Novel nano material for enriching phosphorylated peptide in biological sample as well as preparation method and application of novel nano material

Abstract

The invention relates to the technical field of analysis and testing, in particular to a novel nano material for enriching phosphorylated peptide in a biological sample as well as a preparation method and application of the novel nano material. The novel nano material is an immobilized metal affinity chromatography material based on a bionic honeycomb chitosan membrane, is a biomacromolecule with good biocompatibility, has good hydrophilicity and strong metal chelating ability, and can be used for enriching low-abundance phosphorylated peptide in a biological sample in a high-selectivity manner; the result of the method is beneficial to revealing pathological mechanisms of some serious diseases and realizing early diagnosis. The analysis material provided by the invention is easy to produce and manufacture, the experimental method is relatively simple, and the analysis material can be applied to the fields of clinical medicine, life science and the like; the novel nano material disclosed by the invention has high sensitivity, high selectivity and excellent recycling stability and acid-base stability.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and testing, and in particular to a novel nano material for enriching phosphorylated peptides in biological samples, and a preparation method and application thereof. Background Art

[0002] Protein is an important biological molecule that is responsible for a series of important life processes. Phosphorylation is a reversible protein post-translational modification process that can change the molecular conformation and surface charge distribution of proteins, and affect the activity of enzymes and protein interactions in the body. Therefore, protein phosphorylation plays a vital role in various life activities, such as cell growth, differentiation, proliferation, metabolism and apoptosis. A large number of studies have shown that abnormal phosphorylation is related to the development of many diseases, such as Alzheimer's disease, Parkinson's disease, diabetes, leukemia and cancer. Therefore, accurately analyzing the dynamic process of protein phosphorylation and accurately identifying its phosphorylation sites are of great value for accurate diagnosis of diseases and revealing the pathology of the onset and development of diseases. However, in actual biological samples, phosphorylated peptides, especially those related to diseases, have very low abundance, and are often interfered by other high-abundance protein or polypeptide molecules during the analysis process, which makes the currently commonly used mass spectrometry analysis very difficult. Therefore, selective separation and enrichment of phosphorylated proteins / peptides before the analysis and detection of clinical samples is a prerequisite for their effective identification and analysis.

[0003] Due to the important biological significance of phosphorylated peptides, their analysis and identification have always been a matter of interest. The accurate analysis of phosphorylated peptides first relies on the acquisition of phosphorylated peptides in biological samples, that is, separating and enriching extremely small amounts of phosphorylated peptides from blood or tissues for the next step of analysis and identification. At present, the capture methods of phosphorylated peptides in biological samples include immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), immunoprecipitation and electrostatic interaction methods. Among them, IMAC minimizes the interference of non-phosphorylated peptides by utilizing the acid-base regulation mechanism. It can significantly improve the selectivity, sensitivity and accuracy of phosphorylated peptide enrichment and has become one of the most effective mainstream methods. Summary of the invention

[0004] The purpose of the present invention is to provide a novel nanomaterial for enriching phosphorylated peptides in biological samples and a preparation method and application thereof in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention is to provide a method for preparing a novel nanomaterial for enriching phosphorylated peptides in a biological sample, the steps comprising:

[0007] S1, mixing the chitosan solution and the cross-linking agent solution, and then sequentially subjecting the mixture to ultrasonic treatment, centrifugal treatment, freeze drying treatment, heat curing treatment, washing treatment and drying treatment to obtain a chitosan membrane BHCM;

[0008] S2, mixing the chitosan membrane BHCM with toluene and (3-mercaptopropyl)trimethoxysilane, and then stirring, immersing, rinsing and vacuum drying in sequence to obtain BHCM-SH;

[0009] S3, mixing BHCM-SH, 2-acrylamido-2-methyl-1-propanesulfonic acid, azobisisobutyronitrile, ethanol and vinylphosphonic acid, and reacting them under a nitrogen atmosphere; soaking, rinsing and drying the material after the reaction to obtain BHCM-SH-AMPA / VPA;

[0010] S4, after mixing BHCM-SH-AMPA / VPA with titanium sulfate aqueous solution for reaction, the material is rinsed and vacuum dried to obtain the nanomaterial BHCM-SH-AMPA / VPA-Ti 4+ .

[0011] Preferably, in step S1, the solvent of the chitosan solution is acetic acid.

[0012] Preferably, in step S1, the solute in the cross-linking agent solution is polyethylene glycol diglycidyl ether.

[0013] The second aspect of the present invention is to provide a novel nanomaterial for enriching phosphorylated peptides in biological samples obtained by the above preparation method.

[0014] Preferably, the biological sample includes: animal body fluid, tissue enzymatic hydrolysate and cell enzymatic hydrolysate.

[0015] The third aspect of the present invention is to provide an application of the novel nanomaterial prepared by the above preparation method in enriching phosphorylated peptides in biological samples.

[0016] Preferably, the step of using the novel nanomaterial to analyze phosphorylated peptides in biological samples includes: mixing the novel nanomaterial, loading buffer and the biological sample to be tested, centrifuging to obtain an eluate, and analyzing the eluate using a mass spectrometer.

[0017] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0018] The novel nanomaterial of the present invention is an immobilized metal affinity chromatography material based on a biomimetic honeycomb chitosan membrane, a biomacromolecule with good biocompatibility, good hydrophilicity and strong metal chelating ability, and can be used to highly selectively enrich low-abundance phosphorylated peptides in biological samples to carry out further analytical research, the results of which will help reveal the pathological mechanisms of some major diseases and achieve early diagnosis. The analytical material provided by the present invention is easy to produce and manufacture, the experimental method is relatively simple, and can be applied in the fields of clinical medicine, life sciences, etc.; the novel nanomaterial of the present invention has high sensitivity, high selectivity, excellent cyclic stability and acid-base stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The novel nanomaterial BHCM-SH-AMPA / VPA-Ti 4+ Synthesis route map.

[0020] Figure 2 The novel nanomaterial BHCM-SH-AMPA / VPA-Ti 4+ The property characterization results of Figure 2 (a) is BHCM-SH-AMPA / VPA-Ti 4+ Elemental analysis diagram of Figure 2 (b) BHCM-SH-AMPA / VPA-Ti 4+ EDS spectrum of Figure 2 (c) FT-IR spectra of BHCM, BHCM-SH, and BHCM-SH-AMPA / VPA-Ti 4+ ; Figure 2 (d) is the contact angle between BHCM-SH and water molecules; Figure 2 (e) is the contact angle between BHCM-SH-AMPA / VPA and water molecules; Figure 2 (f) BHCM and BHCM-SH-AMPA / VPA-Ti 4+ Thermogravimetric analysis results.

[0021] Figure 3 In order to use the new nanomaterial BHCM-SH-AMPA / VPA-Ti 4+ Protocol for enrichment of phosphopeptides from biological samples.

[0022] Figure 4 In order to adopt the new nano material BHCM-SH-AMPA / VPA-Ti 4+ Mass spectrometry analysis results of phosphorylated peptides in enriched β-casein biological samples; among them, Figure 4 (a) before enrichment; Figure 4 (b) After enrichment, ★ indicates the mass spectrum peak of the enriched phosphorylated peptide.

[0023] Figure 5 β-casein enzymatic hydrolysate was treated with BHCM-SH-AMPA / VPA-Ti 4+ MALDI-TOF MS spectra of phosphorylated peptides after repeated enrichment; Figure 5 (a) for the first time; Figure 5 (b) the fourth time; Figure 5 (c) the seventh time; Figure 5 (d) is the tenth time. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0026] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0027] Example 1

[0028] This embodiment provides a method for preparing a novel nanomaterial for enriching phosphorylated peptides in a biological sample, the steps comprising:

[0029] S1. Weigh 1g chitosan and dissolve it in 100mL 1% acetic acid solution. After ultrasonic treatment for 30min to ensure complete dissolution, add 1g crosslinker solution and ultrasonic treatment for 15min; centrifuge the combined solution at 7800rpm for 15min to remove bubbles and pour it into a beaker; place the beaker in a -80℃ refrigerator and quickly freeze it for 1h, then transfer it to a vacuum freeze dryer and freeze-dry it for 24h until the solvent is completely removed; then place the beaker in a 60℃ oven and heat and cure it for 6h, wash the product three times with water and ethanol in sequence, and then dry it under vacuum to obtain chitosan membrane BHCM;

[0030] The cross-linking agent solution is 0.1 g of polyethylene glycol diglycidyl ether (PEGDGE) dissolved in 0.9 g of water.

[0031] S2. Weigh 3 g of BHCM, cut it into pieces and put it into a flask, add 50 mL of toluene and 15 mL of (3-mercaptopropyl)trimethoxysilane (MPTMS), and stir at 90°C for 72 h to react; after the reaction, transfer the material to a beaker and soak and rinse it with toluene for several times, and finally, vacuum dry it at 40°C for 8-12 h to obtain BHCM-SH.

[0032] As a chain transfer agent, thiol groups were successfully introduced into the surface of chitosan membranes to provide active sites for subsequent polymer grafting. Meanwhile, the stable CS bonds formed improved the structural stability of the material.

[0033] S3. Weigh 3 g of BHCM-SH, 8 g of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPA) and 200 mg of azobisisobutyronitrile (AIBN) and place them in a flask, then add 30 mL of ethanol and 3 mL of vinylphosphonic acid (VPA), and stir for 24 h under a nitrogen atmosphere to react; after the reaction is completed, transfer the product to a beaker, soak it, and rinse it with water and ethanol for several times. Finally, dry the product for 8-12 h to obtain BHCM-SH-AMPA / VPA.

[0034] Sulfonic acid groups can be protonated and deprotonated at different pH values, thereby adjusting the surface charge of the material, so that the material maintains good stability and function under different pH environments. Phosphate groups can form a chelating structure with multiple coordination sites with metal ions, and the stability of this structure is higher than that of a single coordination site. The introduction of phosphate groups enhances the adsorption capacity of the material for metal ions, thereby improving the cyclic stability of the material. The distribution and interaction of thiol, sulfonic acid and phosphate groups on the surface of the material form a multifunctional adsorption site network. This network not only improves the adsorption selectivity of phosphorylated peptides, but also the interaction of multiple groups enhances the overall stability of the material during the cyclic use process.

[0035] S4, weigh 3g BHCM-SH-AMPA / VPA and put it in a flask, then add 240mL 100mM titanium sulfate aqueous solution, stir at room temperature for 12h to react; after the reaction is completed, transfer the product to a beaker, soak, and rinse with water and ethanol for multiple times, finally, vacuum dry the product at 40°C for 8-12h to obtain the nanomaterial BHCM-SH-AMPA / VPA-Ti 4+ .

[0036] Phosphate groups on Ti 4+ The ions have excellent chelating ability, and this chelation can keep the Ti4+ ions stably bound to the material during the elution of phosphorylated peptides, thus ensuring the recyclability of the material.

[0037] Example 2

[0038] This embodiment provides a nanomaterial BHCM-SH-AMPA / VPA-Ti prepared by the preparation method described in Example 1. 4+ A method for analyzing phosphorylated peptides in human serum, comprising the steps of:

[0039] A serum sample containing 1 mg of protein was dissolved in an ammonium bicarbonate (50 μL, 50 mM) solution and denatured at 100°C for 10 min; after natural cooling, dithiothreitol (DTT, 5 μL, 200 mM) and ammonium bicarbonate (NH4HCO3, 45 μL, 50 mM) were added and shaken at 37°C for 1 h; subsequently, a mixture of iodoacetamide (IAA, 10 μL, 400 mM) and NH4HCO3 (90 μL, 50 mM) was added in a dark environment and alkylated at 37°C for 1 h; finally, 25 μL of trypsin (1 mg / mL) was added and shaken overnight; the obtained biological samples were packaged and freeze-dried and stored at -80°C (each containing 500 μg of protein);

[0040] 1 mg of freeze-dried biological sample was re-dissolved in 200 μL loading buffer (50% acetonitrile ACN + 0.5% trifluoroacetic acid TFA aqueous solution), and then 1 mg of nanomaterial BHCM-SH-AMPA / VPA-Ti was added. 4+ Ultrasound; then, shake at 37°C for 30 minutes, centrifuge to remove the supernatant, and then wash five times with loading buffer (about 200 μL each time, ACN:TFA:H2O=50%:0.5%:49.5%); then add 20 μL elution buffer (ammonia water, 10%), incubate at 37°C for 15 minutes, and collect the eluate by centrifugation; after the eluate is lyophilized and desalted, Nano-LC-MS / MS is used to analyze the phosphopeptides in the reconstituted solution.

[0041] Example 3

[0042] This embodiment provides a nanomaterial BHCM-SH-AMPA / VPA-Ti prepared by the preparation method described in Example 1. 4+ The method for analyzing phosphorylated peptides in a standard protein β-casein enzymatic hydrolysate comprises the following steps:

[0043] β-casein (2 mg) was dispersed in an aqueous solution of ammonium bicarbonate (400 μL) and denatured at 100°C for 10 min. After returning to room temperature, it was enzymatically digested with trypsin (2 mg / mL, 50 μL) for 12 h.

[0044] 1 mg of nanomaterial BHCM-SH-AMPA / VPA-Ti 4+200 μL of loading buffer (ACN: H2O: TFA = 50%: 49.5%: 0.5%) was added, and 2 μL of β-casein enzymatic hydrolysate (5 mg / mL) was added after sonication; then, the mixture was shaken at 37°C for 30 min, the supernatant was removed by centrifugation, and then washed three times with loading buffer; then, 20 μL of elution buffer (ammonia water, 10%) was added, and the eluate was collected by centrifugation after incubation at 37°C for 15 min; finally, the phosphopeptides in the eluate were analyzed by MALDI-TOF MS. The analysis results are shown in Figure 2. Figure 4 As shown, it can be seen that the nanomaterial of the present invention has good selectivity and enrichment efficiency, and has high sensitivity and high selectivity.

[0045] 200 μL of loading buffer and elution buffer were used to precipitate the enriched nanomaterial BHCM-SH-AMPA / VPA-Ti 4+ After washing for 3 times, the β-casein enzymatic hydrolysate was enriched and analyzed again, and the cycle was repeated 10 times. The analysis results are as follows Figure 5 As shown, the nanomaterial BHCM-SH-AMPA / VPA-Ti of the present invention 4+ It has excellent cyclic stability. The phosphoric acid group in the nanomaterial of the present invention has excellent cyclic stability. 4+ The chelation ability of Ti ions is excellent, and this chelation can maintain the Ti 4+ The stable combination of ions and materials ensures the recyclability of the materials.

[0046] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a novel nanomaterial for enriching phosphorylated peptides in biological samples, characterized in that the steps include: S1, mixing the chitosan solution and the cross-linking agent solution, and then sequentially subjecting the mixture to ultrasonic treatment, centrifugal treatment, freeze drying treatment, heat curing treatment, washing treatment and drying treatment to obtain a chitosan membrane BHCM; S2, mixing the chitosan membrane BHCM with toluene and (3-mercaptopropyl)trimethoxysilane, and then stirring, immersing, rinsing and vacuum drying in sequence to obtain BHCM-SH; S3, mixing BHCM-SH, 2-acrylamido-2-methyl-1-propanesulfonic acid, azobisisobutyronitrile, ethanol and vinylphosphonic acid, and reacting them under a nitrogen atmosphere; soaking, rinsing and drying the material after the reaction to obtain BHCM-SH-AMPA / VPA; S4, after mixing BHCM-SH-AMPA / VPA with titanium sulfate aqueous solution for reaction, the material is rinsed and vacuum dried to obtain the nanomaterial BHCM-SH-AMPA / VPA-Ti 4+ .

2. The preparation method according to claim 1, characterized in that: In step S1, the solvent of the chitosan solution is acetic acid.

3. The preparation method according to claim 1, characterized in that: In step S1, the solute in the cross-linking agent solution is polyethylene glycol diglycidyl ether.

4. A novel nanomaterial for enriching phosphorylated peptides in a biological sample obtained by the preparation method according to any one of claims 1 to 3.

5. The novel nanomaterial according to claim 4, characterized in that: The biological samples include: animal body fluid, tissue enzymatic hydrolysate and cell enzymatic hydrolysate.

6. Use of a novel nanomaterial prepared by the preparation method according to any one of claims 1 to 3 in enriching phosphorylated peptides in biological samples.

7. The use according to claim 6, characterized in that: The steps of using the novel nanomaterial to analyze phosphorylated peptides in biological samples include: mixing the novel nanomaterial, a loading buffer and a biological sample to be tested, centrifuging to obtain an eluate, and analyzing it using a mass spectrometer.

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