A purification and enrichment method for detecting endogenous small peptide hormones in plant materials

Abstract

The present invention discloses a method for purifying plant endogenous small peptides based on reverse-phase ion exchange mixed mode solid phase extraction. In this method, plant endogenous small peptides are subjected to solvent extraction, and the extract acts on an adsorption material, and the plant endogenous small peptides are adsorbed on the surface of the material. After eluting to remove matrix impurities, the plant small peptides adsorbed on the solid phase extraction material are eluted to obtain. The method is simple, fast, and easy to operate, and the separation material used is a commercial solid phase extraction column, which is easy to obtain. The entire processing flow is easy to learn and use, and can be conveniently used by relevant scientific researchers, and has broad application prospects in the fields of plant hormone regulation, biomarker screening, etc.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry and relates to a purification and enrichment method for detecting plant endogenous small peptide hormones in plant materials. Background Art

[0002] Small peptides refer to chain polymers formed by natural amino acids connected end to end by peptide bonds. Their structure is between amino acids and proteins. They are usually composed of no more than 50 amino acids and have a molecular weight of less than 10KDa. Active small peptides are widely present in the plant kingdom and play an important role in maintaining and regulating their normal physiological activities. In addition to TomSys, dozens of endogenous active regulatory peptides such as AtPep and ductal differentiation inhibitory factor (TDIF) have been found in plants. They participate in regulating various physiological processes of plant growth and development, especially in short-distance information exchange between cells. They play a key role. Because these endogenous active small peptides have a similar action mode to plant hormones and can exert their physiological functions at extremely low physiological concentrations, they are also called plant small peptide hormones. With the completion of the "Arabidopsis Genome Project" and subsequent sequencing studies of other plant genomes, the discovery, identification and physiological regulation mechanism research of plant small peptide hormones has entered the fast lane, attracting the attention of more and more plant physiologists and becoming a hot research topic in the field of plant physiology.

[0003] It is worth noting that whether it is the discovery and identification of unknown plant active small peptides, the exploration of the regulatory mechanism of existing plant small peptide hormones, or the screening of small peptide biomarkers, they are inseparable from the efficient purification and comprehensive analysis of endogenous plant small peptides. The traditional plant hormone family has a small molecular weight, and each type of plant small peptide hormone contains a similar molecular skeleton or mother nucleus structure, and the physicochemical properties are relatively close, which greatly facilitates the development of analytical methods. Plant small peptide hormones are composed of amino acids. The number, type, sequence and various post-translational modifications of amino acids make the structure and physicochemical properties of plant active peptides extremely diverse, and their detection and analysis face more severe challenges.

[0004] At present, due to its good separation ability, high sensitivity and excellent sequence identification and analysis ability, LC-MS technology is widely used in the detection of plant small peptides. The combination of high-resolution mass spectrometry and multi-stage mass spectrometry technology has greatly improved the analytical ability and accuracy of small peptide structures. However, plant matrices are often complex in composition, and the content of plant active peptides is very low. It is difficult to obtain ideal results by directly detecting plant sample extracts, and components such as inorganic salts in the matrix are prone to irreversible damage to the mass spectrum. Therefore, before detection, endogenous small peptides in the plant matrix need to be enriched and purified. At present, the purification methods of plant small peptides are mostly comprehensive flocculation sedimentation protein removal and later desalting, high-performance liquid chromatography, glucose gel chromatography, rapid column chromatography, immunopurification and other steps. They perform well in the analysis of some plant small peptides, but there is still a lot of room for improvement. First, facing different matrices or different target small peptides, the purification process is different, the required separation materials are different, and some separation materials cannot be obtained through commercial channels. Second, some operations such as immunopurification have high technical requirements for analysts, which brings a certain learning time cost. Therefore, establishing a simple and easy method for purifying plant peptides is the best choice to solve these problems.

[0005] From the structure, it can be seen that the basic building blocks of small peptides are amino acids, and the combination and arrangement of different amino acids give the small peptides a diversity of structures. Different amino acids have different hydrophilicity, hydrophobicity, acidity, and alkalinity, which causes the properties of small peptides to show significant differences. Based on these structural characteristics, plant small peptides can be retained on the separation material through reverse phase or ion exchange. The stronger the hydrophobicity, the stronger the reverse phase retention, and the stronger the hydrophilicity, the stronger the ion exchange. If the separation material is given reverse phase and ion exchange sites at the same time, multiple types of small peptides can be enriched to a greater extent, and suitable elution solvents can be selected to remove impurities while avoiding the loss of target small peptides. Summary of the invention

[0006] The purpose of the present invention is to provide a method for purifying plant endogenous small peptides based on reverse phase-ion exchange mixed mode solid phase extraction.

[0007] The method for purifying plant endogenous small peptides provided by the present invention comprises the following steps:

[0008] 1) adding an extraction solvent to the plant material, mixing well and then leaving it to stand for extraction, removing the residue, and collecting the extract for the following treatment a;

[0009] The treatment a is to load the sample onto an Oasis solid phase extraction column filled with a mixed ion exchange and reverse phase retention adsorbent to obtain a conjugate I;

[0010] 2) The conjugate I obtained in step 1) is sequentially washed and eluted, and the eluate is collected and concentrated to obtain the plant endogenous small peptide.

[0011] The method for screening plant endogenous small peptides provided by the present invention comprises:

[0012] 3) adding an extraction solvent to the plant material, mixing well and then leaving it to stand for extraction, removing the residue, and collecting the extract for the following treatment b;

[0013] The treatment b is to sequentially load the sample onto an Oasis solid phase extraction column filled with a mixed ion exchange and reverse phase retention adsorbent with two complementary retention mechanisms to obtain conjugates II and III;

[0014] 4) The conjugates II and III obtained in step 3) are respectively rinsed and eluted, and the eluates are collected and combined in the same container, and concentrated to obtain the plant endogenous small peptide.

[0015] In step 1) and step 3) of the above method, the plant endogenous small peptide refers to a small peptide that is endogenously present in the plant and has not been proteolytically hydrolyzed, and can be a known plant small peptide hormone or an unknown plant small peptide, more specifically TomSys, TDIF, etc.;

[0016] The plant raw material can specifically be rice, Arabidopsis or tomato, etc.;

[0017] The extraction solvent is a methanol aqueous solution with a mass percentage concentration of 50%-100% or a trifluoroacetic acid aqueous solution with a mass percentage concentration of 0.1%-2%;

[0018] The amount of the plant raw material is no more than 1000 mg, or no more than 200 mg, or no more than 100 mg; specifically, 50 mg-1000 mg, specifically, 50 mg-500 mg, more specifically, 50 mg-200 mg, and more specifically, 100 mg;

[0019] The ratio of the plant material to the extraction solvent is 100 mg: 0.5-5 mL;

[0020] In the extraction step, the extraction method is various conventional methods, such as ultrasound, vortex, centrifugation or static, etc.;

[0021] The extraction includes vortex mixing, ultrasound, overnight placement at 4°C and centrifugation; specifically, the ultrasound time is 1-60 minutes; specifically 2-10 minutes; the ultrasound power is 200-300W; in the centrifugation step, the centrifugal speed is 9000-20000rpm; the centrifugal force is 10000-20000g; the centrifugal time is 10-20 minutes;

[0022] The Oasis solid phase extraction column filled with mixed ion exchange and reverse phase retention adsorbent includes a mixed-mode strong cation exchange column (Mixed-Mode Strong Cation-Exchange, MCX), a mixed-mode weak cation exchange column (Mixed-Mode Weak Cation-Exchange, WCX), a mixed-mode strong anion exchange column (Mixed-Mode Strong Anion-Exchange, MAX) and a mixed-mode weak anion exchange column (Mixed-Mode Weak Anion-Exchange, WAX). The above solid phase extraction columns can all be purchased from Waters.

[0023] The purpose of treatment a is to extract and bind the small peptides in the extract to the solid phase extraction adsorbent by means of reverse phase reaction or ion exchange.

[0024] From steps 1) and 3), it can be seen that the sample pretreatment process of this method only requires 1-2 Oasis solid phase extraction columns for purification, and the solid phase extraction columns used can be easily purchased from commercial channels.

[0025] In the elution steps of step 2) and step 4), the elution solvent is an elution solvent suitable for reverse phase and ion exchange mechanism.

[0026] The elution solvent is selected from water, methanol, acetonitrile, aqueous formic acid solution, aqueous trifluoroacetic acid solution, methanol solution of formic acid and acetonitrile solution of formic acid;

[0027] In the formic acid aqueous solution, formic acid methanol solution and formic acid acetonitrile solution, the mass percentage concentration of formic acid is 0.1-10%, more specifically 5%.

[0028] In the trifluoroacetic acid aqueous solution, the mass percentage concentration of trifluoroacetic acid is 0.1-5%; more specifically 1%;

[0029] In the acetonitrile solution containing formic acid, the mass percentage concentration of formic acid is specifically 0.1-10%, more specifically 5%;

[0030] The elution is specifically performed by eluting with a 5% by mass concentration of formic acid aqueous solution, water, and methanol in sequence;

[0031] In the elution steps of step 2) and step 4), the elution is completed in one step or in steps;

[0032] In the one-step process, the elution reagent used is a methanol solution containing trifluoroacetic acid or ammonia; wherein the MCX column or WAX column uses a methanol solution containing NH 4 Aqueous solution of OH in methanol, NH 4The mass percentage concentration of OH is 1-5%; the mass percentage concentration of methanol is 60-80%, and the remaining component is water;

[0033] The WCX column or MAX column uses a methanol-water solution containing trifluoroacetic acid, wherein the mass percentage concentration of trifluoroacetic acid is 0.1-5%, specifically 2%; the mass percentage concentration of methanol is 60-98%, specifically 80%; and the remaining component is water;

[0034] The step-by-step completion includes: the first step is to use an acidic aqueous solution or an alkaline aqueous solution to destroy the ion exchange effect; the second step is to use a methanol aqueous solution or an acetonitrile aqueous solution as an elution reagent;

[0035] In the first step, the acidic aqueous solution is trifluoroacetic acid aqueous solution or ammonia aqueous solution, wherein the MCX column or WAX column uses ammonia aqueous solution, NH 4 The mass percentage concentration of OH is 1-5%; the WCX column or MAX column uses a trifluoroacetic acid aqueous solution, and the mass percentage concentration of trifluoroacetic acid is 0.1-5%; specifically 2%;

[0036] In the second step, the mass percentage concentration of methanol in the methanol aqueous solution is 60-100%; the mass percentage concentration of acetonitrile in the acetonitrile aqueous solution is 60-100%.

[0037] In addition, the application of the above method in any of the following also falls within the protection scope of the present invention:

[0038] a. Quantitative analysis of plant peptides;

[0039] b. Qualitative analysis of plant peptides;

[0040] c. Screening of endogenous active small peptides from plants;

[0041] d. Identification of endogenous active small peptides in plants.

[0042] The present invention also claims a method for analyzing plant endogenous small peptides, comprising the following steps: dissolving the purified product obtained by the method in a solvent and performing LC-MS / MS detection.

[0043] Specifically, the solvent is selected from at least one of an acetonitrile aqueous solution containing an acid, a methanol aqueous solution containing an acid, and an aqueous solution containing an acid;

[0044] Specifically, the acid is formic acid or trifluoroacetic acid; the mass percentage concentration of the acid in the acetonitrile aqueous solution of the acid, the methanol aqueous solution containing the acid, and the aqueous solution containing the acid is 0.05-2%; specifically 0.1%;

[0045] In the acid-containing acetonitrile aqueous solution, the mass percentage concentration of acetonitrile is 0-10%, specifically 5%;

[0046] In the methanol aqueous solution containing acid, the mass percentage concentration of methanol is 0-10%, specifically 5%.

[0047] The method provided by the present invention is simple, rapid and easy to operate, and the separation material used is a commercial solid phase extraction column, which is easy to obtain. The entire processing flow is easy to learn and use, and can be conveniently used by relevant scientific researchers. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The typical chromatograms of the spike recovery experiment of endomorphin and neuropeptide. The plant matrix is ​​immature rice seeds (100 mg FW), and the spiked amount of morphin and neuropeptide is 100 ng.

[0049] Figure 2 Multiple reaction monitoring chromatograms of endogenous TDIF in Arabidopsis stems and flowers;

[0050] Figure 3 This is the multiple reaction monitoring chromatogram of endogenous TomSys in tomato leaves;

[0051] Figure 4 This is a comparison chart of the enrichment effects of different small peptides by WAX and WCX;

[0052] Figure 5 This is a comparison of the salt response behaviors of candidate compounds of salt-tolerant peptide markers in different rice lines. The ordinate represents the ratio of the compound content after salt treatment to that before salt treatment, and the abscissa represents the molecular weight of the compound. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified. The raw materials described can be obtained from public commercial channels unless otherwise specified. The concentrations described are all mass percentage concentrations unless otherwise specified.

[0054] Example 1: Spiked recovery experiment of endomorphins and neuropeptides in rice matrix

[0055] Grind plant tissue (wild-type rice Nipponbare immature seeds, panicles or seedlings) into powder under liquid nitrogen, weigh 100 mg of plant material powder and place it in a 2 mL centrifuge tube, add 1 mL of 80% methanol, then add 200 ng of endomorphin and neuropeptide, and vortex to mix evenly. Ultrasonicate at 250 W for 2 minutes and place at 4 ° C for 1 hour. Centrifuge at 15000 rpm for 15 minutes, take the supernatant, load the supernatant onto the Oasis WCX solid phase extraction column (6cc, 150 mg), rinse the solid phase extraction column with water, methanol and 2% trifluoroacetic acid aqueous solution in sequence, and then elute the small peptide with 90% methanol aqueous solution containing 2% trifluoroacetic acid. After the collected eluate is dried, it is re-dissolved with 100 μL of aqueous solution containing 0.1% formic acid and 5% acetonitrile, and finally 5 μL is analyzed by LC-HRMS. The results are shown in the figure below. WCX can enrich the target small peptides added to the rice matrix. The total recovery rates of endomorphins in immature rice seeds, panicles and seedlings are 67%, 52% and 48%, and the total recovery rates of neuropeptides in immature rice seeds, panicles and seedlings are 76%, 87% and 73%.

[0056] Example 2: Spiked recovery experiment of endomorphins and neuropeptides in Arabidopsis matrix

[0057] Plant tissues (wild-type Arabidopsis Col-0 flowers, stems, fruit pods and whole seedlings) were ground into powder under liquid nitrogen. 100-200 mg of plant material powder was weighed and placed in a 2 mL centrifuge tube. 1-1.5 mL of 80% methanol was added, and then 200 ng of endomorphin and neuropeptide were added. The mixture was vortexed and mixed evenly. Ultrasonication was performed at 250 W for 2 min. The mixture was placed at 4 ° C for 1 h. Centrifugation was performed at 15000 rpm for 15 min, and the supernatant was taken. The supernatant was loaded onto an Oasis WCX solid phase extraction column (6 cc, 150 mg). The solid phase extraction column was washed with water, methanol and 2% trifluoroacetic acid aqueous solution in sequence, and then the small peptides were eluted with 90% methanol aqueous solution containing 2% trifluoroacetic acid. The collected eluate was dried and re-dissolved with 100 μL of aqueous solution containing 0.1% formic acid and 5% acetonitrile. Finally, 5 μL was analyzed by LC-HRMS. The results showed that the exogenously added target small peptide standards could be successfully detected in different Arabidopsis tissues. In each Arabidopsis tissue, the total recovery rate of WCX for endomorphin was higher than 90%, and the total recovery rate of WCX for neuropeptides was higher than 67%.

[0058] Example 3: Spiked recovery experiment of plant peptide hormones

[0059] Rice flag leaves were ground into powder under liquid nitrogen, 100 mg of plant material powder was weighed and placed in a 2 mL centrifuge tube, 1 mL of 80% methanol was added, and then 200 ng each of TomSys, Atpep1, Pep6, CLE19 (Clavata3 / embryo surrounding region-related 19), CLE40, TDIF, and GmRIC1 (Glycine max Rhizobia-induced CLE1) were added, vortexed and mixed evenly, and ultrasonicated at 250 W for 2 min. The mixture was placed at 4°C overnight. Centrifuge at 15000rpm for 15min, take the supernatant, load the supernatant onto the Oasis WCX solid phase extraction column (3cc, 60mg), wash the solid phase extraction column with water, methanol and 2% trifluoroacetic acid aqueous solution in turn, then elute the small peptide with methanol, dry the collected eluate, re-dissolve it with 100μL of aqueous solution containing 0.1% formic acid and 5% acetonitrile, and finally enter LC-HRMS analysis with 5μL. The spiked recovery of 7 kinds of plant small peptide hormones is shown in Table 1, which shows that WCX has a good enrichment effect on 7 typical plant small peptide hormones.

[0060] Table 1 Recovery results of plant peptide hormone spiked in rice matrix

[0061]

[0062] Example 4: Determination of TDIF in Arabidopsis

[0063] Plant materials (Arabidopsis stems and flowers) were ground to powder under liquid nitrogen. Three portions (named A, B, and C) of 1 g of plant material powder were weighed and placed in 2 mL centrifuge tubes. 10 mL of 1% trifluoroacetic acid aqueous solution was added. Then 0 ng, 1 ng, and 10 ng of TDIF standard were added to samples A, B, and C. The samples were vortexed and mixed evenly. Ultrasonication was performed at 250 W for 10 min. The samples were placed at 4 ° C overnight. Centrifugation was performed at 15000 rpm for 15 min. The supernatant was taken and loaded onto an Oasis WCX solid phase extraction column (6 cc, 150 mg). The solid phase extraction column was washed with water and 0.1% trifluoroacetic acid aqueous solution in turn. The small peptides were eluted with a methanol solution containing 2% trifluoroacetic acid. The collected eluate was dried and reconstituted with 100 μL of an aqueous solution containing 0.1% formic acid and 5% acetonitrile. Finally, 5 μL was analyzed by LC-MS / MS. Results: Endogenous TDIF was detected in both Arabidopsis stems and flowers. Figure 2 ), combined with the spiked results, the endogenous TDIF content was calculated to be 0.18 ng / g (stem) and 0.21 ng / g (flower).

[0064] Example 5. Determination of TomSys in tomato leaves

[0065] Grind mechanically injured tomato leaves under liquid nitrogen until they are powdered, weigh three portions (named A, B, and C) of 1g of plant material powder and place them in 2mL centrifuge tubes, add 10mL of 1% trifluoroacetic acid aqueous solution, then add 0ng, 1ng, and 10ng of TDIF standard to samples A, B, and C, vortex to mix evenly, and sonicate at 250W for 10min. Place at 4°C overnight. Centrifuge at 15000rpm for 15min, take the supernatant, load the supernatant onto an Oasis WCX solid phase extraction column (6cc, 150mg), rinse the solid phase extraction column with water and 0.1% trifluoroacetic acid aqueous solution in turn, then elute the small peptides with a methanol solution containing 2% trifluoroacetic acid, dry the collected eluate, and re-dissolve it with 100μL of an aqueous solution containing 0.1% formic acid and 5% acetonitrile, and finally enter LC-MS / MS analysis with 5μL. The results are as follows Figure 3 As shown, endogenous TomSys can be detected in tomato leaves. Combined with the spiked results, the endogenous TomSys content can be calculated to be 1.07 ng / g.

[0066] Example 6: Comparison of small peptide purification effects of WAX and WCX in rice immature seed matrix

[0067] Weigh 300 mg of ground immature rice seeds and put them into a 7 mL centrifuge tube. Add 3 mL of 80% methanol and 400 ng of small peptide standards with different sequence structures, vortex to mix evenly, and ultrasonicate at a power of 250 W for 2 minutes. Place at 4°C for 1 hour. Centrifuge at 15,000 rpm for 15 minutes, take the supernatant, divide the supernatant into two equal parts, and load them onto Oasis WCX solid phase extraction columns (6cc, 150 mg) and Oasis WAX solid phase extraction columns (6cc, 150 mg), respectively. Elute the solid phase extraction columns with water, methanol, water, and a 2% trifluoroacetic acid aqueous solution in sequence, then elute the small peptides with methanol. After the collected eluate is dried, it is re-dissolved with 100 μL of an aqueous solution containing 0.1% trifluoroacetic acid, and finally 5 μL is subjected to LC-HRMS analysis. Figure 4 As shown in the figure, for small peptides with different sequence structures, the enrichment effects of WAX and WCX are significantly different. WAX is suitable for the more acidic Caspase-1, while WCX is more suitable for the neutral or alkaline Endomorphin and Neuropeptide. It can be seen that WAX and WCX have complementary enrichment behaviors for small peptides, so that their combined use will help to obtain more comprehensive information on plant endogenous small peptides, thereby providing necessary technical support for the discovery and screening of plant small peptide hormones.

[0068] Example 7: Screening of small peptide markers for salt tolerance in rice

[0069] Weigh 100 mg of the ground rice stem base and put it into a 2 mL centrifuge tube. Add 1 mL of 80% methanol, vortex to mix evenly, and ultrasonicate at 250 W for 2 minutes. Place at 4°C overnight. Centrifuge at 15000 rpm for 15 minutes, take the supernatant, load it onto the Oasis WCX solid phase extraction column (6cc, 150 mg), collect the flow-through and load it onto the Oasis WAX solid phase extraction column (6cc, 150 mg). For the WCX column, wash the solid phase extraction column with water, methanol, water, and a 2% mass percent trifluoroacetic acid aqueous solution in sequence, and then elute the small peptide with methanol. For the WAX ​​column, wash with water, methanol, water, and 5% NH 4 The solid phase extraction column was eluted with an aqueous solution of OH in turn, and then the small peptide was eluted with methanol. The collected WAX and WCX eluates were combined, concentrated and dried with nitrogen, and then reconstituted with 100 μL of an aqueous solution containing 0.1% trifluoroacetic acid, and the last 5 μL was analyzed by LC-HRMS. By comparing the composition and changes of small peptides in different strains before and after salt treatment, more than 10 candidate small peptide compounds that may be related to salt tolerance were finally found. The salt response differences of some compounds are shown in Figure 5 .

[0070] Example 8. Comparison of the purification effects of MAX and WCX columns on CLE family peptides

[0071] Grind rice flag leaves under liquid nitrogen until powdery, weigh 200 mg of plant material powder and place it in a 7 mL centrifuge tube, add 2 mL of 80% methanol, then add 400 ng each of CLE19, CLE40, TDIF and CLV3, vortex mix evenly, and sonicate at 250 W for 2 min. Place at 4 ° C overnight. Centrifuge at 20000 rpm for 10 min, take the supernatant, divide the supernatant into two parts, and load them onto WCX column (3 cc, 60 mg) and MAX column (3 cc, 60 mg) respectively. Elute the solid phase extraction column with water and methanol in turn, and then elute the small peptide with an aqueous solution containing 2% trifluoroacetic acid and 80% methanol. After the collected eluate is dried, it is re-dissolved with 100 μL of an aqueous solution containing 0.1% formic acid and 5% acetonitrile, and finally 5 μL is analyzed by LC-HRMS. The comparison of the enrichment effects of MAX and WCX is shown in the table below. It can be seen that the enrichment effect of WCX on the four CLE family plant peptide hormones is significantly better than that of MAX.

[0072] Table 2 Comparison of the enrichment effects of MAX and WCX on CLE small peptides

[0073]

[0074] Example 9: Comparison of small peptide purification effects using WAX, MCX, MAX and WCX columns in rice flag leaf matrix

[0075] Weigh 150 mg of ground rice flag leaves and put them into a 2 mL centrifuge tube. Add 1.5 mL of 1% trifluoroacetic acid aqueous solution, vortex to mix evenly, and ultrasonicate at 250 W for 2 minutes. Place at 4 ° C for 1 hour. Centrifuge at 20000 rpm for 15 minutes, take the supernatant, add an appropriate amount of 1% trifluoroacetic acid aqueous solution to make the volume 15 mL, take out 4 mL of the solution, divide it into four parts, add 100 ng of small peptide standard to each part, load it on WAX, MCX, MAX and WCX columns respectively, rinse with water, MCX and WAX are washed with 5% NH 4 OH and 80% MeOH in water, WCX and MAX were eluted with 2% trifluoroacetic acid and 80% MeOH in water, and the eluted fractions were collected.

[0076] After the liquid was blown dry, it was re-dissolved with 100 μL of aqueous solution containing 0.1% formic acid and 5% acetonitrile, and the last 5 μL was analyzed by LC-HRMS. The results are shown in the following table. The four columns have different enrichment effects on small peptides with different structures and properties. Reasonable combination of anion and cation columns will cover more small peptides, which can be applied to the related research of small peptide screening and small peptide interaction.

[0077] Table 3. Comparison of small peptide purification effects of four small columns

[0078]

[0079] The above embodiments disclose the specific operation contents of the present invention, but the present invention is not limited to the above implementation modes and embodiments, and any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention.

Claims

1. A method for purifying plant endogenous small peptides, comprising: 1) Add the extraction solvent to the plant material, mix well and then let it stand for extraction. After removing the residue, collect the extract and perform the following treatment a; The extraction solvent is a methanol aqueous solution with a mass percentage concentration of 50%-100%; The treatment a is to load the sample onto an Oasis solid phase extraction column filled with a mixed ion exchange and reverse phase retention adsorbent to obtain a conjugate I; The Oasis solid phase extraction cartridge with mixed ion exchange and reverse phase retention adsorbent is a mixed mode weak cation exchange cartridge; 2) washing and eluting the conjugate I obtained in step 1) in sequence, collecting and concentrating the eluate to obtain the plant endogenous small peptide; The reagents used for the washing and elution are any of the following: a. The eluting reagents are water, methanol and a 2% trifluoroacetic acid aqueous solution; the eluting reagent is methanol; b. The eluting reagents are water and methanol; the eluting reagent is an aqueous solution containing 2% trifluoroacetic acid and 80% methanol by mass percentage; The plant endogenous small peptides are TomSys, AtPep1, Pep6, CLE19, CLE40, TDIF and GmRIC1.

2. The method according to claim 1, characterized in that: In the step 1), the amount of the plant raw material does not exceed 1000 mg; The ratio of the plant material to the extraction solvent is 100 mg: 0.5-5 mL; The extraction included vortex mixing, sonication, overnight storage at 4 °C and centrifugation.

3. The method according to claim 2, characterized in that: The amount of the plant material does not exceed 200 mg; In the ultrasound, the time is 1-60 min; the ultrasound power is 200-300 W; In the centrifugation step, the centrifugal speed is 9000-20000 rpm; the centrifugal force is 10000-20000 g; and the centrifugal time is 10-20 min.

4. The method according to claim 3, characterized in that: The amount of the plant material does not exceed 100 mg; The ultrasound lasts for 2-10 min.

5. Application of the method according to any one of claims 1 to 4 in any of the following: a. Quantitative analysis of plant peptides; b. Qualitative analysis of plant peptides; c. Identification of endogenous active small peptides in plants.

6. A method for analyzing endogenous small peptides in plants, comprising the following steps: dissolving the purified product obtained by any method of claims 1 to 4 in a solvent, and performing LC-MS / MS detection.

7. The method according to claim 6, characterized in that: The solvent is selected from at least one of an acetonitrile aqueous solution containing an acid and an aqueous solution containing an acid; The acid is formic acid or trifluoroacetic acid; the mass percentage concentration of the acid in the acetonitrile aqueous solution of the acid and the aqueous solution containing the acid is 0.1%; In the acid-containing acetonitrile aqueous solution, the mass percentage concentration of acetonitrile is 5%.

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