Magnetic nanomaterial capable of simultaneously enriching phosphopeptides and glycopeptides, and preparation method and application thereof

Abstract

The application relates to a magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides and a preparation method and application thereof. A dendrimer PAMAM is modified on Fe3O4 magnetic beads, then a negative charged functional group and titanium ions Ti 4+ are modified on the surface of the dendrimer PAMAM, and the magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides is obtained. The magnetic nanomaterial is mixed with a sample to be detected after enzymolysis in an enrichment solution system, after solid-liquid separation and washing, supernatant containing non-phosphorylated peptides and non-glycopeptides is removed; mixing reaction is carried out in an elution solution system, and supernatant containing phosphorylated peptides and glycopeptides is obtained, sugar chains are removed through enzymolysis, and a sample simultaneously enriched with phosphorylated peptides and glycopeptides is obtained. The magnetic nanomaterial is simple in synthesis and high in magnetic response capability, when the magnetic nanomaterial is applied to enrichment of phosphorylated peptides and glycopeptides, the anti-interference capability is improved, and the magnetic nanomaterial has advantages of high efficiency and high selectivity and can be widely applied to the field of biological medicine sample analysis.

Description

Technical Field

[0001] This invention relates to the field of nanomaterial preparation and application technology, and in particular to a magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides, its preparation method and application. Background Technology

[0002] Protein phosphorylation and glycosylation, as two of the most typical and well-regarded post-translational modifications of proteins, play important regulatory roles in cellular processes. For example, aberrant phosphorylation of Tau protein is observed in Alzheimer's disease, and aberrant glycosylation is a protein marker in many cancers. Therefore, studying protein phosphorylation and glycosylation and their interactions at the molecular level is of great significance for revealing their regulatory mechanisms in life processes. Mass spectrometry has provided strong technical support for proteomics research, but due to the reversible dynamics and low abundance of phosphorylation and glycosylation modifications, the efficiency of mass spectrometry analysis is easily limited by dynamic range and low peptide ionization efficiency, posing a significant challenge to directly analyzing phosphopeptides and glycoproteins in complex biological samples. Therefore, before mass spectrometry analysis, it is necessary to enrich low-abundance phosphorylated peptides and glycopeptides to increase their detection count and intensity.

[0003] Current methods for enriching phosphorylated peptides include metal ion affinity chromatography and metal oxide affinity chromatography, while methods for enriching glycopeptides include boric acid method, lectin method, hydrazine chemical method, and hydrophilic interaction chromatography. However, there are relatively few methods developed for the co-enrichment of phosphorylated peptides and glycopeptides, and these methods mainly suffer from limitations such as complex synthesis of enrichment materials, poor anti-interference ability during enrichment, and low sensitivity.

[0004] Patent CN109855929A discloses a method for multi-mode capture and continuous strong elution of glycosylated and phosphorylated peptides. The specific steps are as follows: A strongly magnetically responsive nanomaterial modified with titanium ions and cysteine ​​is prepared into a dispersion with an enrichment buffer solution. This dispersion is then mixed with the target glycosylated peptide and a standard phosphorylated peptide and incubated for 40-50 minutes. With the assistance of a magnet, the material is washed with an enrichment buffer solution. Elution buffer I is prepared with acetonitrile (1%-80% by volume) and trifluoroacetic acid (0.1%-5% by volume), and elution buffer II is prepared with ammonia (1%-10% by volume). The two eluents are used for target spotting and then mixed for mass spectrometry analysis. However, this method involves a complex material synthesis process and requires multiple elution steps.

[0005] Patent CN113828284A discloses a method for preparing and using multi-metal multifunctional magnetic nanomaterials. The specific steps are as follows: a hydrophilic metal-organic framework (MOF) matrix porous material is synthesized in situ on the surface of magnetic Fe3O4 nanoparticles; phosphate groups are introduced through the electrostatic interaction between the amino groups of the hydrophilic ligands in the MOF material and phytic acid; and metal ions are further immobilized to prepare the multi-metal multifunctional magnetic nanomaterial. The MOF uses one or more transition metals such as zirconium and titanium as the metal center; another transition metal ion is immobilized by phytic acid. The metal oxide core, the metal-oxygen clusters in the MOF material, and the phytic acid-immobilized metal ions simultaneously provide a large number of phosphorylated peptide affinity sites. The introduction of hydrophilic groups such as amino groups and phosphate groups enables high selectivity and efficient enrichment of glycosylated peptides. Simultaneous capture and stepwise selective elution achieve the simultaneous enrichment and separation of phosphorylated and glycosylated peptides. However, this method involves a complex material synthesis process and poor anti-interference ability during the enrichment process.

[0006] Therefore, in order to achieve the co-enrichment and elution of phosphorylated peptides and glycopeptides in complex biological samples, there is an urgent need to develop a simple and efficient new co-enrichment method that can guarantee the coverage and selectivity of phosphorylated peptides and glycopeptides. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of complex synthesis process of enrichment materials, poor anti-interference ability and low sensitivity in existing methods for co-enrichment of phosphorylated peptides and glycopeptides, and to provide a magnetic nanomaterial that can simultaneously enrich phosphorylated peptides and glycopeptides, as well as its preparation method and application.

[0008] The magnetic nanomaterials provided in this application are simple to synthesize, have abundant activation sites, and have a fast response speed. They can be used for the efficient enrichment, separation, and analysis of low stoichiometry phosphorylated peptides and glycopeptides in complex biological samples, and have the characteristics of high specificity, high selectivity, and simple operation.

[0009] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides a method for preparing magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides, comprising: Dendritic macromolecules PAMAM were modified onto Fe3O4 magnetic beads, and then negatively charged functional groups and titanium ions (Ti) were added to the surface of the dendritic macromolecules PAMAM. 4+ This yielded magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides.

[0010] In one embodiment of the present invention, the modification method between Fe3O4 magnetic beads and dendritic macromolecule PAMAM is as follows: using the cross-linking reaction between carboxyl and amino groups, dendritic macromolecule PAMAM is modified onto Fe3O4 magnetic beads.

[0011] In one embodiment of the present invention, the surface of the Fe3O4 magnetic beads is modified with carboxyl groups, and the dendritic macromolecule PAMAM is an amino-terminated polyamide amine. By utilizing the cross-linking reaction between the carboxyl groups and the amino groups, the dendritic macromolecule PAMAM can be modified onto the Fe3O4 magnetic beads.

[0012] In one embodiment of the invention, the dendritic macromolecule PAMAM is a commercially available fifth-generation amino-terminated polyamide amine.

[0013] In one embodiment of the present invention, the negatively charged functional group is selected from any one or a combination of several of carboxyl, aldehyde, or phosphate groups.

[0014] In one embodiment of the invention, the titanium ions react with negatively charged functional groups through chelation.

[0015] In one embodiment of the present invention, the method for preparing magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides further employs the following scheme: amino-containing dendritic macromolecules PAMAM are modified onto Fe3O4 magnetic beads via a crosslinking reaction between carboxyl and amino groups; then, compounds with negatively charged functional groups are modified onto the amino groups of the dendritic macromolecules PAMAM; finally, Ti... 4+ Magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides are obtained by modifying the material surface through chelation and interaction with negatively charged functional groups.

[0016] In one embodiment of the present invention, a method for preparing magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides specifically includes the following steps: S1: Carboxyl-modified Fe3O4 magnetic beads were dispersed in 2-morpholine ethanesulfonic acid buffer solution, N-hydroxysuccinimide ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, activated, washed, dendritic macromolecule PAMAM solution was added, reacted, and the product was washed to obtain Fe3O4@PAMAM. S2: Disperse Fe3O4@PAMAM in phytic acid solution, stir and react. After the reaction is complete, wash the product to obtain Fe3O4@PAMAM@PA. S3: Dissolve Fe3O4@PAMAM@PA in a solution containing Ti 4+ The reaction was carried out in a solution with stirring. After the reaction was completed, the product was washed to obtain the magnetic nanomaterials Fe3O4@PAMAM@PA-Ti. 4+ This refers to magnetic nanomaterials that can simultaneously enrich phosphorylated peptides and glycopeptides.

[0017] In one embodiment of the present invention, in step S1, the ratio of carboxyl-modified Fe3O4 to 2-morpholine ethanesulfonic acid buffer solution is (1-100) mg : (0.1-10) mL, the concentration of N-hydroxysuccinimide ester is 0.05-0.5 M, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.02-0.5 M; the pH of 2-morpholine ethanesulfonic acid buffer solution is 5.0-6.5, the dendritic macromolecular PAMAM solution is a methanol solution with a PAMAM mass fraction of 1%-10%, and when the carboxyl-modified Fe3O4 is (1-100) mg, the amount of dendritic macromolecular PAMAM solution used is 5-200 μL.

[0018] In one embodiment of the present invention, in step S1, the activation conditions are treatment at 20-50°C for 10-120 min.

[0019] In one embodiment of the present invention, in step S2, the ratio of the amount of Fe3O4@PAMAM to the amount of phytic acid solution is (1-100) mg: (1-100) mL, and the concentration of the phytic acid is 1-50 mg / mL.

[0020] In one embodiment of the present invention, step S3 contains Ti 4+ The solution is a Ti(SO4)2 solution.

[0021] In one embodiment of the present invention, in step S3, the ratio of the amount of Fe3O4@PAMAM@PA to Ti(SO4)2 solution is (1-100) mg: (1-100) mL, and the concentration of Ti(SO4)2 is 20-500 mM.

[0022] More specifically, in the preparation process of the aforementioned magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides, the following conditions were met: 10 mg of carboxyl-modified Fe3O4, 1 ml of 2-morpholine ethanesulfonic acid buffer solution, pH 5.5, 0.15 M concentration of N-hydroxysuccinimide ester, 0.1 M concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and activation conditions of 37°C. C, 30 min, PAMAM solution is soluble in methanol and has a mass fraction of 5%, PAMAM solution volume is 20 L; wherein PAMAM is a commercially available 5th generation amino-terminated polyamide amine; in step S2, when Fe3O4@PAMAM solid powder is used, the volume of phytic acid solution is 20 mL, and the concentration is 8.4-16.8 mg / mL; in step S3, when Fe3O4@PAMAM@PA is used, the volume of Ti(SO4)2 solution is 20 mL, and the concentration is 100-200 mM.

[0023] In a second aspect, the present invention provides a magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides, wherein the magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides is prepared based on the above-described preparation method.

[0024] A third aspect of this invention provides the application of magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides, wherein the magnetic nanomaterials are used to simultaneously enrich phosphorylated peptides and glycopeptides in a sample. This results in samples containing phosphorylated peptides and glycopeptides that can be used for mass spectrometry analysis. In other words, the magnetic nanomaterials provided in this application capable of simultaneously enriching phosphorylated peptides and glycopeptides can be used in analytical chemistry and post-translational modification proteomics, among other technical fields.

[0025] In a fourth aspect, the present invention provides a method for co-enriching phosphorylated peptides and glycopeptides. The method involves mixing the magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides with the enzymatically digested sample in an enrichment solution system. After solid-liquid separation and washing, the supernatant containing non-phosphorylated peptides and non-glycopeptides is removed. The mixture is then mixed in an elution solution system to obtain a supernatant containing both phosphorylated peptides and glycopeptides. The glycans are then removed by enzymatic cleavage to obtain a sample simultaneously enriched with phosphorylated peptides and glycopeptides.

[0026] In one embodiment of the present invention, the method for co-enriching phosphorylated peptides and glycopeptides specifically includes the following steps: S1: Activate the magnetic nanomaterials that can simultaneously enrich phosphorylated peptides and glycopeptides using an enrichment solution to obtain a material suspension; S2: Mix the material suspension described in step S1 with the sample to be tested after enzymatic hydrolysis, enrich it, and remove the supernatant containing non-phosphorylated peptides and non-glycopeptides after solid-liquid separation and washing to obtain the material containing phosphorylated peptides and glycopeptides. S3: Mix the material containing phosphorylated peptides and glycopeptides described in step S2 with the elution solution, elute, and obtain a supernatant containing phosphorylated peptides and glycopeptides through solid-liquid separation. S4: The supernatant containing phosphorylated peptides and glycopeptides described in step S3 is deglycosylated by enzyme digestion to remove the glycan chains, thereby obtaining a sample enriched with both phosphorylated peptides and glycopeptides. The sample enriched with both phosphorylated peptides and glycopeptides can be directly used for mass spectrometry analysis.

[0027] In one embodiment of the present invention, in step S1, the enrichment solution is a mixed solution of organic solvent, acid and water: the volume ratio of organic solvent is 70-90%, the volume ratio of acid is 0.1-5%, and the pH range of the enrichment solution is 0.5-3.

[0028] In one embodiment of the present invention, the organic solvent is selected from one or more of acetonitrile, methanol, and ethanol, and the acid is selected from one or more of formic acid, acetic acid, trifluoroacetic acid, etc.

[0029] Preferably, in step S1, the enrichment solution is a mixed solution of acetonitrile, trifluoroacetic acid, and water, wherein the components are 90% acetonitrile and 3% trifluoroacetic acid.

[0030] In one embodiment of the present invention, in step S2, the sample to be tested after enzymatic hydrolysis refers to the sample to be tested after hydrolysis with a mass spectrometry-grade enzyme. The mass spectrometry-grade enzyme is selected from one or more of Trypsin, LysC, or Arg. The mass spectrometry-grade enzyme is located in a buffer solution, and the buffer solution of the mass spectrometry-grade enzyme is an aqueous solution of ammonium bicarbonate.

[0031] In one embodiment of the present invention, in step S2, the sample to be tested includes: (1) a peptide standard containing phosphorylated peptides and glycopeptides; (2) a complex sample such as tissue, cell, body fluid, or mixed protein containing phosphorylated peptides and glycopeptides; and (3) a mixed sample of the above (1) and (2).

[0032] In one embodiment of the present invention, in step S2, when the sample to be tested is a complex biological sample, before enzymatic digestion, the complex biological sample is further subjected to lysis, reduction, and alkylation. The lysis reagent used for lysis is a surfactant, the reduction reagent used for reduction is tris(2-carboxyethyl)phosphine (TCEP) or urea, and the alkylation reagent used for alkylation is chloroacetamide (CAA), iodoacetamide (IAA), or N-ethylmaleimide. The concentration of the reduction reagent is 5-20 mmol / L, and the concentration of the alkylation reagent is 20-80 mmol / L.

[0033] In one embodiment of the present invention, in step S3, the elution solution is an alkaline solution or an alkaline solution / organic solvent mixture. The alkaline solution has a mass concentration of 5-10% and a pH range of 10-12. In the alkaline solution / organic solvent mixture, the volume ratio of the organic solvent is 0-70%, and the pH range is 10-12.

[0034] In one embodiment of the present invention, in step S3, the organic solvent in the elution solution is selected from one or more of acetonitrile, methanol, and ethanol, and the alkaline solution is selected from one or more of sodium carbonate aqueous solution, sodium bicarbonate aqueous solution, ammonium bicarbonate aqueous solution, and ammonia solution.

[0035] Preferably, in step S3, the elution solution is 10% ammonia water by volume.

[0036] In one embodiment of the present invention, the enrichment temperature range in step S2 is 25-45°C. C, the reaction time range is 30-60 min.

[0037] In one embodiment of the present invention, in step S3, the reaction temperature range is 25-45°C. C, the reaction time range is 30-60 min.

[0038] In one embodiment of the present invention, in step S4, the enzyme used for enzymatic digestion is selected from one or more of PNGase F, PNGase H or sialyl glycosidase, and the enzyme buffer solution is an aqueous solution of ammonium bicarbonate; the temperature range for enzymatic digestion of sugar chains is 10-60°C, and the reaction time range is 1-24 h.

[0039] The technical principle of this invention lies in the fact that hydrophilic groups such as amino and hydroxyl groups can be used to specifically enrich glycopeptides, while abundant titanium ion modification can be used to specifically enrich phosphorylated peptides. The magnetic nanomaterials provided in this application, capable of simultaneously enriching phosphorylated peptides and glycopeptides, contain both negatively charged functional groups (suitable for specifically enriching glycopeptides) and titanium ions (suitable for specifically enriching phosphorylated peptides) on Fe3O4 magnetic beads. Therefore, the magnetic nanomaterials provided in this application can simultaneously enrich phosphorylated peptides and glycopeptides.

[0040] In an enrichment solution environment with specific temperature, pH, and organic phase ratio, the magnetic nanomaterials of this application can selectively bind phosphorylated peptides and glycopeptides, while other impurity peptides remain in the supernatant. Non-phosphorylated and glycosylated peptides can be removed through magnetic solid-liquid separation and washing. Furthermore, the binding between phosphorylated peptides and glycopeptides and the magnetic nanomaterials is disrupted by the elution solution, thereby allowing the phosphorylated peptides and glycopeptides to reappear in the supernatant.

[0041] Compared with the prior art, the present invention has the following beneficial effects: (1) The magnetic nanomaterials provided by the present invention, which can simultaneously enrich phosphorylated peptides and glycopeptides, are simple to synthesize and have strong magnetic response capabilities, which can shorten the overall process; the magnetic nanomaterials have a large number of hydrophilic groups such as hydroxyl and amino groups, which ensure the enrichment efficiency of glycopeptides while improving the anti-interference ability; the magnetic nanomaterials are loaded with abundant titanium ions, which is beneficial to the enrichment of phosphorylated peptides.

[0042] (2) Based on magnetic nanomaterials that can simultaneously enrich phosphorylated peptides and glycopeptides, this invention exhibits advantages such as high efficiency and high selectivity in the enrichment and separation of phosphorylated peptides and glycopeptides when co-enriching phosphorylated peptides and glycopeptides, and can achieve co-enrichment and one-step elution of phosphorylated peptides and glycopeptides.

[0043] (3) This invention can be used for complex sample analysis, including but not limited to the whole phosphorylation and glycosylation proteomics analysis of model organisms, and can be widely applied in the field of biomedical sample analysis. Attached Figure Description

[0044] Figure 1This is a schematic diagram illustrating the preparation and enrichment method of the magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides according to the present invention.

[0045] Figure 2 The images show the infrared spectrum and Zeta potential of the magnetic nanomaterials prepared in Example 1.

[0046] Figure 3 As in Example 2, after co-enrichment and elution, - A schematic diagram of mass spectrometry signals of phosphorylated peptides and glycopeptides in the protein hydrolysate of casein and horseradish peroxidase.

[0047] Figure 4 This is a comparison of mass spectrometry signal intensities after optimizing the enrichment solution and elution solution in Example 2.

[0048] Figure 5 The low concentration obtained after co-enrichment and elution in Example 3 - A schematic diagram of mass spectrometry signals of phosphorylated peptides and glycopeptides in the protein hydrolysate of casein and horseradish peroxidase.

[0049] Figure 6 As in Example 4, after co-enrichment and elution, - A schematic diagram of the mass spectrometry signals of phosphorylated peptides and glycopeptides after casein and horseradish peroxidase protein were doped with bovine serum albumin protein hydrolysate. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0051] This invention provides a method for preparing magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides, and a method for co-enriching phosphorylated peptides and glycopeptides using such nanomaterials. (Reference) Figure 1 , Figure 1 Table a presents a preparation process for magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides. Figure 1 Table b presents a procedure for co-enriching phosphorylated peptides and glycopeptides using this method.

[0052] The preparation method of magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides specifically includes the following steps: S1: Carboxyl-modified Fe3O4 magnetic beads were dispersed in 2-morpholine ethanesulfonic acid buffer solution, N-hydroxysuccinimide ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, activated, washed, dendritic macromolecule PAMAM solution was added, reacted, and the product was washed to obtain Fe3O4@PAMAM. S2: Disperse Fe3O4@PAMAM in phytic acid solution, stir and react. After the reaction is complete, wash the product to obtain Fe3O4@PAMAM@PA. S3: Dissolve Fe3O4@PAMAM@PA in a solution containing Ti 4+ The reaction was carried out in a solution with stirring. After the reaction was completed, the product was washed to obtain the magnetic nanomaterials Fe3O4@PAMAM@PA-Ti. 4+ This refers to magnetic nanomaterials that can simultaneously enrich phosphorylated peptides and glycopeptides.

[0053] In step S1, the ratio of carboxyl-modified Fe3O4 to 2-morpholine ethanesulfonic acid buffer solution is (1–100) mg : (0.1–10) mL. The concentration of N-hydroxysuccinimide ester is 0.05–0.5 M, and the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.02–0.5 M. The pH of the 2-morpholine ethanesulfonic acid buffer solution is 5.0–6.5. The dendritic macromolecular PAMAM solution is a methanol solution with a PAMAM mass fraction of 1%–10%. When the amount of carboxyl-modified Fe3O4 is (1–100) mg, the volume of the dendritic macromolecular PAMAM solution is 5–200 μL. In step S1, the activation conditions are treatment at 20–50 °C for 10–120 min. In step S2, the ratio of Fe3O4@PAMAM to phytic acid solution is (1-100) mg : (1-100) mL, and the concentration of phytic acid is 1-50 mg / mL. In step S3, Ti is contained... 4+ The solution is a Ti(SO4)2 solution. In step S3, the ratio of Fe3O4@PAMAM@PA to Ti(SO4)2 solution is (1-100) mg : (1-100) mL, and the concentration of Ti(SO4)2 is 20-500 mM.

[0054] This invention also provides a method for co-enriching phosphorylated peptides and glycopeptides, specifically comprising the following steps: S1: Activate the magnetic nanomaterials that can simultaneously enrich phosphorylated peptides and glycopeptides using an enrichment solution to obtain a material suspension; S2: Mix the material suspension described in step S1 with the sample to be tested after enzymatic hydrolysis, enrich it, and remove the supernatant containing non-phosphorylated peptides and non-glycopeptides after solid-liquid separation and washing to obtain the material containing phosphorylated peptides and glycopeptides. S3: Mix the material containing phosphorylated peptides and glycopeptides described in step S2 with the elution solution, elute, and obtain a supernatant containing phosphorylated peptides and glycopeptides through solid-liquid separation. S4: The supernatant containing phosphorylated peptides and glycopeptides described in step S3 is deglycosylated by enzyme digestion to remove the glycan chains, thereby obtaining a sample enriched with both phosphorylated peptides and glycopeptides. The sample enriched with both phosphorylated peptides and glycopeptides can be directly used for mass spectrometry analysis.

[0055] In step S1, the enrichment solution is a mixed solution of organic solvent, acid, and water: the volume ratio of organic solvent is 70-90%, the volume ratio of acid is 0.1-5%, and the pH range of the enrichment solution is 0.5-3. The organic solvent is selected from one or more of acetonitrile, methanol, and ethanol, and the acid is selected from one or more of formic acid, acetic acid, trifluoroacetic acid, etc.

[0056] In step S2, the sample to be tested after enzymatic hydrolysis refers to the sample to be tested after hydrolysis with a mass spectrometry-grade enzyme. The mass spectrometry-grade enzyme is selected from one or more of Trypsin, LysC, or Arg. The mass spectrometry-grade enzyme is located in a buffer solution, and the buffer solution of the mass spectrometry-grade enzyme is an aqueous solution of ammonium bicarbonate.

[0057] In step S2, the sample to be tested includes: (1) peptide standards containing phosphorylated peptides and glycopeptides; (2) complex samples such as tissues, cells, body fluids, and mixed proteins containing phosphorylated peptides and glycopeptides; and (3) a mixed sample of the above (1) and (2). In step S2, when the sample to be tested is a complex biological sample, before enzymatic digestion, the complex biological sample is also lysed, reduced, and alkylated. The lysing reagent used for lysing is a surfactant, the reducing reagent used for reduction is tris(2-carboxyethyl)phosphine (TCEP) or urea, and the alkylating reagent used for alkylation is chloroacetamide (CAA), iodoacetamide (IAA), or N-ethylmaleimide. The concentration of the reducing reagent is 5-20 mmol / L, and the concentration of the alkylating reagent is 20-80 mmol / L.

[0058] In step S3, the elution solution is an alkaline solution or a mixture of an alkaline solution and an organic solvent. The alkaline solution has a mass concentration of 5-10% and a pH range of 10-12. In the alkaline solution / organic solvent mixture, the volume ratio of the organic solvent is 0-70%, and the pH range is 10-12. In the elution solution, the organic solvent is selected from one or more of acetonitrile, methanol, and ethanol, and the alkaline solution is selected from one or more of sodium carbonate aqueous solution, sodium bicarbonate aqueous solution, ammonium bicarbonate aqueous solution, and ammonia solution.

[0059] In step S2, the enrichment temperature range is 25-45°C, and the enrichment reaction time range is 30-60 min. In step S3, the elution temperature range is 25-45°C, and the elution reaction time range is 30-60 min.

[0060] In step S4, the enzyme used for digestion is selected from one or more of PNGase F, PNGase H, or sialyl glycosidase, and the enzyme buffer solution is an aqueous solution of ammonium bicarbonate; the temperature range for digestion of sugar chains is 10-60°C, and the reaction time range is 1-24 h.

[0061] In the following embodiments, unless otherwise specified, the raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0062] Example 1: This embodiment provides a method for preparing magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides, the specific steps of which are as follows: (1) Preparation of Fe3O4@PAMAM: 10 mg of Fe3O4 solid powder was dispersed in 1 mL of 2-morpholinoethanesulfonic acid (MES) buffer solution (pH=5.5), and N-hydroxysuccinimide ester (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) were added to make the final concentration of N-hydroxysuccinimide ester (NHS) 0.15 M and the final concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) 0.1 M. 37 Activate the carboxyl group at C for 30 min, then wash the activated product three times with phosphate (PBS) buffer, and add 20 5% PAMAM (dissolved in methanol) was reacted at room temperature for 16 h. The product was washed three times with PBS to obtain Fe3O4@PAMAM.

[0063] (2) Preparation of Fe3O4@PAMAM@PA: 10 mg of Fe3O4@PAMAM solid powder was dispersed in 20 mL of phytic acid (PA) solution with a concentration of 12.6 mg / mL, and the mixture was mechanically stirred at room temperature for 6 h. After the reaction was completed, the product was washed three times with water to obtain Fe3O4@PAMAM@PA.

[0064] (3) Fe3O4@PAMAM@PA-Ti 4+ Preparation: 10 mg of Fe3O4@PAMAM@PA was dissolved in 20 mL of 150 mM Ti(SO4)2 solution, and the mixture was mechanically stirred at room temperature for 2 h. After the reaction was complete, the product was washed three times with water to obtain Fe3O4@PAMAM@PA-Ti. 4+ .

[0065] Characterization of the magnetic nanomaterials prepared in this embodiment: like Figure 2As shown in Figure a, Fourier transform infrared spectroscopy (FTIR) was used to confirm the Fe3O4@PAMAM@PA-Ti 4+ The preparation results are located at 1550 cm. -1 and 1128 cm -1 The characteristic absorption peaks are attributed to the NH bending vibration and PO stretching vibration of the amide bond, respectively. (1550 cm⁻¹) -1 The presence of this indicates that an amide bond was successfully formed between the carboxyl-modified Fe3O4 (i.e., Fe3O4-COOH in the figure) and the amino group on PAMAM, meaning that PAMAM was covalently grafted onto the Fe3O4 surface. 1128 cm -1 The appearance of the characteristic absorption peaks indicates that the phosphate groups in the phytic acid (PA) molecule have been successfully introduced into the material surface, confirming the successful modification of PAMAM and PA.

[0066] like Figure 2 As shown in Figure b, the zeta potential was used to monitor Fe3O4@PAMAM@PA-Ti 4+ In each reaction step of the synthesis process, the initial Zeta potential of Fe3O4 was -28.3 mV. After covalent grafting of PAMAM, the large number of amino groups restored the potential to positive. Subsequently, the negatively charged phosphate groups on the PA molecule caused the potential to turn negative. Finally, Ti was chelated... 4+ This resulted in a significant increase in potential exceeding 34 mV. The potential changes at each step were consistent with the expected changes in surface chemistry, demonstrating the successful occurrence of each modification step. This was particularly true for Ti. 4+ The dramatic positive shift in potential after chelation indicates that a large number of titanium ions are stably fixed on the material surface, and Ti 4+ An effective coordination relationship is formed between it and the phosphate group.

[0067] based on Figure 2 Figures a and b show that FTIR spectroscopy confirms the successful modification of the Fe3O4 surface by PAMAM (via amide bonds) and phytic acid (via PO bonds). The zeta potential change fully verifies each modification step (PAMAM grafting, PA bonding, Ti...) 4+ The gradual realization of chelation, especially Ti 4+ The immobilized material surface carries a strong positive charge, which facilitates the efficient capture of negatively charged phosphorylated peptides through electrostatic and coordination interactions. These results collectively demonstrate that this embodiment successfully prepared Fe3O4@PAMAM@PA-Ti. 4+ Composite materials are rationally designed and have clear and controllable synthesis pathways.

[0068] Example 2: This embodiment provides Fe3O4@PAMAM@PA-Ti prepared based on Example 1. 4+Magnetic nanomaterials are used to co-enrich phosphorylated peptides and glycopeptides.

[0069] This embodiment utilizes Fe3O4@PAMAM@PA-Ti 4+ In the process of co-enriching phosphorylated peptides and glycopeptides with magnetic nanomaterials, the pH and organic phase ratio of the enrichment solution and elution solution were optimized.

[0070] Using Fe3O4@PAMAM@PA-Ti 4+ The specific steps for co-enriching phosphorylated peptides and glycopeptides with magnetic nanomaterials are as follows: (1) Preparation of standard protease hydrolysate: Take 2 mg Casein and horseradish peroxidase were dissolved in 1 mL of ammonium bicarbonate solution (50 mM, pH=8.3) and then... Denature at high temperature (C) for 10 min. After cooling to room temperature, proceed with the reaction with trypsin and... - Add trypsin at a ratio of 1:40 (w / w) of casein or trypsin to horseradish peroxidase for enzymatic hydrolysis, and then hydrolyze at 37°C. The reaction was carried out at C for 16 h, and the resulting protein hydrolysate was used for the following experimental procedures.

[0071] (2) Fe3O4@PAMAM@PA-Ti 4+ Simultaneous enrichment and elution of phosphorylated peptides and glycopeptides: Casein and horseradish peroxidase hydrolysate were mixed at a 1:1 concentration ratio, with concentrations of 100 fmol / L. L. Prepare a mixed peptide solution. Use an enrichment solution (a mixture of organic solvent, acid, and water: organic solvent volume ratio 70-90%, acid volume ratio 0.1-5%, pH range 0.5-3) to concentrate Fe3O4@PAMAM@PA-Ti. 4+ The material was activated and prepared into a 10 mg / mL suspension. 80 L mixed peptide solution and 20 L material suspension phase mixing, at 37 The reaction was carried out under shaking at C for 30 min. After the reaction was complete, the material was washed three times with the enrichment solution, and the material and supernatant were separated using a magnet. After washing, 10 g of the enrichment solution was added to the material. L elution solution (alkaline solution or alkaline / organic solvent mixture: 5-10% by mass of alkaline solution, 0-70% by volume of organic solvent, pH range 10-12), at 37 The reaction was shaken at C for 30 min, the materials were separated using a magnet, the supernatant was collected, and the enriched phosphorylated peptides and glycopeptides were analyzed using MALDI-TOF MS.

[0072] like Figure 3 As shown, this embodiment can produce 100 fmol / Eleven phosphorylated peptides and 17 glycopeptides were co-enriched in the L-mixed standard peptides.

[0073] (3) Optimization of the enrichment solution: In the enrichment solution, the proportion of acetonitrile in the organic phase was changed to 70%, 80%, and 90%, and the amount of trifluoroacetic acid added was changed to 3%, 1%, and 0.1%, while other conditions remained the same as in Example 2(2). The results are as follows: Figure 4 As shown in a, based on the mass spectrometry results, the enrichment solution with 90% acetonitrile and 3% trifluoroacetic acid has the best comprehensive enrichment ability for phosphorylated peptides and glycopeptides, and is therefore identified as the optimal enrichment solution condition.

[0074] (4) Optimization of elution solutions: Considering both pH and hydrophilicity, 30% acetonitrile and 0.1% TFA, 50% acetonitrile and 1% TFA, and 10% ammonia were selected as elution solutions, with other conditions the same as in Example 2(2). The results are as follows: Figure 4 As shown in b, based on the mass spectrometry results, the elution solution with 10% ammonia water has the best overall elution capacity for phosphorylated peptides and glycopeptides, and is therefore considered the optimal elution solution condition.

[0075] Example 3: This embodiment verifies the co-enrichment sensitivity of standard phosphorylated peptides and glycopeptides.

[0076] This embodiment provides Fe3O4@PAMAM@PA-Ti prepared based on Example 1. 4+ The specific steps for using magnetic nanomaterials to co-enrich phosphorylated peptides and glycopeptides are as follows: (1) Preparation of standard protease hydrolysate: - The preparation of casein and horseradish peroxidase hydrolysate is the same as in Example 2.

[0077] (2) Fe3O4@PAMAM@PA-Ti 4+ Detection limit test for co-enrichment of phosphorylated peptides and glycopeptides: A series of serially diluted mixed peptide solutions were prepared using the enrichment solution (10 fmol / μL, 1 fmol / μL, 0.1 fmol / μL, 0.01 fmol / μL, 0.001 fmol / μL), and 80 μL of each solution was taken. L and 20 The L-magnetic nanomaterial mixing reaction, the specific operation process is the same as in Example 2. For example... Figure 5 As shown, at 1 fmol - In the mixed protein hydrolysate of casein and horseradish peroxidase, one phosphorylated peptide signal and one glycopeptide signal can be detected simultaneously, indicating that the detection limit of this method for phosphorylated peptides and glycopeptides is as low as 1 fmol.

[0078] Example 4: This embodiment verifies the selectivity when co-enriching standard phosphorylated peptides and glycopeptides.

[0079] This embodiment provides Fe3O4@PAMAM@PA-Ti prepared based on Example 1. 4+ The specific steps for using magnetic nanomaterials to co-enrich phosphorylated peptides and glycopeptides are as follows: (1) Preparation of protease hydrolysate: The preparation process for casein, horseradish peroxidase hydrolysate, and bovine serum albumin hydrolysate is the same as in Example 2.

[0080] (2) Fe3O4@PAMAM@PA-Ti 4+ Selectivity assay for co-enrichment of phosphorylated peptides and glycopeptides: Casein and horseradish peroxidase hydrolysate were mixed at a 1:1 ratio to prepare a mixed peptide solution. Then, bovine serum albumin hydrolysate was added to the mixed peptide solution at different concentrations ranging from 5000:1 to 100:1 to prepare a series of test solutions doped with non-phosphorylated and glycosylated peptides. 80 μL of each was taken... L of the test solution and 20 The L-magnetic nanomaterial mixing reaction, the specific operation process is the same as in Example 2. For example... Figure 6 As shown, in the test solution after doping bovine serum albumin digest and mixed peptide solution at a concentration ratio of 2000:1, the phosphorylated peptides and glycopeptides detected by the present invention have outstanding signal intensity and quantity in the mass spectrum, indicating that the method has good selectivity for the detection of phosphorylated peptides and glycopeptides.

[0081] Example 5: This embodiment verifies that the method of the present invention has a good effect on the co-enrichment of phosphorylated peptides and glycopeptides in model organisms.

[0082] This embodiment provides Fe3O4@PAMAM@PA-Ti prepared based on Example 1. 4+ The specific steps for using magnetic nanomaterials to co-enrich phosphorylated peptides and glycopeptides are as follows: (1) Sample preparation: The selected model organism was *C. elegans*. *C. elegans* were collected in solution and centrifuged. After removing the supernatant, 200 μL of solution was added. L of lysis buffer (4 M guanidine hydrochloride, 100 mM triethylammonium bicarbonate, pH 7.0) was sonicated on ice for 6 min. Then, the mixture was centrifuged at 20000 g for 20 min at 4 °C, and the supernatant was collected. Then, 150 μL of the supernatant was taken... The supernatant was reduced with 5 mM tris(2-carboxyethyl)phosphine at 37 °C for 20 min, and then alkylated with 10 mM iodoacetamide in the dark at room temperature for 15 min. Finally, trypsin (1 / 25, w / w) was added, and the mixture was incubated at 37 °C. The reaction was carried out at C for 16 h, and the resulting protein hydrolysate was used for the following experimental procedures.

[0083] (2) Using Fe3O4@PAMAM@PA-Ti 4+ Co-enrichment of phosphorylated peptides and glycopeptides from *C. elegans*: Take 80-150... L protein hydrolysate and 20-40 The L-magnetic nanomaterial mixing reaction was performed using the same procedure as in Example 2. After collecting the supernatant, the sugar chains were cleaved using PNGase F enzyme (the enzyme buffer solution was an ammonium bicarbonate aqueous solution), and the reaction was carried out at 37°C. The reaction was carried out at C for 16 h, followed by desalting (C18 solid-phase extraction), lyophilization, and redissolution in 0.1% mass spectrometry grade formic acid aqueous solution. The enriched phosphorylated peptides and glycopeptides were analyzed using a nano-LC MS / MS instrument to determine the types and expression levels of phosphorylated peptides and glycopeptides.

[0084] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides, characterized in that, Dendritic macromolecules PAMAM were modified onto Fe3O4 magnetic beads, and then negatively charged functional groups and titanium ions (Ti) were added to the surface of the dendritic macromolecules PAMAM. 4+ This yielded magnetic nanomaterials capable of simultaneously enriching phosphorylated peptides and glycopeptides.

2. The method for preparing a magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides according to claim 1, characterized in that, Includes the following steps: S1: Carboxyl-modified Fe3O4 magnetic beads were dispersed in 2-morpholine ethanesulfonic acid buffer solution, N-hydroxysuccinimide ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, activated, washed, dendritic macromolecule PAMAM solution was added, reacted, and the product was washed to obtain Fe3O4@PAMAM. S2: Disperse Fe3O4@PAMAM in phytic acid solution, stir and react. After the reaction is complete, wash the product to obtain Fe3O4@PAMAM@PA. S3: Dissolve Fe3O4@PAMAM@PA in a solution containing Ti 4+ The reaction was carried out in a solution with stirring. After the reaction was completed, the product was washed to obtain the magnetic nanomaterials Fe3O4@PAMAM@PA-Ti. 4+ This refers to magnetic nanomaterials that can simultaneously enrich phosphorylated peptides and glycopeptides.

3. The method for preparing a magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides according to claim 2, characterized in that, In step S1, the ratio of carboxyl-modified Fe3O4 to 2-morpholine ethanesulfonic acid buffer solution is (1-100) mg : (0.1-10) mL. The concentration of N-hydroxysuccinimide ester is 0.05-0.5 M, and the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 0.02-0.5 M. The pH of the 2-morpholine ethanesulfonic acid buffer solution is 5.0-6.5, and the dendritic macromolecular PAMAM solution is a methanol solution with a PAMAM mass fraction of 1%-10%. When the amount of carboxyl-modified Fe3O4 is (1-100) mg, the amount of dendritic macromolecular PAMAM solution used is 5-200 mL. L; In step S2, the ratio of Fe3O4@PAMAM to phytic acid solution is (1 - 100) mg: (1 - 100) mL, and the concentration of phytic acid is 1 - 50 mg / mL. In step S3, Ti is present 4+ The solution is a Ti(SO4)2 solution, and the ratio of Fe3O4@PAMAM@PA to Ti(SO4)2 solution is (1 - 100) mg : (1 - 100) mL, and the concentration of Ti(SO4)2 is 20 - 500 mM.

4. A magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides, characterized in that, It is prepared by any one of the preparation methods according to claims 1-3.

5. An application of the magnetic nanomaterial capable of simultaneously enriching phosphorylated peptides and glycopeptides as described in claim 4, characterized in that, The magnetic nanomaterials are used to simultaneously enrich phosphorylated peptides and glycopeptides in a sample.

6. A method for co-enriching phosphorylated peptides and glycopeptides, characterized in that, The magnetic nanomaterials described in claim 4, which can simultaneously enrich phosphorylated peptides and glycopeptides, are mixed and reacted with the enzymatically hydrolyzed test sample in an enrichment solution system. After solid-liquid separation and washing, the supernatant containing non-phosphorylated peptides and non-glycopeptides is removed. The mixture was reacted in an elution solution system to obtain a supernatant containing phosphorylated peptides and glycopeptides. The glycan chains were removed by enzymatic cleavage to obtain a sample enriched with both phosphorylated peptides and glycopeptides.

7. The method for co-enriching phosphorylated peptides and glycopeptides according to claim 6, characterized in that, Includes the following steps: S1: Activate the magnetic nanomaterials that can simultaneously enrich phosphorylated peptides and glycopeptides using an enrichment solution to obtain a material suspension; S2: Mix the material suspension described in step S1 with the sample to be tested after enzymatic hydrolysis, enrich it, and remove the supernatant containing non-phosphorylated peptides and non-glycopeptides after solid-liquid separation and washing to obtain the material containing phosphorylated peptides and glycopeptides. S3: Mix the material containing phosphorylated peptides and glycopeptides described in step S2 with the elution solution, elute, and obtain a supernatant containing phosphorylated peptides and glycopeptides through solid-liquid separation. S4: The supernatant containing phosphorylated peptides and glycopeptides described in step S3 is deglycosylated by enzyme digestion to remove the glycan chains, thereby obtaining a sample enriched with both phosphorylated peptides and glycopeptides. The sample enriched with both phosphorylated peptides and glycopeptides can be directly used for mass spectrometry analysis.

8. A method for co-enriching phosphorylated peptides and glycopeptides according to claim 6 or 7, characterized in that, The enrichment solution is a mixture of organic solvent, acid, and water: the volume ratio of organic solvent is 70-90%, the volume ratio of acid is 0.1-5%, and the pH range of the enrichment solution is 0.5-3; the organic solvent is selected from one or more of acetonitrile, methanol, and ethanol, and the acid is selected from one or more of formic acid, acetic acid, trifluoroacetic acid, etc. The enrichment temperature range in step S2 is 25-45℃, and the enrichment reaction time range is 30-60 min.

9. A method for co-enriching phosphorylated peptides and glycopeptides according to claim 6 or 7, characterized in that, The sample to be tested after enzymatic hydrolysis refers to the sample to be tested after hydrolysis with a mass spectrometry-grade enzyme. The mass spectrometry-grade enzyme is selected from one or more of Trypsin, LysC, or Arg. The mass spectrometry-grade enzyme is located in a buffer solution, and the buffer solution of the mass spectrometry-grade enzyme is an aqueous solution of ammonium bicarbonate. The samples to be tested include: (1) peptide standards containing phosphorylated peptides and glycopeptides; (2) complex samples such as tissues, cells, body fluids, and mixed proteins containing phosphorylated peptides and glycopeptides; and (3) mixed samples of the above (1) and (2). When the sample to be tested is a complex biological sample, before enzymatic digestion, the complex biological sample is also subjected to lysis, reduction and alkylation. The lysis reagent used for lysis is a surfactant, the reduction reagent used for reduction is tris(2-carboxyethyl)phosphine or urea, and the alkylation reagent used for alkylation is chloroacetamide, iodoacetamide or N-ethylmaleimide.

10. A method for co-enriching phosphorylated peptides and glycopeptides according to claim 6 or 7, characterized in that, The elution solution is an alkaline solution or a mixture of alkaline solution and organic solvent; the mass concentration of the alkaline solution is 5-10%, and the pH range is 10-12; in the mixture of alkaline solution and organic solvent, the volume ratio of organic solvent is 0-70%, and the pH range is 10-12. In the elution solution, the organic solvent is selected from one or more of acetonitrile, methanol, and ethanol, and the alkaline solution is selected from one or more of sodium carbonate aqueous solution, sodium bicarbonate aqueous solution, ammonium bicarbonate aqueous solution, and ammonia solution. In step S3, the elution temperature range is 25-45℃, and the elution reaction time range is 30-60 min. The enzyme used for cleavage is selected from one or more of PNGase F, PNGase H, or sialyl glycosidase, and the enzyme buffer solution is an aqueous solution of ammonium bicarbonate; the temperature range for cleavage of sugar chains is 10-60°C, and the reaction time range is 1-24 h.

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