CN109535223B - Method for separating and enriching phosphorylated peptide by using double-titanium functionalized magnetic nano material - Google Patents
Method for separating and enriching phosphorylated peptide by using double-titanium functionalized magnetic nano material Download PDFInfo
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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Abstract
The invention provides a method for specifically separating and enriching phosphorylated peptide from a double-titanium functionalized magnetic nano material, which comprises the steps of dispersing the double-titanium functionalized magnetic nano material in a phosphorylated peptide sample solution by means of ultrasonic action, incubating for 10-60 minutes at 37 ℃, fully washing the phosphorylated peptide sample solution, eluting for 10-60 minutes at 37 ℃ by using ammonia water with the volume ratio of 0.5-20% as an eluent, taking the eluent as a target, and performing mass spectrometry. The invention realizes the enrichment of monophosphoryl peptide and polyphosphoryl peptide by controlling the synthesis conditions of the magnetic titanium dioxide/titanium ion functional material, can realize large-scale identification of protein phosphorylation sites by combining with a chromatographic mass spectrometry technology, thereby establishing a phosphorylation site database of specific diseases, and providing data support and thought inspiration for a plurality of diseases related to protein phosphorylation.
Description
Technical Field
The invention belongs to a method for separating and enriching phosphorylated peptides, and particularly relates to a method for specifically separating and enriching phosphorylated peptides by using a double-titanium functionalized magnetic nano material.
Background
Reversible phosphorylation of proteins plays a very important role in a variety of biological processes, especially in cellular signaling processes. Studies have shown that different degrees of phosphorylation (monophosphorylation or polyphosphorylation) of proteins play different roles in biological processes: monophosphorylation often acts as an activator of tumor suppressor factors, while polyphosphates are generally responsible for the hierarchical regulation of potassium channels or the regulation of cell cycle turnover, among other things. Thus, more comprehensive identification and analysis of phosphoproteomics is particularly important for understanding the role protein phosphorylation plays in biological processes. In the past decades, with the rapid development of mass spectrometry, the means of phosphorylated protein/peptide fragment analysis based on mass spectrometry strategy has received more and more attention. However, a major challenge faced by this assay is that the presence of large amounts of non-phosphorylated proteins/peptides in the actual sample severely inhibits mass spectrometry signals. Therefore, it would be desirable to use an efficient separation and enrichment strategy to analyze more and different degrees of protein phosphorylation prior to mass spectrometry.
Among all methods for separating and enriching phosphorylated peptide fragments, metal oxide affinity chromatography and fixed metal ion affinity chromatography are two more common methods for enriching, and the methods mainly rely on the formation of different coordination between phosphate groups on peptide fragments and metal ions/metal oxides to realize the separation and enrichment with a sample system. The phosphorylated peptide fragments obtained by the two methods have respective preferences, for example, the immobilized metal ion affinity chromatography prefers to enrich the more basic and hydrophilic polyphosphorylated peptide, and the metal oxide affinity chromatography prefers to monophosphoylated peptide fragments. At present, although methods such as step-by-step multiple elution or chromatography combination are developed to enhance the separation and analysis of phosphorylated peptide fragments, the methods generally have the defects of complex operation, low flux and the like, and the development of a multifunctional nano material which integrates the advantages of metal oxide affinity chromatography and fixed metal ion affinity chromatography is particularly important for large-scale phosphorylated proteomics analysis.
Disclosure of Invention
The invention aims to provide a method for specifically separating and enriching phosphorylated peptide by using a double-titanium functionalized magnetic nano material, which synthesizes the double-titanium functionalized magnetic nano material with strong magnetic responsiveness for the first time and uses the double-titanium functionalized magnetic nano material in separation and purification of phosphorylated peptide fragments. Under the action of an external magnetic field, the double-titanium functionalized magnetic nano material capturing the phosphorylated peptide segment can be quickly separated from a complex sample solution, so that the time required by the whole enrichment and elution processes is greatly shortened. Meanwhile, the material which combines the advantages of the affinity chromatography of the fixed metal ions and the affinity chromatography of the metal oxides has good enrichment effect on peptide fragments with different degrees of phosphorylation.
The method for separating and enriching the phosphorylated peptide by the specificity of the double-titanium functionalized magnetic nano material comprises the following specific steps of dispersing the double-titanium functionalized magnetic nano material in a phosphorylated peptide sample solution by means of ultrasonic action, incubating for 10-60 minutes at 37 ℃, fully washing with the phosphorylated peptide sample solution, eluting for 10-60 minutes at 37 ℃ by using ammonia water with the volume ratio of 0.5-20% as an eluent, taking the eluent as a target, and performing mass spectrometry.
The invention discloses a synthesis method of a double-titanium functionalized magnetic nano material, which comprises the following specific steps:
(1) dissolving ferric trichloride in ethylene glycol, adding anhydrous sodium acetate after full dissolution, transferring the mixture to a hydrothermal reaction kettle after full stirring and ultrasonic treatment, heating the mixture for 8 to 16 hours at the temperature of 100-350 ℃, cooling the mixture to room temperature, respectively and fully washing the obtained product by deionized water and anhydrous ethanol, and carrying out vacuum drying at the temperature of 50 to 100 ℃;
(2) uniformly dispersing the product obtained in the step (1) in a solvent, adding a titanium dioxide precursor, stirring the obtained mixed solution at the temperature of 20-80 ℃ for reaction for 12-48 hours, and respectively and fully washing with deionized water and absolute ethyl alcohol;
(3) dissolving adenosine nucleoside disodium triphosphate salt in an alkaline solvent, stirring for 5-30 minutes at 0-20 ℃, adding gamma-glycidoxypropyltrimethoxysilane, and stirring for reaction for 6-18 hours at 10-80 ℃;
(4) adjusting the pH value of the solution obtained in the step (3) to 3-7 by using acid, adding the product obtained in the step (2), stirring for 1-6 hours at 50-95 ℃, and fully washing by using deionized water and absolute ethyl alcohol;
(5) and (3) uniformly dispersing the product obtained in the step (4) in a solvent containing titanium salt, reacting for 6-24 hours at 20-80 ℃, fully washing the obtained product with deionized water, and drying in vacuum at 40-75 ℃ to obtain the target product.
In the invention, the solvent in the step (2) is one or more of methanol, ethanol or ethanol/deionized water mixed solution.
In the invention, the titanium dioxide precursor in the step (2) is one or more of tetraisopropyl titanate, n-butyl titanate, titanium sulfate or titanium tetrachloride.
In the invention, one or more of sodium hydroxide, sodium carbonate or ammonia water is also added before the titanium dioxide precursor is added in the step (2).
In the invention, the mass ratio of the product obtained in the step (1) in the step (2) to the titanium dioxide precursor is 30:1-15: 1.
In the invention, the alkaline solvent in the step (3) is one or more of sodium hydroxide aqueous solution, sodium carbonate aqueous solution or ammonia water.
In the invention, the mass ratio of the gamma-glycidoxypropyltrimethoxysilane to the adenosine triphosphate disodium salt in the step (3) is 1:5-1: 1.
In the invention, the acid in the step (4) is one or more of concentrated hydrochloric acid, sulfuric acid or nitric acid.
In the invention, the titanium salt in the step (5) is one or more of titanium sulfate, titanium tetrachloride or titanium nitrate.
The method for specifically separating and enriching phosphorylated peptides has the following advantages:
1. the double-titanium functionalized magnetic nano material has good magnetic responsiveness, has strong interaction with phosphorylated peptides, and can separate and enrich the phosphorylated peptides more sensitively and more efficiently.
2. Titanium dioxide on the double-titanium functionalized magnetic nano material is combined with titanium ions, so that the capture of peptide fragments phosphorylated to different degrees is facilitated, and the material shows good enrichment capacity on phosphorylated peptides in complex biological samples.
3. The double-titanium functionalized magnetic nano material is applied to the research of protein posttranslational modification, deep coverage can be realized on the posttranslational modified peptide segment of phosphorylated peptide by combining the advantages of metal oxide affinity chromatography and fixed metal ion affinity chromatography, and the phosphorylated protein can be identified in a large scale and the phosphorylation sites can be determined by combining nano-LC MS/MS.
Drawings
FIG. 1 is a scanning electron microscope photograph of the dual titanium functionalized magnetic nanomaterial of example 1;
FIG. 2 is a TEM photograph of the dual-Ti functionalized magnetic nanomaterial of example 1;
FIG. 3 is an X photoelectron spectrum of the dual titanium functionalized magnetic nanomaterial of example 1;
fig. 4 is an enriched mass spectrum of the di-titanium functionalized magnetic nanomaterial in example 2 on monophosphorylated standard peptides and di-phosphorylated standard peptides. The diagram A is a mass spectrogram of the phosphorylated standard peptide which is not enriched by the double-titanium functionalized magnetic nano material, the diagram B is a mass spectrogram of the phosphorylated standard peptide which is enriched by the magnetic titanium dioxide nano material, and the diagram C is a mass spectrogram of the phosphorylated standard peptide which is enriched by the double-titanium functionalized magnetic nano material;
FIG. 5 is a mass spectrum of the double-titanium functionalized magnetic nanomaterial of example 3 on separation and enrichment of phosphorylated peptides in a standard phosphorylated protein β -casein enzymatic hydrolysate. The graph A is a mass spectrogram of the beta-casein phosphopeptide which is not enriched, the graph B is a mass spectrogram of the phosphopeptide in the magnetic titanium dioxide nano material enriched beta-casein enzymatic hydrolysate, and the graph C is a mass spectrogram of the phosphopeptide in the double-titanium functionalized magnetic nano material enriched beta-casein enzymatic hydrolysate;
FIG. 6 is a selective mass spectrum of the double-titanium functionalized magnetic nanomaterial of example 4 on enrichment of phosphorylated peptides in β -casein enzymatic hydrolysate. The figure A is a mass spectrogram of a mixed enzymolysis product of beta-casein and non-phosphorylated protein BSA without enrichment, and the figure B is a mass ratio of the beta-casein to the non-phosphorylated protein BSA of 1: and (3) at 100 ℃, enriching a mass spectrogram of phosphorylated peptides in the beta-casein enzymolysis product by using a double-titanium functionalized magnetic nano material, wherein a chart C shows that the mass ratio of the two is 1: at 400, using a double-titanium functionalized magnetic nano material to enrich a mass spectrogram of phosphorylated peptides in a beta-casein enzymolysis product, wherein a diagram D shows that the mass ratio of the two is 1: at 800, enriching a mass spectrogram of phosphorylated peptides in a beta-casein enzymolysis product by using a double-titanium functionalized magnetic nano material;
FIG. 7 is a mass spectrum of phosphorylated peptides in saliva enriched with the bis-titanium functionalized magnetic nanomaterial of example 5. The figure A is a mass spectrogram of phosphorylated peptide fragments in saliva directly analyzed without enrichment, the figure B is a mass spectrogram of phosphorylated peptide in saliva enriched by the magnetic titanium dioxide nano material, and the figure C is a mass spectrogram of phosphorylated peptide in saliva enriched by the double-titanium functionalized magnetic nano material;
FIG. 8 is a mass spectrum of mono/poly-phosphorylated peptide in milk enriched with bis-titanium functionalized magnetic nanomaterial of example 6. The graph A is a mass spectrogram of phosphorylated peptide segments in milk directly analyzed without enrichment, the graph B is a mass spectrogram of phosphorylated peptide in magnetic titanium dioxide nano material enriched milk, and the graph C is a mass spectrogram of phosphorylated peptide in double-titanium functionalized magnetic nano material enriched milk.
Detailed Description
The invention realizes the separation and enrichment of phosphorylated peptides in a complex sample by utilizing the interaction of a double-titanium functionalized magnetic nano material and the phosphorylated peptides, and a specific implementation mode is introduced below.
Example 1: synthesis of double-titanium functionalized magnetic nano material
(1) 1.35 g FeCl3·6H2Magnetically stirring O in 75 mL of glycol until the solution is clear, adding 3.6 g of NaAc, fully stirring and ultrasonically treating, transferring to a hydrothermal reaction kettle, heating for 16 hours at 200 ℃, washing the product with deionized water and ethanol for three times after the reaction kettle is cooled, and vacuum-drying at 50 ℃;
(2) ultrasonically dispersing 80 mg of the product obtained in the step (1) in 200 ml of ethanol containing 0.72 ml of ammonia water, dropwise adding 1.6 ml of n-butyl titanate, reacting the obtained mixed solution at 45 ℃ for 24 hours, and fully washing with deionized water and absolute ethyl alcohol;
(3) dissolving 2.5 g adenine nucleoside triphosphate disodium salt in 2M sodium carbonate solution, stirring at 0 deg.C for 10 min, adding 0.8 ml gamma-glycidyl ether oxypropyl trimethoxysilane, stirring at 65 deg.C, and reacting for 12 hr;
(4) adjusting the pH value of the solution obtained in the step (3) to 6 by using concentrated hydrochloric acid, adding the product obtained in the step (2), stirring for 2 hours at 95 ℃, and fully washing by using deionized water;
(5) and (3) uniformly dispersing the product obtained in the step (4) in a titanium sulfate solution, reacting for 2 hours at 25 ℃, fully washing the obtained product with deionized water, and drying in vacuum at 40 ℃ to obtain the double-titanium functionalized magnetic nano material.
The prepared double-titanium functionalized magnetic nano material is detected by a scanning electron microscope, and the detection conditions are as follows: a small amount of dried material is adhered on an insulating adhesive tape under the working voltage of 15kV, and after gold spraying and vacuum pumping, the dried material is observed by a scanning electron microscope under a 8-micrometer scale bar, and the detection result is shown in figure 1.
Detecting the prepared double-titanium functionalized magnetic nano material by using a transmission electron microscope, wherein the detection conditions are as follows: under 200kV working voltage, a small amount of dried material is uniformly dispersed in absolute ethyl alcohol, the micro-grid mesh is soaked by the mixed solution, the dried material is inserted into an instrument for vacuumizing, a projection electron microscope image is observed under a 100 nanometer scale, and the detection result is shown in figure 2.
FIG. 3 is an X photoelectron spectrum of the dual titanium functionalized magnetic nanomaterial.
Example 2: the double-titanium functionalized magnetic nano material obtained in the example 1 is used as a solid phase adsorbent for enriching monophosphorylated target peptides and double-phosphorylated target peptides
(1) Preparation of a sample: 1 mg of the monophosphorylated or bisphosphorylated standard peptide is dissolved in 1 ml of 50 mM NH4HCO3In solution;
(2) and (3) ultrasonically dispersing 100 mu g of the double-titanium functionalized magnetic nano material in 100 mu L of loading solution containing 0.1 mu L of the peptide solution obtained in the step (1), and incubating for 20 min at 37 ℃. The sample was rinsed three times with 100 μ L of the loading solution. Eluting with 10 μ L0.4M ammonia water for 30 min;
(3) mass spectrometry analysis: and (3) taking 1 mu L of eluent in the step (2) and 1 mu L of matrix solution as a point target, naturally drying, and performing mass spectrometry, wherein a mass spectrogram is shown in figure 4.
And (3) analysis results: as can be seen from FIG. 4, the signal peak intensities of the monophosphorylated and diphosphorylated standard peptides are greatly improved after the enrichment of the material.
Example 3: the double-titanium functionalized magnetic nano material obtained in the example 1 is used as a solid phase adsorbent for separating and enriching phosphorylated peptides in phosphorylated protein beta-casein
(1) Preparation of a sample: 1 mg beta-casein at 50 mM NH4HCO3Carrying out enzymolysis for 16 h at 37 ℃ in the solution;
(2) and (3) ultrasonically dispersing 100 mu g of the double-titanium functionalized magnetic nano material in 100 mu L of sample liquid containing 100 fmol/mu L of beta-casein enzymolysis product obtained in the step (1), and incubating for 20 min at 37 ℃. The sample was rinsed three times with 100 μ L of the loading solution. Eluting with 10 μ L0.4M ammonia water for 30 min;
(3) mass spectrometry analysis: and (3) taking 1 mu L of eluent in the step (2) and 1 mu L of matrix solution as a point target, naturally drying, and performing mass spectrometry, wherein a mass spectrogram is shown in figure 5.
And (3) analysis results: as can be seen from FIG. 5, phosphorylated peptides derived from the enzymatic hydrolysis product of phosphorylated protein β -casein were captured by the material, and the interference caused by non-phosphorylated peptides in the stock solution was largely eliminated.
Example 4: the double-titanium functionalized magnetic nano material obtained in the example 1 is used as a solid phase adsorbent for simulating separation and enrichment of phosphorylated peptides in a complex environment
(1) Respectively preparing a mixed enzymolysis product of beta-casein and non-phosphorylated protein BSA into a simulated complex sample system with increased complexity according to the mass ratio of 1:100, 1:400 and 1: 800;
(2) and (3) ultrasonically dispersing 100 mu g of the double-titanium functionalized magnetic nano material in 100 mu L of loading solution containing the solution prepared in the step (1), and incubating for 30 min at 37 ℃. The sample was rinsed three times with 200 μ L of the loading solution. Eluting with 8 μ L0.8M ammonia water for 30 min;
(3) mass spectrometry analysis: and (3) taking 1 mu L of eluent in the step (1) and 1 mu L of matrix solution as a point target, naturally drying, and performing mass spectrometry, wherein a mass spectrogram is shown in FIG. 6.
And (3) analysis results: as can be seen from FIG. 6, the impurity peaks from the non-phosphorylated protein BSA were removed after enrichment, and the phosphorylated peptide in the phosphorylated protein β -casein enzymatic hydrolysate was captured by the material, so that the signal peak intensity was greatly enhanced.
Example 5: the double-titanium functionalized magnetic nano material obtained in example 1 is used as a solid phase adsorbent for separation and enrichment of phosphorylated peptides in saliva
(1) 100 mu g of the double-titanium functionalized magnetic nano material is dispersed in 100 mu L of sample solution containing 5 mu L of saliva by ultrasonic dispersion, and incubated for 30 min at 37 ℃. The sample was rinsed three times with 100 μ L of the loading solution. Eluting with 8 μ L0.4M ammonia water for 30 min;
(2) mass spectrometry analysis: and (3) taking 1 mu L of eluent in the step (1) and 1 mu L of matrix solution as a point target, naturally drying, and performing mass spectrometry, wherein a mass spectrogram is shown in FIG. 7.
And (3) analysis results: as can be seen from FIG. 7, the material still can exhibit good enrichment effect on mono/polyphosphorylated peptide fragments in the complex environment of saliva.
Example 6: the double-titanium functionalized magnetic nano material obtained in the example 1 is used as a solid phase adsorbent for separating and enriching phosphorylated peptides in milk
(1) 100 mu g of the double-titanium functionalized magnetic nano material is dispersed in 100 mu L of sample liquid containing 5 mu L of milk by ultrasonic dispersion, and incubated for 30 min at 37 ℃. The sample was rinsed three times with 100 μ L of the loading solution. Eluting with 8 μ L0.4M ammonia water for 30 min;
(2) mass spectrometry analysis: and (3) taking 1 mu L of eluent in the step (1) and 1 mu L of matrix solution as a point target, naturally drying, and performing mass spectrometry, wherein a mass spectrogram is shown in FIG. 8.
And (3) analysis results: as can be seen from FIG. 8, the material still can exhibit good enrichment effect on mono/polyphosphorylated peptide fragments in the complex environment of milk.
Example 7: synthesis of double-titanium functionalized magnetic nano material
(1) 1.35 g FeCl3·6H2Magnetically stirring O in 75 mL of glycol until the solution is clear, adding 3.6 g of NaAc, fully stirring and ultrasonically treating, transferring to a hydrothermal reaction kettle, heating for 12 hours at 180 ℃, washing the product with deionized water and ethanol for three times after the reaction kettle is cooled, and vacuum-drying at 40 ℃;
(2) ultrasonically dispersing 50 mg of the product obtained in the step (1) in 500 ml of ethanol, dropwise adding 1.5 ml of tetraisopropyl titanate, reacting the obtained mixed solution at 45 ℃ for 20 hours, and fully washing with deionized water and absolute ethyl alcohol;
(3) dissolving 2 g of adenosine disodium triphosphate in a sodium hydroxide solution, stirring at 0 ℃ for 30 min, adding 1 ml of gamma-glycidoxypropyltrimethoxysilane, and stirring at 65 ℃ for reacting for 16 hours;
(4) adjusting the pH value of the solution obtained in the step (3) to 5 by using concentrated hydrochloric acid, adding the product obtained in the step (2), stirring for 3 hours at 90 ℃, and fully washing by using deionized water;
(5) and (3) uniformly dispersing the product obtained in the step (4) in a titanium sulfate solution, reacting for 2 hours at 45 ℃, fully washing the obtained product with deionized water, and drying in vacuum at 50 ℃ to obtain the double-titanium functionalized magnetic nano material.
Example 8: synthesis of double-titanium functionalized magnetic nano material
(1) 1.5 g of FeCl3·6H2Magnetically stirring O in 80 mL of glycol until the solution is clear, adding 3.6 g of NaAc, fully stirring and ultrasonically treating, transferring to a hydrothermal reaction kettle, heating for 16 hours at 200 ℃, washing the product with deionized water and ethanol for three times after the reaction kettle is cooled, and vacuum-drying at 40 ℃;
(2) dispersing 50 mg of the product obtained in the step (1) in 200 ml of methanol containing 0.05 mmol of sodium hydroxide by ultrasonic wave, adding 0.5 mg of titanium sulfate, reacting the obtained solution at 30 ℃ for 20 h, and fully washing with deionized water and absolute ethyl alcohol;
(3) dissolving 5 g of adenosine disodium triphosphate in 0.1M ammonia water, stirring at 0 ℃ for 30 min, adding 0.7 ml of gamma-glycidyl ether oxypropyltrimethoxysilane, and stirring at 65 ℃ for reaction for 16 hours;
(4) adjusting the pH value of the solution obtained in the step (3) to 5 by using 5M sulfuric acid, adding the product obtained in the step (2), stirring for 3 hours at 90 ℃, and fully washing by using deionized water;
(5) and (3) uniformly dispersing the product obtained in the step (4) in a titanium tetrachloride solution, reacting for 2 hours at 45 ℃, fully washing the obtained product with deionized water, and drying in vacuum at 50 ℃ to obtain the double-titanium functionalized magnetic nano material.
Example 9: synthesis of double-titanium functionalized magnetic nano material
(1) 1 g of FeCl3·6H2Magnetically stirring O in 60 mL of glycol until the solution is clear, adding 3.2 g of NaAc, sufficiently stirring and ultrasonically treating, transferring to a hydrothermal reaction kettle, heating at 200 ℃ for 12 hours, cooling the reaction kettle,washing the product with deionized water and ethanol for three times, and vacuum drying at 40 deg.C;
(2) ultrasonically dispersing 10 mg of the product obtained in the step (1) into ethanol/water mixed solution (volume ratio is 95: 5) containing 0.5 g of sodium carbonate, dropwise adding 0.5 ml of titanium tetrachloride solution, reacting the obtained mixed solution at 25 ℃ for 30 hours, and fully washing with deionized water and absolute ethyl alcohol;
(3) dissolving 2 g of adenosine disodium triphosphate in a sodium hydroxide solution, stirring at 0 ℃ for 30 min, adding 1 ml of gamma-glycidoxypropyltrimethoxysilane, and stirring at 65 ℃ for reacting for 16 hours;
(4) adjusting the pH value of the solution obtained in the step (3) to 5 by using 5M nitric acid, adding the product obtained in the step (2), stirring for 3 hours at 90 ℃, and fully washing by using deionized water;
(5) and (3) uniformly dispersing the product obtained in the step (4) in a titanium nitrate solution, reacting for 2 hours at 20 ℃, fully washing the obtained product with deionized water, and drying in vacuum at 50 ℃ to obtain the double-titanium functionalized magnetic nano material.
Claims (9)
1. A method for separating and enriching phosphorylated peptides by using the specificity of a double-titanium functionalized magnetic nano material is characterized by comprising the following specific steps of dispersing the double-titanium functionalized magnetic nano material in a phosphorylated peptide sample solution by means of ultrasonic action, incubating for 10-60 minutes at 37 ℃, fully washing with the phosphorylated peptide sample solution, eluting for 10-60 minutes at 37 ℃ by using ammonia water with the volume ratio of 0.5-20% as an eluent, taking the eluent as a target, and performing mass spectrometry;
the method for synthesizing the double-titanium functionalized magnetic nano material comprises the following specific steps:
(1) dissolving ferric trichloride in ethylene glycol, adding anhydrous sodium acetate after full dissolution, transferring the mixture to a hydrothermal reaction kettle after full stirring and ultrasonic treatment, heating the mixture for 8 to 16 hours at the temperature of 100-350 ℃, cooling the mixture to room temperature, respectively and fully washing the obtained product by deionized water and anhydrous ethanol, and carrying out vacuum drying at the temperature of 50 to 100 ℃;
(2) uniformly dispersing the product obtained in the step (1) in a solvent, adding a titanium dioxide precursor, stirring the obtained mixed solution at the temperature of 20-80 ℃ for reaction for 12-48 hours, and respectively and fully washing with deionized water and absolute ethyl alcohol;
(3) dissolving adenosine nucleoside disodium triphosphate salt in an alkaline solvent, stirring for 5-30 minutes at 0-20 ℃, adding gamma-glycidoxypropyltrimethoxysilane, and stirring for reaction for 6-18 hours at 10-80 ℃;
(4) adjusting the pH value of the solution obtained in the step (3) to 3-7 by using acid, adding the product obtained in the step (2), stirring for 1-6 hours at 50-95 ℃, and fully washing by using deionized water and absolute ethyl alcohol;
(5) and (3) uniformly dispersing the product obtained in the step (4) in a solvent containing titanium salt, reacting for 6-24 hours at 20-80 ℃, fully washing the obtained product with deionized water, and drying in vacuum at 40-75 ℃ to obtain the target product.
2. The method for specifically separating and enriching phosphopeptide of a double-titanium functionalized magnetic nanomaterial according to claim 1, wherein the solvent in the step (2) is one or more of methanol, ethanol or ethanol/deionized water mixed solution.
3. The method for specifically separating and enriching phosphorylated peptides by using the double-titanium functionalized magnetic nanomaterial according to claim 1, wherein the titanium dioxide precursor in the step (2) is one or more of tetraisopropyl titanate, n-butyl titanate, titanium sulfate and titanium tetrachloride.
4. The method for specifically separating and enriching phosphorylated peptides by using the double-titanium functionalized magnetic nanomaterial according to claim 1, wherein one or more of sodium hydroxide, sodium carbonate and ammonia water is/are further added before the titanium dioxide precursor is added in the step (2).
5. The method for specifically separating and enriching phosphorylated peptides by using the double-titanium functionalized magnetic nanomaterial as claimed in claim 1, wherein the mass ratio of the product obtained in the step (1) to the titanium dioxide precursor in the step (2) is 30:1-15: 1.
6. The method for specifically separating and enriching phosphopeptide of a double-titanium functionalized magnetic nanomaterial according to claim 1, wherein the alkaline solvent in step (3) is one or more of an aqueous sodium hydroxide solution, an aqueous sodium carbonate solution or an aqueous ammonia solution.
7. The method for specific separation and enrichment of phosphorylated peptides by using the bis-titanium functionalized magnetic nanomaterial according to claim 1, wherein the mass ratio of the gamma-glycidoxypropyltrimethoxysilane to the adenosine disodium triphosphate in the step (3) is 1:5 to 1: 1.
8. The method for specifically separating and enriching phosphorylated peptides by using the double-titanium functionalized magnetic nanomaterial according to claim 1, wherein the acid in the step (4) is one or more of concentrated hydrochloric acid, sulfuric acid and nitric acid.
9. The method for specifically separating and enriching phosphorylated peptides by using the double-titanium functionalized magnetic nano material according to claim 1, wherein the titanium salt in the step (5) is one or more of titanium sulfate, titanium tetrachloride or titanium nitrate.
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