 2019-10-18 中国海洋大学 The extraction of the endogenous peptide of one seed oyster and identification method
CN110346479B (en) * 2019-08-06 2021-11-05 中国海洋大学 Method for extracting and identifying oyster endogenous peptide
CN112868881A (en) * 2021-02-15 2021-06-01 巨野恒丰果蔬有限公司 Extraction and preparation method of wheat polypeptide

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Application publication date: 20190419