A preparation method of immobilized trypsin applicable to proteomics research
By preparing immobilized trypsin through layer-by-layer self-assembly modification of the surface of gold nanoparticles and covalent bonding, the stability and activity issues of free trypsin during application and storage were solved, enabling its effective application in proteomics research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU UNIV
- Filing Date
- 2022-11-22
- Publication Date
- 2026-06-09
AI Technical Summary
Free trypsin exhibits decreased catalytic activity, self-dissolution, and self-aggregation during application and storage, making it difficult to reuse and affecting the effectiveness of proteomics research.
Using gold nanoparticles as a carrier, active amino acid residues were linked to the surface of the gold nanoparticles through layer-by-layer self-assembly modification and covalent bonding to prepare immobilized trypsin, thereby improving the enzyme's stability and catalytic activity.
Stable immobilization of trypsin was achieved, avoiding autolysis and self-aggregation, maintaining high catalytic activity, and making it suitable for repeated use in proteomics research.
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Figure CN115725562B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of immobilized enzyme preparation technology, specifically relating to a method for preparing immobilized trypsin that can be applied to proteomics research. Background Technology
[0002] Enzymes are natural biocatalysts with advantages such as mild reaction conditions, high catalytic efficiency, good substrate selectivity, and environmental friendliness. However, free enzyme preparations also have many limitations, including poor environmental stability, short shelf life, and difficulty in recycling. Enzyme immobilization technology, by using physical or chemical methods to confine enzymes within a certain space, can improve enzyme stability and allows for enzyme recovery and reuse. Furthermore, through careful design of immobilization methods and carrier materials, the catalytic activity and substrate specificity of enzymes can be preserved to the greatest extent. Therefore, well-developed immobilized enzymes have been widely used in the food industry, environmental protection, and biomedicine in recent years.
[0003] Shotgun proteomics based on mass spectrometry is one of the main methods in proteomics research. The process involves first digesting the proteome sample with a site-specific protease to form a peptide matrix, followed by high-performance liquid chromatography (HPLC) separation and mass spectrometry (MS) detection. Site-specific protease digestion of the protein sample is a prerequisite and foundation for MS detection. Trypsin is a commonly used serine protease in proteomics research. Trypsin digestion produces short peptides ending in a basic arginine or lysine, which is suitable for peptide fragmentation, forming strong b and y ion pairs suitable for existing identification algorithms. Furthermore, this enzyme is reasonably priced and widely used in practice. However, liquid-phase trypsin exhibits autolysis, unfolding, and self-aggregation effects, causing the catalytic activity of the natural enzyme to decline rapidly during application and storage, making it difficult to reuse. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention aims to solve one of the aforementioned problems; and to provide a method for preparing immobilized trypsin that can be applied to proteomics research.
[0005] In nanomaterials, gold nanoparticles are widely used in biology, chemistry, and medicine due to their controllable morphology, excellent optical properties, easy surface modification, and good biocompatibility. Their synthesis methods are simple, and surface modification with suitable organic compounds or biological ligands is relatively easy. After layer-by-layer self-assembly modification of gold nanoparticles, active amino acid residues are covalently linked to the surface of the gold nanoparticle carrier, achieving stable immobilization of trypsin. The trypsin-gold nanoparticle copolymer prepared by covalent bonding exhibits strong stability, with almost no leakage of the immobilized trypsin. The immobilization of trypsin effectively solves the problems of self-dissolution and self-aggregation, decreased catalytic activity, and difficulty in reusing natural enzymes during application and storage.
[0006] To achieve a technical objective, a method for preparing immobilized trypsin applicable to proteomics research is provided, with the following specific steps:
[0007] (1) First, chloroauric acid is dissolved in deionized water to obtain chloroauric acid solution; then the chloroauric acid solution is heated under constant temperature and refluxed for a period of time. Under constant temperature conditions, trisodium citrate solution is added and heated for a period of time. After heating is stopped, it is placed at room temperature and stirred to obtain reaction solution. After the reaction solution is cooled to room temperature, sodium citrate modified gold nanoparticle solution is obtained.
[0008] (2) Modification of the surface of gold nanoparticles by layer-by-layer self-assembly.
[0009] (a) Centrifuge the sodium citrate-modified gold nanoparticle solution obtained in step (1) to remove the supernatant and obtain sodium citrate-gold nanoparticle solid. Then wash the sodium citrate-gold nanoparticle with deionized water and redisperse it in an equal volume of deionized water to obtain a purified sodium citrate-gold nanoparticle colloidal solution.
[0010] Subsequently, the sodium citrate-gold nanoparticle colloidal solution was added dropwise to the polyallylamine hydrochloride solution. After stirring in the dark at 20–40 °C for 10–60 min, the mixture was centrifuged and washed to remove unreacted polyallylamine hydrochloride, yielding polyallylamine hydrochloride-modified gold nanoparticles, i.e., amino-modified gold nanoparticles. The amino-modified gold nanoparticles were then washed with deionized water and redispersed in ultrapure water to obtain an amino-modified gold nanoparticle dispersion.
[0011] (b) The amino-modified gold nanoparticle solution obtained in step (a) is added dropwise to the sodium polyacrylate solution. After stirring at 20-40°C for 10-60 min in the dark, the solution is centrifuged and washed to remove unreacted sodium polyacrylate, and carboxyl-modified gold nanoparticles are obtained. The carboxyl-modified gold nanoparticles are then redispersed in phosphate buffer to obtain a carboxyl-modified gold nanoparticle colloidal solution.
[0012] (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxythiosuccinimide solution to the carboxyl-modified gold nanoparticle colloidal solution prepared in step (2). After shaking for 10 to 30 minutes, add dropwise to trypsin solutions of different concentrations. Stir continuously under certain temperature conditions. After the reaction, centrifuge and wash to obtain the solid product, which is trypsin-gold nanoparticles, thus obtaining immobilized trypsin with gold nanoparticles as the carrier.
[0013] Preferably, in step (1), the concentration of the chloroauric acid solution is 1-2 mM; the concentration of the trisodium citrate solution is 2-3 mM; the specific temperature condition is 170°C, the constant temperature heating and reflux time is 10 min; the continued heating time is 10 min; and the stirring time at room temperature is 60 min.
[0014] Preferably, in step (1), the molar ratio of chloroauric acid in the chloroauric acid solution to trisodium citrate in the trisodium citrate solution is 1:(1-5).
[0015] Preferably, in step (2) (a), the volume ratio of polyallylamine hydrochloride solution to sodium citrate-gold nanoparticle colloidal solution is 1:(1-3); the concentration of the polyallylamine hydrochloride solution is 10-20 mg / mL.
[0016] Preferably, in step (2) (a), the amino-modified gold nanoparticles are redispersed in ultrapure water to obtain an amino-modified gold nanoparticle dispersion with a concentration of 1 to 10 nM.
[0017] Preferably, in step (2) (b), the concentration of the sodium polyacrylate solution is 10-20 mg / mL; the volume ratio of the amino-modified gold nanoparticle solution to the sodium polyacrylate solution is 1:1; the concentration of the carboxyl-modified gold nanoparticle colloidal solution is 1-10 nM; and the concentration of the phosphate buffer solution is 20 mM with a pH of 6.8.
[0018] Preferably, in step (3), the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 12 mM; the concentration of the N-hydroxythiosuccinimide solution is 60 mM; and the concentration of the trypsin is 1 mg / mL to 10 mg / mL.
[0019] Preferably, in step (3), the amounts of the modified gold nanoparticle colloidal solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxythiosuccinimide solution and trypsin solution are in the following relationship: 760 μL: 20 μL: 20 μL: 200 μL; the temperature condition is 25 °C, and the reaction time is 1 to 5 h with continuous stirring.
[0020] Performance testing:
[0021] Trypsin-gold nanoparticles were redispersed in 1 mL of ammonium bicarbonate buffer (pH 8.5) to prepare a 10-fold concentrated solution of trypsin-gold nanoparticles. This concentrated solution was used to digest and hydrolyze the model protein myoglobin at 37°C. After hydrolysis, the immobilized trypsin-gold nanoparticles were removed by centrifugation to terminate the reaction, thus obtaining the immobilized trypsin-digested polypeptide fragment product. The myoglobin digestion solution was detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) for qualitative and quantitative analysis of the protein.
[0022] In an optional embodiment of the present invention, in the study of protein enzymatic hydrolysis by trypsin-gold nanoparticles, the molar ratio of immobilized enzyme to myoglobin is 1:(20-200), and the protein hydrolysis reaction time is 5 min to 20 h.
[0023] Beneficial effects:
[0024] (1) In this invention, polyallylamine hydrochloride and sodium polyacrylate are used to modify gold nanoparticles for layer-by-layer self-assembly. The large amount of charge carried by the two modifiers significantly improves the stability of gold nanoparticles in solution. In addition, after modification of gold nanoparticles with sodium polyacrylate, a large number of carboxyl groups are placed on the surface, providing a large number of binding sites for trypsin immobilization and increasing the enzyme loading capacity.
[0025] (2) The present invention limits the molar ratio of chloroauric acid to trisodium citrate to 1:(1-5). This ratio has an important influence and is directly related to the size of the gold nanoparticles. If the ratio is too low, the synthesized gold nanoparticles will be too small. The small particle size may block the enzyme active site and affect the activity of the immobilized enzyme. If the ratio is too high, the gold nanoparticles will be too large and the enzyme-gold nanoparticle dispersibility will be weakened.
[0026] In addition, the present invention also limits the amount of the two modifiers, polyallylamine hydrochloride and sodium polyacrylate. If the concentration of the two modifiers is too low during the reaction, the amount of carboxyl groups introduced after modification will be small, resulting in a reduction in the amount of enzyme immobilized; if the concentration is too high, the polymer modification layer formed will be thick and may form certain pores, and the enzyme trapped inside the pores will not be able to play its role. Attached Figure Description
[0027] Figure 1 The results show the dynamic light scattering (DLS) and zeta potential measurements of the sodium citrate-gold nanoparticles, amino-modified gold nanoparticles, carboxyl-modified gold nanoparticles, and trypsin-gold nanoparticles prepared in Example 1.
[0028] Figure 2The images show the total ion chromatograms of myoglobin before and after hydrolysis using trypsin-gold nanoparticles. The top image shows the total ion chromatogram of the myoglobin solution before hydrolysis using trypsin-gold nanoparticles, and the bottom image shows the total ion chromatogram of the peptide mixture generated after hydrolysis of myoglobin using trypsin-gold nanoparticles.
[0029] Figure 3 The figure shows the effect of different ratios of trypsin and myoglobin used in the proteolytic process on sequencing coverage. Detailed Implementation
[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments, and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0031] Example 1:
[0032] (1) Before the reaction begins, 25.25 mg of chloroauric acid is dissolved in 50 mL of deionized water. The solution is refluxed at 170 °C for 10 min. Under constant conditions, 6.25 mL of 2.28 mM trisodium citrate solution is added to the solution. The reaction is continued to be heated for 10 min. Then the heating is stopped and the solution is placed at room temperature and stirred for 60 min. The temperature of the reaction solution is cooled to room temperature to obtain a solution of gold nanoparticles modified with sodium citrate. The solution is stored at 4 °C for later use.
[0033] (2) Subsequently, the gold nanoparticles were modified by layer-by-layer self-assembly. First, polyallylamine hydrochloride was used as the first layer modifier of the gold nanoparticles.
[0034] (a) The sodium citrate-modified gold nanoparticle solution was centrifuged and washed to remove unreacted sodium citrate, and then redispersed in an equal volume of deionized water to obtain a purified sodium citrate-gold nanoparticle colloidal solution.
[0035] Under continuous stirring, 10 mL of sodium citrate-gold nanoparticle colloidal solution was added dropwise to 10 mL of 20 mg / mL polyallylamine hydrochloride solution. After stirring and reacting for 30 min at 25 °C in the dark, the mixture was centrifuged at 12000 rpm for 15 min. Unreacted polyallylamine hydrochloride was removed by centrifugation to obtain amino-modified gold nanoparticle solids.
[0036] After washing with deionized water, the amino-modified gold nanoparticles were redispersed in 10 mL of ultrapure water to obtain an amino-modified gold nanoparticle dispersion with a concentration of 4.8 nM.
[0037] (b) 10 mL of amino-modified gold nanoparticle dispersion was added dropwise to 10 mL of 20 mg / mL sodium polyacrylate solution. The mixture was stirred for 30 min at 25 °C in the dark, followed by centrifugation and washing to remove unreacted sodium polyacrylate, yielding carboxyl-modified gold nanoparticle solids. The carboxyl-modified gold nanoparticles were then redispersed in 10 mL of 20 mM phosphate buffer (pH 6.8) to obtain a colloidal solution with a concentration of 4.0 nM.
[0038] (3) Preparation of immobilized trypsin
[0039] Prepare a 12 mM solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and a 60 mM solution of N-hydroxythiosuccinimide, respectively. The 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution should be prepared fresh for each use.
[0040] 20 μL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 20 μL of N-hydroxythiosuccinimide were added to 760 μL of carboxyl-modified gold nanoparticle colloidal solution, respectively. After shaking for 20 min, the carboxyl-modified gold nanoparticle colloidal solution was added dropwise to 200 μL of trypsin solution of different concentrations (1 mg / mL to 10 mg / mL), and the reaction was carried out by stirring at 25 °C for 2 h.
[0041] The sample was centrifuged at 12,000 rpm for 10 min, and washed with deionized water to remove unreacted trypsin. The resulting solid product was trypsin-gold nanoparticles, i.e., immobilized trypsin with gold nanoparticles as the carrier. The trypsin-gold nanoparticles were then redispersed in 1 mL of 20 mM phosphate buffer (pH 6.8) to obtain a dispersion, which was stored at 4 °C for later use.
[0042] Example 2: Characterization of trypsin-gold nanoparticles
[0043] The gold nanoparticles obtained after each surface modification step were characterized using ultraviolet spectrophotometry to determine the plasmon resonance (SPR) absorption peak of the gold nanoparticles. The measurement wavelength range was from 300 nm to 800 nm to monitor the size changes and dispersion state of the nanoparticles. The SPR absorption peak wavelength of the sodium citrate-gold nanoparticle colloidal solution was 527 nm, while the SPR absorption peak wavelength of the gold nanoparticle solution after double-layer modification was 528 nm, indicating that the size of the gold nanoparticles had a certain variation and good dispersion. In addition, this invention also used dynamic light scattering (DLS) technology to characterize the particle size distribution of the gold nanoparticles. The stability of the gold nanoparticles was characterized by measuring the Zeta potential on the surface of the gold nanoparticles. Each measurement was performed in a 173° backscattering detection mode. Each sample in the above experiments was diluted 5 times with water, and the measurements were performed in triplicate.
[0044] like Figure 1 As shown in the figure, Citrate-GNP represents sodium citrate-gold nanoparticles, GNP / PAH represents amino-modified gold nanoparticles, GNP / PAH / PAA represents carboxyl-modified gold nanoparticles, and Trypsin-GNP represents trypsin-gold nanoparticles. The average hydrated diameter of Citrate-GNP was 34 nm, and its Zeta potential was -44.4 mV. After modification with polyallylamine hydrochloride, the particle size increased to 133 nm. Due to the large number of amino groups on the surface of the modified gold nanoparticles, the Zeta potential became +60.5 mV. After a second layer of modification with sodium polyacrylate, the particle size of the gold nanoparticles increased to 134 nm, but the increase was small. This was mainly because sodium polyacrylate was modified onto the surface of the gold nanoparticles through electrostatic interaction with polyallylamine hydrochloride. The electrostatic interaction was strong, and the binding was very tight, resulting in a certain degree of compression of the volume of the surface-modified polymer, which led to an insignificant change in particle size. Due to the large number of carboxyl groups on the surface, its Zeta potential became -42.5 mV. Finally, after trypsin was immobilized on the surface of the gold nanoparticles, the particle size increased to 182 nm, and the Zeta potential was -35.9 mV.
[0045] Example 3: Determination of the amount of immobilized trypsin
[0046] After trypsin immobilization, trypsin-gold nanoparticles were precipitated by centrifugation and washed three times with ultrapure water. The supernatant and the wash buffer of the trypsin-gold nanoparticles were collected. The absorbance of unbound trypsin in the supernatant and wash buffer after reaction with Coomassie Brilliant Blue G-250 staining solution was measured using the Bradford method to calculate the concentration of unimmobilized trypsin. The amount of immobilized trypsin was obtained by calculating the difference between the initial total amount of trypsin and the amount of unreacted enzyme.
[0047] Specific determination process: First, prepare Coomassie Brilliant Blue G-250 staining solution by dissolving 10 mg of Coomassie Brilliant Blue G-250 in 5 mL of 95% ethanol, adding 10 mL of 85% phosphoric acid, and then adding deionized water to make up to 100 mL.
[0048] Prepare a standard trypsin solution of 0.01–1 mg / mL, add the staining solution and react with trypsin for 5 min. Measure the absorbance of each tube at wavelengths of 595 nm and 465 nm, respectively. Use the ratio of absorbance at 595 nm to 465 nm as the ordinate to construct a standard curve.
[0049] Add 5 mL of dye binding solution to 200 μL of trypsin immobilization supernatant, washing buffer, and blank buffer solution, respectively. After 5 min of incubation, the solution changes from reddish-brown to blue. Measure the absorbance of the sample at 595 nm and 465 nm, respectively, and calculate the ratio of the two absorbance values. Use the standard curve to calculate the concentration of unimmobilized trypsin. The final immobilized trypsin concentration is 1.8 μmol / L.
[0050] Example 4: Enzymatic hydrolysis of proteins by trypsin-gold nanoparticles
[0051] First, myoglobin was dissolved in deionized water to prepare a protein stock solution with a concentration of 1 mg / mL.
[0052] Add 10 μL of 80 mM urea and 10 μL of 50 mM tris(2-carbonylethyl) phosphate hydrochloride to 100 μL of myoglobin solution, heat to 60 °C and react for 30 min to destroy the disulfide bonds in the protein structure.
[0053] After cooling to room temperature, add 10 μL of 150 mM iodoacetamide to the reaction solution and react at room temperature in the dark for 30 min.
[0054] Subsequently, 10 mL of trypsin-gold nanoparticles were taken, and after centrifugation to remove the supernatant, they were redispersed in 1 mL of ammonium bicarbonate buffer (pH 8.5) to obtain a 10-fold concentrated solution of trypsin-gold nanoparticles.
[0055] The concentrated trypsin-gold nanoparticle solution was mixed with myoglobin to form solutions with different enzyme-to-protein molar ratios (from 1:20 to 1:200). The hydrolysis reaction temperature was 37℃, and the protein hydrolysis reaction time was controlled from 5 min to 20 h.
[0056] Centrifuge the reaction solution, take the supernatant, filter it through a 0.22 μm filter membrane, and then inject it into a liquid chromatography-mass spectrometry system for analysis.
[0057] High performance liquid chromatography-electrospray ionization-time-of-flight tandem mass spectrometry was used to analyze protein enzymatic hydrolysis products.
[0058] A Phenomenex Aeris Peptide C18 (3.6 μm 150 x 2.1 mm ID) column was used as the liquid chromatography column. The mobile phase consisted of a gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile solution containing 0.1% formic acid (B). The flow rate was 0.3 mL / min and the injection volume was 10 μL to separate the protein enzymatic hydrolysis products.
[0059] The obtained mass spectrometry data were compared with the database using MASCOT MS / MS.
[0060] Figure 2 The total ion chromatograms (TICs) of myoglobin before and after hydrolysis using trypsin-gold nanoparticles are shown. Before hydrolysis, a myoglobin peak can be found at a retention time of 7.18 min. Reconstructing the QTOF high-resolution data using the Bio Toolkit plugin yielded a protein molecular weight of 16951.7, confirming this peak as myoglobin. After hydrolysis, the peak intensity at 7.18 min decreases significantly, becoming extremely low. Simultaneously, numerous polypeptide fragment peaks appear between retention times of 2.5 and 7 min, indicating that myoglobin has been efficiently hydrolyzed by trypsin-gold nanoparticles. Therefore, [the following is a continuation of the previous paragraph]. Figure 2 This demonstrates that the immobilized trypsin prepared by this invention has high proteolytic power and can be applied to proteomics research.
[0061] Figure 3 The results show the optimized ratio of trypsin to myoglobin (MYB) during protein hydrolysis. When the molar ratio of myoglobin to immobilized trypsin was 20, the sequencing coverage of myoglobin obtained by MASCOT MS / MS library search and alignment was the highest, exceeding 40%. Furthermore, comparisons revealed that under all ratio conditions, the sequencing coverage using the immobilized enzyme hydrolysate was significantly higher than that using the free enzyme hydrolysate. This is mainly due to the interference caused by the autolysis of the free trypsin itself. Therefore, these results demonstrate that immobilized trypsin can provide more accurate protein sequencing coverage in protein analysis applications, effectively avoiding the autolysis effect of free enzymes during protein hydrolysis.
[0062] In summary, the immobilization method described in this invention has significant advantages in terms of reaction principle, selection of immobilization materials, choice of reaction modifier ratio, and application effects in protein research. The prepared trypsin-gold nanoparticles are small in size, providing dispersion properties similar to free enzymes, and exhibit high hydrolysis efficiency for proteins, while effectively avoiding autolysis that occurs during protein hydrolysis by free enzymes, making them suitable for proteomics research.
[0063] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.
Claims
1. A method for preparing immobilized trypsin applicable to proteomics research, characterized in that, Includes the following steps: (1) First, chloroauric acid is dissolved in deionized water to obtain a chloroauric acid solution; then, the chloroauric acid solution is heated under constant temperature and refluxed for a period of time. Under constant temperature conditions, trisodium citrate solution is added and heated for a period of time. After heating is stopped, it is placed at room temperature and stirred to obtain a reaction solution. After the reaction solution is cooled to room temperature, a solution of gold nanoparticles modified with sodium citrate is obtained; wherein, the molar ratio of chloroauric acid to trisodium citrate is 1: (1~5). (2) Surface modification of gold nanoparticles through layer-by-layer self-assembly: (a) Centrifuge the sodium citrate-modified gold nanoparticle solution obtained in step (1) to remove the supernatant and obtain sodium citrate-gold nanoparticle solid. Then wash the sodium citrate-gold nanoparticle with deionized water and redisperse it in an equal volume of deionized water to obtain a purified sodium citrate-gold nanoparticle colloidal solution. Subsequently, the sodium citrate-gold nanoparticle colloidal solution was added dropwise to the polyallylamine hydrochloride solution, the concentration of which was 10-20 mg / mL, and the volume ratio of the polyallylamine hydrochloride solution to the sodium citrate-gold nanoparticle colloidal solution was 1:(1-3). After stirring in the dark at 20-40 °C for 10-60 min, the mixture was centrifuged and washed to remove unreacted polyallylamine hydrochloride, yielding polyallylamine hydrochloride-modified gold nanoparticles, i.e., amino-modified gold nanoparticles. The amino-modified gold nanoparticles were then washed with deionized water and redispersed in ultrapure water to obtain an amino-modified gold nanoparticle dispersion. (b) The amino-modified gold nanoparticle solution obtained in step (a) is added dropwise to a sodium polyacrylate solution with a concentration of 10-20 mg / mL and a volume ratio of 1:1 between the amino-modified gold nanoparticle solution and the sodium polyacrylate solution. After stirring for 10-60 min in the dark at 20-40 °C, the mixture is centrifuged and washed to remove unreacted sodium polyacrylate, thereby obtaining carboxyl-modified gold nanoparticles. The carboxyl-modified gold nanoparticles were then redispersed in phosphate buffer to obtain a colloidal solution of carboxyl-modified gold nanoparticles. (3) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxythiosuccinimide solution to the carboxyl-modified gold nanoparticle colloidal solution prepared in step (2). After shaking for 10-30 min, add dropwise to trypsin solutions of different concentrations. Stir continuously under certain temperature conditions. After the reaction, centrifuge and wash to obtain the solid product, which is trypsin-gold nanoparticles, thus obtaining immobilized trypsin with gold nanoparticles as the carrier.
2. The method for preparing immobilized trypsin applicable to proteomics research according to claim 1, characterized in that, In step (1), the concentration of the chloroauric acid solution is 1~2 mM; the concentration of the trisodium citrate solution is 2~3 mM.
3. The method for preparing immobilized trypsin applicable to proteomics research according to claim 1, characterized in that, In step (1), the certain temperature condition is 170℃, the constant temperature heating and reflux time is 10min; the continued heating time is 10min; the stirring time under normal temperature conditions is 60min.
4. According to claim 1, in step (2) (a), the amino-modified gold nanoparticles are redispersed in ultrapure water to obtain an amino-modified gold nanoparticle dispersion with a concentration of 1~10 nM.
5. A method for preparing immobilized trypsin applicable to proteomics research according to claim 1, characterized in that, In step (2) (b), the concentration of the carboxyl-modified gold nanoparticle colloidal solution is 1~10 nM; the concentration of the phosphate buffer is 20 mM and the pH is 6.
8.
6. A method for preparing immobilized trypsin applicable to proteomics research according to claim 1, characterized in that, In step (3), the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution is 12 mM; the concentration of the N-hydroxythiosuccinimide solution is 60 mM; and the concentration of the trypsin is 1 mg / mL to 10 mg / mL.
7. A method for preparing immobilized trypsin applicable to proteomics research according to claim 1, characterized in that, In step (3), the amounts of the modified gold nanoparticle colloidal solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, N-hydroxythiosuccinimide solution and trypsin solution are in the following order: 760 μL: 20 μL: 20 μL: 200 μL.
8. A method for preparing immobilized trypsin applicable to proteomics research according to claim 1, characterized in that, In step (3), the temperature condition is 25℃, and the reaction time is 1~5 h with continuous stirring.