CN116068190A - Metal organic framework modified magnetic nano probe and synthesis method and application thereof - Google Patents
Metal organic framework modified magnetic nano probe and synthesis method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of advanced nano materials, and particularly relates to a metal-organic framework modified magnetic nano probe, a synthesis method and application thereof. According to the invention, firstly, ferroferric oxide magnetic nano-microspheres are loaded on graphene oxide by utilizing a self-assembly strategy, and then N is used for preparing the ferroferric oxide magnetic nano-microspheres 2 Calcining under atmosphere; dispersing the obtained graphene material loaded with the ferroferric oxide microspheres in an aqueous solution in which sodium hydroxide, aluminum chloride hexahydrate and 3, 5-pyrazoledicarboxylic acid are dissolved for reaction, and finally magnetically separating to obtain the metal organic framework modified magnetic nano probe taking aluminum ions as the center. The nano probe has large specific surface area, strong hydrophilicity and proper pore size, shows good stability in enrichment of standard glycosylated peptide sample, has excellent volume exclusion effect and ultra-low sensitivity, and can be applied to analysis of glycosylated proteomics in clinical serum samples.
Description
Technical Field
The invention belongs to the technical field of advanced nanomaterials, and in particular relates to a metal-organic framework modified magnetic nanoprobe taking aluminum ions as central ions, a synthesis method thereof and application of glycosylated peptide enrichment in clinical samples.
Background
Liver cancer (Hepatocellular Carcinoma, HCC) is one of the most common malignant tumors worldwide, and the incidence rate of liver cancer is the fourth most frequently seen among all malignant tumors in our country, and the death rate is the second most frequently seen. In recent years, the risk of liver cancer caused by obesity, diabetes, metabolic syndrome, non-alcoholic fatty liver and the like is increasing, as well as being influenced by the risk factors such as hepatitis virus infection, liver cirrhosis, aflatoxin exposure, alcohol and the like. The study proves that the prognosis of the patient is closely related to the diagnosis time, and the survival rate of the early diagnosis and treatment liver cancer patient for 5 years is over 70 percent, which is far higher than that of the late liver cancer patient (less than 16 percent). Clinically, early liver cancer diagnosis comprises imaging, pathology, detection of serum tumor markers and the like. However, the liver cancer is hidden, the early imaging detection is atypical and is not easy to find (the focus is small, and the ultrasonic detection sensitivity is only 63%). Alpha Fetoprotein (AFP) is a serum biomarker applied to clinically detecting liver cancer at present, but the sensitivity and specificity of AFP are poor, and the clinical requirements cannot be completely met. Therefore, it is urgent and important to find new liver cancer biomarkers for rapid, accurate, noninvasive early screening of liver cancer.
Protein glycosylation is a very common posttranslational modification of proteins in biological cells, determines functions and metabolic pathways of the proteins, and plays an irreplaceable role in the process of life activities such as metabolism, signal transduction, cell proliferation and division in human bodies. Abnormal glycosylation of proteins is closely related to occurrence and development of diseases such as cancers, so that glycosylated proteins are specifically identified, dynamic changes, localization, distribution and the like of the glycosylated proteins are analyzed, novel disease biomarkers are found, and early screening, diagnosis and treatment of diseases are realized. At present, the glycoprotein is usually analyzed and detected by mass spectrometry technology on glycosylated peptide fragments of enzymatic hydrolysis products, however, the glycosylated peptide fragments are difficult to analyze and detect by directly using mass spectrometry due to the weak ionization capacity and low abundance of the glycosylated peptide fragments, complex samples and the like. Thus, enrichment and isolation of glycosylated peptide fragments in complex biological samples prior to mass spectrometry is critical for successful analysis of glycosylated proteins.
Metal Organic Frameworks (MOFs) materials are a class of porous materials constructed by coordination self-assembly of organic ligands with central metal ions or metal clusters. MOFs material has become a research hotspot in various fields because of the characteristics of large surface area, strong designability, easily-adjusted pore size and environment, easy functionalization and the like. In the field of analytical chemistry research, the efficient enrichment and screening of target molecules can be achieved by selecting suitable MOFs porous materials. The invention synthesizes the magnetic nano probe modified by the metal organic framework with strong magnetic response and large specific surface area and taking the aluminum ion as the center ion for the first time and is applied to the separation and enrichment of glycosylated peptide. The whole process is quick and simple through magnetic separation. Meanwhile, the hydrophilic pore canal in the Al-MOFs metal organic framework has strong affinity effect on the target glycosylated peptide, after enrichment by the nano probe, the mass spectrum signal of the target glycosylated peptide is obviously enhanced, and due to the unique porous structure of the Al-MOFs material, the nano probe can successfully remove large-size interfering substances, so that the enrichment efficiency of the glycosylated peptide is effectively improved. The method is applied to analysis of clinical samples (serum of liver cancer patients and normal people), and satisfactory identification results are obtained in research on glycosylated peptides in serum.
Disclosure of Invention
The invention aims to provide a metal organic framework modified magnetic nano probe which takes aluminum ions as central ions and has the advantages of convenient and simple synthesis, good stability and good biocompatibility, a synthesis method thereof and application thereof in glycosylated peptide selective enrichment detection.
The invention provides a synthesis method of a metal-organic framework modified magnetic nano probe, which comprises the following specific steps:
(1) Dispersing 50-200mg of ferroferric oxide magnetic beads with the size of 10-30nm in 10-30 mL n-hexane solution containing 20-50mg of graphene oxide, performing ultrasonic dispersion for 1-4 hours, and magnetically separating out materials;
(2) Calcining the material obtained in the step (1) at a high temperature of 400-600 ℃ for 2-6h, and marking the obtained material as GO@Fe 3 O 4 ；
(3) Dissolving 0.5-1 g of 3, 5-pyrazoledicarboxylic acid and 0.2-0.5-g sodium hydroxide in 50-150 mL pure water solution, performing ultrasonic treatment for 0.5-2h, and preheating at 100deg.C for 30min;
(4) Adding 0.8-2.1g of aluminum chloride hexahydrate into the solution obtained in the step (3), and carrying out ultrasonic treatment until the aluminum chloride hexahydrate is dissolved;
(5) Dispersing the material obtained in the step (2) in the solution obtained in the step (4), reacting at 25-100 ℃ for 2-24h, magnetically separating out a product, washing with deionized water and absolute ethyl alcohol for 2-5 times, and vacuum drying at 40-80 ℃ to obtain the metal organic framework modified magnetic nano probe taking aluminum ions as central ions, namely GO@Fe 3 O 4 @MOF-303。
In the step (3) of the invention, the weight ratio of the 3, 5-pyrazoledicarboxylic acid to the sodium hydroxide is as follows: 1:0.3-0.5;
in the step (4) of the invention, the weight ratio of the aluminum chloride hexahydrate to the 3, 5-pyrazoledicarboxylic acid is as follows: 1.3-2.5:1, a step of;
in step (5) of the present invention, the reaction temperature is preferably 50 to 100 ℃.
The magnetic nano probe modified by the metal organic framework with the aluminum ion as the center ion has the advantages of large specific surface area, high magnetic response effect, good hydrophilic pore canal structure and stable biocompatibility. The method has high separation effect, strong enrichment selectivity and ultralow sensitivity on glycosylated peptides, and can be used for selectively separating, enriching and identifying glycosylated peptides by mass spectrometry, and the method comprises the following specific steps: fully mixing a metal organic framework modified magnetic nano probe taking aluminum ions as central ions with a target glycosylated peptide solution, adding the mixture into 80-95% acetonitrile/1.0-2.0% trifluoroacetic acid buffer solution, uniformly dispersing, and incubating in a 35-40 ℃ enzymolysis instrument; magnetic probes are separated through magnetism, the magnetic probes are washed by 80-95% acetonitrile/1.0-2.0% trifluoroacetic acid buffer solution for 3-5 times, and the magnetic probes are eluted by 25-30% acetonitrile/0.10-0.15% trifluoroacetic acid; and (3) taking 0.8-1.2 mu L of eluent to be spotted on a MALDI-TOF-MS target plate, naturally drying, then dripping 0.8-1.2 mu L of 2, 5-dihydroxybenzoic acid (DHB) solution with the concentration of 15-25 mg/mL on the liquid drop of the analyte to form a thin-layer matrix, and drying and then carrying out mass spectrometry analysis.
Specifically, the magnetic nano probe modified by the metal organic framework with the aluminum ion as the center ion can be used for enrichment identification of glycopeptides in clinical serum samples (liver cancer patients and normal people) and analysis of glycosylated proteomics. After selectively enriching the synthesized metal organic framework modified magnetic nano probe with the aluminum ion as the center ion, carrying out mass spectrum high-flux detection, so that the signal intensity of the glycosylated peptide which cannot be identified originally is greatly improved, and a clear glycosylated peptide signal peak with high signal to noise ratio is obtained. The enrichment strategy has been practiced in several experiments with standard HRP proteolytic solution, HRP proteolytic solution and Bovine Serum Albumin (BSA) and HRP protein mixed solution, normal human serum, etc.
The invention has the beneficial effects that:
the synthesis method of the metal organic framework modified magnetic nano probe with the aluminum ion as the center ion is convenient and simple to operate, and the magnetic nano microsphere is successfully introduced, so that the material and the solution are subjected to rapid magnetic separation, and the experimental operation steps are greatly simplified; the hydrophilic pore structure with proper size of the material can separate and enrich glycosylated peptide in a large scale, successfully eliminates interference of other impurities such as macromolecular proteins, achieves a satisfactory effect in the identification of clinical complex samples (serum), and can effectively distinguish liver cancer from normal people by carrying out differential analysis on the identified glycosylated proteins. Experimental results fully prove that the magnetic nano probe prepared by the invention has good stability and good biocompatibility, is suitable for complex biological samples such as human serum, various tissue extracts, various cell proteins and the like, and has wide application prospect in large-scale analysis of glycosylated peptide samples. The nano probe can be applied to analysis and identification of glycosylated proteins in serum/urine samples of liver cancer patients in different types and periods, and can be used as a potential biomarker for finding out related liver cancer detection.
Drawings
FIG. 1 is a flow chart of the synthesis of a metal organic framework modified magnetic nanoprobe with aluminum ions as the center ions.
FIG. 2 is (a) GO@Fe in example 1 3 O 4 Transmission electron microscope photographs; (b) For example 1 GO@Fe 3 O 4 Transmission electron micrograph of @ MOF-303.
FIG. 3 is a diagram of example 2 at 10 -6 And (3) mass spectrograms of the HRP enzymolysis liquid (a) and (b) subjected to selective enrichment by using the metal organic framework modified magnetic nano probe taking aluminum ions as the center.
Fig. 4 shows that the mass ratio of example 3 is 1:1000 mass spectrograms of 1000 HRP proteolytic liquid, HRP protein and BSA protein mixed solution (a) and (b) before selective enrichment and (c) and (d) after selective enrichment of the metal organic framework modified magnetic nano probe with aluminum ion as the center.
Fig. 5 is a mass spectrum diagram of HRP enzymatic hydrolysate with different concentrations after selective enrichment of magnetic nanoprobes modified with metal organic frameworks centered on aluminum ions in example 4: (a) 10 fmol/. Mu.L, (b) 1 fmol/. Mu.L, (c) 0.5 fmol/. Mu.L and (d) 0.1 fmol/. Mu.L.
FIG. 6 shows enrichment and separation of glycosylated peptide fragments in clinical serum samples (liver cancer patients and normal persons) by combining the metal organic framework modified magnetic nanoprobe with aluminum ions as central ions in example 5, (a) PLS-DA model-based analysis and (b) thermogram analysis.
Detailed Description
Example 1: the synthesis method of the metal organic framework modified magnetic nano probe is shown in a figure 1, and specifically comprises the following steps:
(1) Dispersing 100 mg ferroferric oxide magnetic beads with the size of 15 nm in 25 mL normal hexane solution in which 25 mg graphene oxide is dissolved, performing ultrasonic dispersion for 2 hours, and performing magnetic separation to obtain a material;
(2) The material obtained in the step (1) is processedMaterial N 2 Calcining for 2 hours at 500 ℃ in atmosphere to obtain the graphene oxide material (GO@Fe) loaded with the ferroferric oxide pellets 3 O 4 ）；
(3) Dissolving 0.75 g of 3, 5-pyrazoledicarboxylic acid and 0.26. 0.26 g sodium hydroxide in 75 mL pure water solution, performing ultrasonic treatment for 1h, and preheating at 100 ℃ for 30min;
(4) Adding 1.04 g aluminum chloride hexahydrate into the solution obtained in the step (3), and carrying out ultrasonic treatment until the solid is dissolved;
(5) The material GO@Fe obtained in the step (2) is processed 3 O 4 Dispersing the powder in the solution obtained in the step (4), reacting for 15 hours at 100 ℃ and then magnetically separating to obtain a product, and then washing the product with deionized water and absolute ethyl alcohol for 3 times respectively to obtain the metal organic framework modified magnetic nano probe GO@Fe with aluminum ions as central ions 3 O 4 Vacuum drying overnight at 50deg.C;
GO@Fe 3 O 4 a transmission electron micrograph is shown in fig. 2 (a); GO@Fe 3 O 4 A transmission electron micrograph of @ MOF-303 is shown in FIG. 2 (b).
Example 2: the metal organic framework modified magnetic nano probe prepared in the example 1 is used for separating and enriching glycosylated peptide in standard HRP enzymatic hydrolysate and detecting MALDI-TOF-MS.
(1) Preparation of standard HRP proteolytic liquid: accurately weighing 1 mg standard protein HRP, dissolving in 25 mM ammonium bicarbonate buffer, boiling for 8 minutes, diluting to 1 mg/mL with 25 mM ammonium bicarbonate buffer, and then according to the mass ratio to protein of 1: adding proper amount of trypsin into the mixture, and carrying out enzymolysis for 16 hours at 37 ℃ overnight;
(2) Washing the 5 mg metal organic framework modified magnetic nano probe with aluminum ions as central ions with a buffer solution containing 95% acetonitrile/1.0% trifluoroacetic acid for 3 times, dispersing in 500 mu L of the buffer solution containing 95% acetonitrile/1.0% trifluoroacetic acid, and performing ultrasonic dispersion until the solution is uniform to prepare 10 mg/mL solution;
(3) Enrichment of glycosylated peptides: taking 50 mu L of the solution obtained in the step (2), and adding 50 mu L of 95% acetonitrile/1.0% trifluoroacetic acid buffer; then HRP standard proteolytic liquid is added to make the peptide fragment contentIs 10 -6 M, incubation reaction for 30min at 37 ℃; the glycopeptide-enriched magnetic probe was obtained by magnetic separation, washed three times with 200. Mu.L of 95% acetonitrile/1.0% trifluoroacetic acid buffer to remove non-glycopeptides and other impurities, then eluted with 10. Mu.L of 30% acetonitrile/0.1% trifluoroacetic acid buffer for 15 minutes, and magnetically separated to obtain an eluted peptide fragment solution.
(4) And (3) point target: taking 1 mu L of the eluent in the step (3) to be spotted on a MALDI-TOF-MS target plate, naturally drying the target plate in the air at room temperature, taking 1 mu L of 2, 5-dihydroxybenzoic acid solution (20 mg/mL of 50% ACN solution) as a matrix to be dropped on the liquid drop of the analyte, generating a thin matrix layer, and drying. Mass spectrometry is performed afterwards.
As can be seen in fig. 3, 10 before enrichment -6 The glycosylated peptide fragment is difficult to identify in the HRP standard proteolytic liquid of M, the mass spectrum is mainly composed of non-glycopeptides (figure 3 (a)), however, after the magnetic nano-probe modified by the metal organic framework with aluminum ions as the center ions acts, 25 glycosylated peptide peaks of HRP proteins appear in the mass spectrum, and the signal peaks of the glycosylated peptides are remarkably improved (figure 3 (b)).
Example 3: the metal organic framework modified magnetic nano probe taking aluminum ions as center ions obtained in the embodiment 1 is used for enrichment and MALDI-TOF-MS detection of HRP enzymatic hydrolysate and mixed solution of HRP protein and Bovine Serum Albumin (BSA) protein.
(1) According to the mass ratio of the protein 1:1000:1000 mixing HRP enzymolysis solution with HRP and BSA protein solution, adding 2 mu L of standard mixed solution into 150 mu L of 95% acetonitrile/1.0% trifluoroacetic acid buffer solution, adding 50 mu L of dispersion liquid (concentration is 10 mg/mL) of metal organic framework modified magnetic nano probe taking aluminum ions as the center, and incubating for 30 minutes at 37 ℃; the probe was separated magnetically, washed three times with 200. Mu.L of 95% acetonitrile/1.0% trifluoroacetic acid buffer, and eluted with 10. Mu.L of 30% acetonitrile/0.1% trifluoroacetic acid buffer for 15 minutes, followed by magnetic separation.
(2) And (3) point target: and (3) 1 mu L of the eluent in the step (2) is spotted on a MALDI-TOF-MS target plate, naturally dried in the air at room temperature, and then 1 mu L of 2, 5-dihydroxybenzoic acid solution (20 mg/mL of 50% ACN solution) is taken as a matrix to be dropped on the liquid drop of the analyte, so as to generate a thin matrix layer, and mass spectrometry analysis is carried out after drying.
As can be seen from fig. 4, glycosylated peptide Duan Feng was difficult to identify in HRP enzymatic hydrolysate and HRP protein and BSA protein mixed solution in a mass ratio of 1:1000:1000 prior to enrichment (fig. 4 (a)); however, after enrichment of the mixed solution with the metal organic framework modified magnetic nanoprobe with aluminum ion as the center ion, a large number of glycosylated peptide peaks are clearly shown in the mass spectrum (fig. 4 (c)). In the mass spectrometry linear mode, HRP protein and BSA protein were identified in the supernatant before enrichment (fig. 4 (b)), while no signal peak was observed for any protein in the eluate after enrichment (fig. 4 (d)).
Example 4: the metal organic framework modified magnetic nano probe with aluminum ions as central ions obtained in the embodiment 1 is used for enrichment and MALDI-TOF-MS detection of ultralow-concentration HRP enzymatic hydrolysate.
(1) Enrichment of glycosylated peptide fraction 50. Mu.L of dispersion of metal organic framework modified magnetic nanoprobe with aluminum ion as center ion (concentration 10 mg/mL) was added to 50. Mu.L of 95% acetonitrile/1.0% trifluoroacetic acid buffer; diluting the standard proteolytic liquid with 25 mM ammonium bicarbonate buffer solution to make the final concentration of proteolytic liquid be 0.1 fmol/. Mu.L, and incubating at 37 deg.C for 45 min; magnetic separation, after washing three times with 200. Mu.L of 95% acetonitrile/1.0% trifluoroacetic acid buffer, eluting with 10. Mu.L of 30% acetonitrile/0.1% trifluoroacetic acid buffer for 15 minutes, magnetic separation.
(2) And (3) point target: and (3) 1 mu L of the eluent in the step (1) is spotted on a MALDI-TOF-MS target plate, naturally dried in the air at room temperature, and then 1 mu L of 2, 5-dihydroxybenzoic acid solution (20 mg/mL of 50% ACN solution) is taken as a matrix to be dropped on the liquid drop of the analyte, so as to generate a thin matrix layer, and mass spectrometry analysis is carried out after drying.
As can be seen from fig. 5, even when the HRP proteolytic liquid concentration was as low as 0.1 fmol/μl, 6 glycosylated peptides Duan Feng derived from HRP protein could be clearly observed in the mass spectrum after enrichment of the magnetic nanoprobe modified with a metal organic framework with aluminum ion as the center ion (fig. 5 (d)).
Example 5: the metal organic framework modified magnetic nano probe with aluminum ions as central ions obtained in the embodiment 1 is used for enrichment and nano-LC-MS/MS detection of glycosylated peptides in serum enzymolysis solution of clinical samples.
(1) Preparing clinical serum enzymolysis liquid: 1. mu.L of human serum was diluted to 20. Mu.L with deionized water and centrifuged at 14000 rmp for 15 min. The supernatant was collected, 10 mM dithiothreitol was added and reacted at 60℃for 30 minutes, followed by 20 mM indoleacetic acid being added and reacted at 37℃for 60 minutes in the absence of light. After cooling, acetone is added according to the volume ratio of the protein to the acetone solution of 1:6, protein precipitation is carried out at-20 ℃, and the reaction is carried out for 12 hours overnight. Before proteolysis, 50 mM ammonium bicarbonate solution is used for dilution, so that the final protein solubility is 2 mg/mL, and the ratio of protein to protein is 1:30 adding proper amount of trypsin into the supernatant, and carrying out enzymolysis for 16 hours at 37 ℃.
(2) Enrichment of glycosylated peptide fragments: 200 mu L of enzymolysis liquid of human serum and 100 mu L of dispersion liquid (the concentration is 10 mg/mL) of metal organic framework modified magnetic nano probe taking aluminum ions as central ions are added into 150 mu L of 95% acetonitrile/1.0% trifluoroacetic acid buffer solution, and the mixture is incubated and spun for 30 minutes at 37 ℃; magnetic separation, after washing three times with 300. Mu.L of 95% acetonitrile/1.0% trifluoroacetic acid buffer, eluting with 20. Mu.L of 30% acetonitrile/0.1% trifluoroacetic acid buffer for 15 minutes, magnetic separation.
(3) Completely lyophilizing the eluted peptide fragment sample, dissolving the dry powder with 12 μl of chromatographic mobile phase A (0.1% formic acid aqueous solution), centrifuging at 17000 rpm for 15 min, and sucking the supernatant; 8. Mu.L of sample was taken, and the chromatographic mobile phase B (0.1% acetonitrile in formic acid) was set from 2% to 45% for linear separation, followed by elution analysis at a flow rate of 300 nL/min.
Enrichment is carried out by using a metal organic framework modified magnetic nano probe taking aluminum ions as central ions, 274 glycosylated peptide fragments and 101 glycosylated proteins are identified in total in normal human serum, 265 glycosylated peptide fragments and 102 glycosylated proteins are identified in total in serum samples of liver cancer patients. And the difference of the identified glycosylated proteins between the dissected samples based on the PLS-DA model can clearly distinguish the serum samples of normal human serum from liver cancer patients (FIG. 6).
Example 6: the specific steps of the method for synthesizing the metal-organic framework modified magnetic nanoprobe by taking aluminum ions as the center ions are the same as those of the embodiment 1, and the difference is that: the amounts of 3, 5-pyrazoledicarboxylic acid and sodium hydroxide in example 1 (3) were reduced to 0.5g and 0.175g, respectively, and the amount of aluminum chloride hexahydrate in example 1 (4) was reduced to 0.695. 0.695 g.
Example 7: the magnetic nano probe modified by the metal organic framework with the aluminum ion as the center ion obtained in the example 6 is used for separating and enriching glycosylated peptides in standard HRP enzymatic hydrolysate and detecting MALDI-TOF-MS, and the specific steps are the same as (1), (2), (3) and (4) in the example 2.
After enrichment of the metal organic framework modified magnetic nanoprobe with aluminum ions as central ions obtained in example 6, only 18 glycosylated peptide peaks of HRP proteins appear in a mass spectrum, which indicates that the reduction of the coating amount of MOF-303 has adverse effect on enrichment effect.
Example 8: the specific procedure of the method for synthesizing the metal-organic framework modified magnetic nanoprobe using aluminum ion as the center ion is the same as that of example 1.
Example 9: the metal organic framework modified magnetic nano probe taking aluminum ions as center ions obtained in the embodiment 8 is used for separating and enriching glycosylated peptides in standard HRP enzymatic hydrolysate and detecting MALDI-TOF-MS.
(1) The standard HRP proteolytic liquid was prepared as in (1) of example 2;
(2) Washing the 5 mg metal organic framework modified magnetic nano probe with aluminum ions as central ions with a buffer solution containing 85% acetonitrile/1.0% trifluoroacetic acid for 3 times, dispersing in 500 mu L of the buffer solution containing 85% acetonitrile/1.0% trifluoroacetic acid, and performing ultrasonic dispersion until the solution is uniform to prepare 10 mg/mL solution;
(3) Enrichment of glycosylated peptides: taking 50 mu L of the solution obtained in the step (2), and adding 50 mu L of 85% acetonitrile/1.0% trifluoroacetic acid buffer; then adding HRP standard proteolytic liquid to make the peptide fragment content 10-6M, and incubating at 37 deg.C for 30min; the glycopeptide-enriched magnetic probe was obtained by magnetic separation, washed three times with 200. Mu.L of 85% acetonitrile/1.0% trifluoroacetic acid buffer to remove non-glycopeptides and other impurities, then eluted with 10. Mu.L of 30% acetonitrile/0.1% trifluoroacetic acid buffer for 15 minutes, and magnetically separated to obtain an eluted peptide fragment solution.
(4) And (3) point target: as in example 2 (4)
After enrichment of the magnetic nano-probe modified by the metal organic framework with aluminum ions as the center ions obtained in the embodiment 8, only 16 glycosylated peptide peaks of HRP proteins appear in a mass spectrum, which indicates that the enrichment elution condition has adverse effect on the enrichment effect of the probe.
Claims (6)
1. The synthesis method of the metal organic framework modified magnetic nano probe with the aluminum ion as the center ion is characterized by comprising the following specific steps:
(1) Dispersing 50-200mg of ferroferric oxide magnetic beads with the size of 10-30nm in 10-30 mL n-hexane solution containing 20-50mg of graphene oxide, performing ultrasonic dispersion for 1-4 hours, and magnetically separating out materials;
(2) Calcining the material obtained in the step (1) at a high temperature of 400-600 ℃ for 2-6h, wherein the obtained material is denoted as GO@Fe 3 O 4 ；
(3) Dissolving 0.5-1 g of 3, 5-pyrazoledicarboxylic acid and 0.2-0.5g of sodium hydroxide in 50-150 mL pure water solution, performing ultrasonic treatment for 0.5-2h, and then preheating at 100 ℃ for 30min;
(4) Adding 0.8-2.1g of aluminum chloride hexahydrate into the solution obtained in the step (3), and carrying out ultrasonic treatment until the aluminum chloride hexahydrate is dissolved;
(5) Dispersing the material obtained in the step (2) in the solution obtained in the step (4), reacting for 2-24 hours at 25-100 ℃, magnetically separating out a product, washing with deionized water and absolute ethyl alcohol for 2-5 times, and vacuum drying at 40-80 ℃ to obtain the metal organic framework modified magnetic nano probe taking aluminum ions as central ions, namely GO@Fe 3 O 4 @MOF-303。
2. The method according to claim 1, wherein the weight ratio of 3, 5-pyrazoledicarboxylic acid to sodium hydroxide in the step (3) is: 1:0.3-0.5.
3. The method according to claim 1, wherein the weight ratio of aluminum chloride hexahydrate to 3, 5-pyrazoledicarboxylic acid in step (4) is: 1.3-2.5:1.
4. a metal organic framework-modified magnetic nanoprobe having an aluminum ion as a center ion obtained by the synthesis method of any one of claims 1 to 3.
5. The use of the metal organic framework modified magnetic nanoprobe with aluminum ions as center ions in glycosylated peptide selective separation, enrichment and mass spectrometry identification as claimed in claim 4, wherein the use is as follows: fully mixing a metal organic framework modified magnetic nano probe taking aluminum ions as central ions with a target glycosylated peptide solution, adding the mixture into 80-95% acetonitrile/1.0-2.0% trifluoroacetic acid buffer solution, uniformly dispersing, and incubating in a 35-40 ℃ enzymolysis instrument; magnetic probes are separated through magnetism, the magnetic probes are washed by 80-95% acetonitrile/1.0-2.0% trifluoroacetic acid buffer solution for 3-5 times, and the magnetic probes are eluted by 25-30% acetonitrile/0.10-0.15% trifluoroacetic acid; and (3) taking 0.8-1.2 mu L of eluent to be spotted on a MALDI-TOF-MS target plate, naturally drying, then dripping 0.8-1.2 mu L of 2, 5-dihydroxybenzoic acid (DHB) solution with the concentration of 15-25 mg/mL on the liquid drop of the analyte to form a thin-layer matrix, and drying and then carrying out mass spectrometry analysis.
6. Use of the metal organic framework modified magnetic nanoprobe with aluminum ions as center ions according to claim 4 for enrichment identification of glycopeptides of clinical serum samples and glycosylated proteomics analysis thereof.
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