Method for enriching plasma / serum low-abundance protein

Abstract

The invention discloses a plasma / serum low-abundance protein enrichment method. The plasma / serum low-abundance protein enrichment method comprises the following steps: step 1, performing surface modification on nano ferroferric oxide particles by using a surfactant; step 2, enriching low-abundance proteins in the blood plasma; and step 3, detecting and analyzing the enriched protein. According to the method, nano ferroferric oxide is subjected to surface modification by using a surfactant to change the hydrophilic-hydrophobic property and surface charge of the nano ferroferric oxide, and then the nano ferroferric oxide is used for enriching the low-abundance protein of the plasma. According to the method, all raw materials are commercially available, and only simple ultrasonic and oscillation incubation treatment is needed in the modification process, so that the use cost is greatly reduced, and the operation convenience is improved. Compared with an enrichment method for removing abundance protein based on an antibody, the method has the advantages of good enrichment effect, low cost, simple preparation method, compatibility with automatic operation and the like.

Description

Technical Field

[0001] The present invention relates to the field of low-abundance protein enrichment, and more particularly to a method for enriching low-abundance proteins in plasma / serum. Background Art

[0002] Blood connects tissues and organs throughout the body, constantly exchanging substances and information with them. Therefore, the biological information in blood represents a comprehensive representation of the biological information of the entire body. Plasma / serum, as essential components of blood, contains circulating proteins that carry crucial information reflecting comprehensive physiological and pathological changes in the human body. Measuring protein composition and quantitative changes in plasma / serum provides crucial data support for disease diagnosis and prognosis, drug development and efficacy monitoring, and physiological status monitoring. In protein analysis, plasma and serum are generally equivalent, and the following background and technical descriptions will use "plasma" instead of "plasma / serum." However, plasma proteins are characterized by a complex composition (estimated to contain over 10,000 proteins), a wide dynamic range of protein concentrations (over 10 orders of magnitude), and a high proportion of highly abundant proteins (the 20 most abundant proteins, such as albumin, lipoproteins, and immunoglobulins, account for over 99% of the total, while the remaining 10,000 or so proteins make up less than 1%). Proteins associated with disease, originating from tissue leakage, are often of lower abundance. Due to the signal masking and interference of high-abundance proteins on low-abundance proteins, the detection of these low-abundance proteins using biological and chemical methods has encountered great challenges, especially in the use of liquid chromatography-mass spectrometry tandem technology (LC-MS / MS) for plasma proteomics. Therefore, in order to detect these low-abundance proteins in plasma, it is necessary to separate high-abundance proteins from low-abundance proteins to achieve the enrichment of low-abundance proteins and remove the interference of high-abundance proteins. To achieve this goal, a variety of separation and enrichment methods have been developed, including chemical precipitation, antibody affinity, peptide ligand affinity, and low-abundance protein enrichment methods based on nanomaterials. However, these methods all have some shortcomings. Chemical precipitation methods, such as ammonium sulfate precipitation, use the phenomenon that high-abundance proteins are more likely to aggregate with each other in high-concentration salt solutions to form large particles and precipitate, thereby reducing the proportion of high-abundance proteins in the solution. However, its effect is poor, and the proportion of high-abundance proteins in the supernatant after precipitation remains high. Antibody affinity methods use specific antibodies (such as anti-albumin and anti-IgG antibodies) to bind and remove high-abundance proteins. Companies such as Thermo Fisher Scientific offer kits that can remove up to 20 high-abundance proteins, but their cost is high and some low-abundance proteins may be lost due to cross-reactions. Peptide ligand affinity methods use a hexapeptide ligand library to selectively bind to low-abundance proteins, then remove the supernatant and release the low-abundance proteins. This method requires the optimization and screening of specific affinity peptides for each protein, which is complex and costly. Nanomaterial-based enrichment methods use nanomaterials to specifically adsorb low-abundance proteins in plasma, remove the supernatant, and then elute and detect the adsorbed proteins. This method has better enrichment effects than the above methods and is relatively simple to operate. However, several nanomaterials reported so far (such as DMB and NaY) are difficult to prepare, costly, and have limited enrichment effects. Therefore, it is necessary to develop enrichment methods that are more effective, less expensive, and more convenient to use. Summary of the Invention

[0003] The present invention aims to provide a low-cost, simple and efficient method for enriching low-abundance proteins in plasma, so as to improve the detection sensitivity of low-abundance proteins and the convenience of using the method, while reducing the enrichment cost.

[0004] To address the limitations of current methods for enriching low-abundance proteins in plasma, this method prepares surfactant-modified nanoferric oxide and uses it for the enrichment of low-abundance proteins in plasma. This method, combined with systematic optimization of enrichment parameters, achieves efficient enrichment. The enriched protein mixture can be used for proteomic analysis, Western blotting, and SDS-PAGE.

[0005] The specific technical solutions of the present invention are as follows:

[0006] A method for enriching low-abundance proteins in plasma / serum, comprising the following steps:

[0007] Step 1. Surface modification of nano-ferroferric oxide particles using surfactants

[0008] Surfactants: anionic surfactants (sodium dodecyl sulfate (SDS), sodium dodecyl sulfonate (SDSO), sodium dodecylbenzenesulfonate (SDBS)), cationic surfactants (cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium bromide (DTAB), etc.), zwitterionic surfactants (3-(3-cholaminylpropyl)dimethylamino-1-propanesulfonic acid (CHAPS), 3-sulfopropylhexadecyldimethyl betaine (HPS), 3-sulfopropyltetradecyldimethyl betaine (MSB), etc.).

[0009] Nano-ferroferric oxide: particle size is 20-1000nm, spherical or amorphous nano-ferroferric oxide particles.

[0010] 1.1. Preparation of surfactant solution: Take an appropriate amount of surfactant and mix it with ultrapure water to make a 0.1%-5% (W / V) solution;

[0011] 1.2 Surface modification of nano-ferroferric oxide: 30 mg of nano-ferroferric oxide was weighed and placed in a 15 mL centrifuge tube. 10 mL of the above-mentioned surfactant solution was added and vortexed to mix. The suspension was then sonicated in an ultrasonic disruptor at 20% power for 15 minutes and incubated at room temperature for 45 minutes. The suspension was then adsorbed on a magnetic rack for 3 minutes, and the liquid portion was removed. The solid portion was washed twice with 10 mL of deionized water by rotation and oscillation for 5 minutes each. The modified ferroferric oxide nanoparticles were stored in deionized water. Before use, they were prepared into a 3 mg / mL suspension in deionized water and sonicated again at 20% power for 5 minutes.

[0012] Step 2. Enrichment of low-abundance plasma proteins

[0013] 2.1. Plasma samples were taken out of the -80°C freezer and thawed on ice. Vortex-mixed and centrifuged at 16,000 × g at 4°C for 15 min.

[0014] 2.2. Place 50 μL of the intermediate clear layer of the centrifuged plasma sample into a new 1.5 mL centrifuge tube. Add 200 μL of dilution buffer (containing 1 mM EDTA, 10 mM TRIS, 0.05% CHAPS, and 150 mM KCl in water), vortex to mix, and then rapidly centrifuge for 2 seconds.

[0015] Wherein, EDTA: ethylenediaminetetraacetic acid; TRIS: tris(hydroxymethyl)aminomethane; CHAPS: 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid; KCl: potassium chloride.

[0016] 2.3. Take 100 μL of the modified nano-ferroferric oxide particles with a concentration of 3 mg / mL and add them to the plasma solution diluted in step 2. Then, place the sample on a shaker with the settings set at 24°C and 900 rpm and incubate for 30 minutes.

[0017] 2.4 After incubation, centrifuge rapidly for 2 seconds, then place the sample on a magnetic rack for adsorption for 5 minutes and remove the liquid portion. Add 200 μL of 150 mM KCl solution to the solid portion and wash it on a magnetic rack for 5 minutes using the same parameters as in step 3. Then adsorb it on a magnetic rack for another 5 minutes, remove the wash solution, and repeat the wash three times. After the final wash and removal of the wash solution, the enrichment process is complete. Different eluents may be used to elute the adsorbed protein in accordance with different subsequent analytical detection methods.

[0018] Step 3. Detection and analysis of enriched proteins

[0019] 3.1. For Western blotting analysis and SDS-PAGE silver staining, add 50 μL of 3% (w / v) SDS solution to the enriched sample. Sonicate in a water bath for 5 minutes to dissociate the adsorbed proteins into the solution. Then, adsorb on a magnetic rack for 5 minutes, and aspirate the solution for subsequent sample processing and analysis.

[0020] 3.2 For proteomic analysis, 50 μL of an aqueous solution containing 10 mM TCEP, 40 mM CAA, and 50 mM TEAB was added and sonicated for 5 minutes, followed by heating at 95°C for 10 minutes. The sample tube was then placed on ice to cool. 60 μL of chromatography-grade acetonitrile was added to the sample solution, and the sample tube was shaken at 900 rpm at 24°C for 5 minutes. The tube was removed and centrifuged at 16,000 × g at 4°C for 10 minutes. The supernatant was discarded, and 50 μL of a 50 mM TEAB solution containing 0.01% DDM (w / v) was added. The nanoparticles were then sonicated in a water bath for 2 minutes to disperse. Then, 1 μg of mass spectrometry-grade trypsin was added to the sample solution, and enzymatic digestion was performed on a shaker at 37°C and 1500 rpm for 4-16 hours. After digestion, the sample was desalted and heat-dried to produce a dry peptide powder. Finally, the dry powder was stored in a -80°C or -20°C refrigerator. Before testing, it was redissolved with 0.1% (V / V) formic acid aqueous solution and then detected and analyzed using a liquid chromatography-mass spectrometer.

[0021] Wherein, TCEP: tris(2-carboxyethyl)phosphine; CAA: chloroacetamide; TEAB: tetraethylammonium bromide; DDM: dodecyl-β-D-maltoside.

[0022] Technical effects and advantages of the present invention:

[0023] This method uses surfactants to modify the surface of nano-ferroferric oxide to alter its hydrophilicity and surface charge, which is then used to enrich low-abundance proteins in plasma. All raw materials for this method are commercially available, and the modification process requires only simple sonication and incubation with shaking, significantly reducing costs and improving operational convenience. Compared to antibody-based enrichment methods for high-abundance proteins, this method offers advantages such as improved enrichment efficiency, low cost, simple preparation, and compatibility with automated operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Comparison of SDS-PAGE silver staining of proteins enriched using the method of the present invention and commercial antibody depletion kits (Neat: unenriched raw plasma, HMNP: proteins enriched using the method of the present invention, TOP-14: proteins enriched after depletion using Thermo Fisher Scientific's TOP-14 depletion kits);

[0025] Figure 2Comparison of the number of proteins enriched in Fe3O4 nanoparticles modified with four surfactants by proteomic analysis (CTAC: cetyltrimethylammonium chloride-modified Fe3O4 nanoparticles, HPS: 3-sulfopropylhexadecyldimethylbetaine-modified Fe3O4 nanoparticles, SDS: sodium dodecyl sulfate-modified Fe3O4 nanoparticles, SDBS: sodium dodecylbenzenesulfonate-modified Fe3O4 nanoparticles);

[0026] Figure 3 Comparison of the number of proteins enriched by the present method and commercial antibody depletion kits using proteomic analysis (Neat: unenriched raw plasma, HMNP: proteins enriched by the present method, TOP-14: proteins enriched after depletion using Thermo Fisher Scientific's TOP-14 depletion kits);

[0027] Figure 4 Comparison of the number of low-abundance proteins identified by proteomic analysis of proteins enriched using the method of the present invention and commercial antibody depletion kits (Neat: unenriched raw plasma, HMNP: proteins enriched using the method of the present invention, TOP-14: proteins enriched after depletion using Thermo Fisher Scientific's TOP-14 depletion kits). DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] Example 1

[0030] A method for enriching low-abundance proteins in plasma / serum, comprising the following steps:

[0031] Step 1. Surface modification of nano-ferroferric oxide particles using surfactants

[0032] Surfactants: anionic surfactants (sodium dodecyl sulfate (SDS), sodium dodecyl sulfonate (SDSO), sodium dodecylbenzenesulfonate (SDBS)), cationic surfactants (cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium bromide (DTAB), etc.), zwitterionic surfactants (3-(3-cholaminylpropyl)dimethylamino-1-propanesulfonic acid (CHAPS), 3-sulfopropylhexadecyldimethyl betaine (HPS), 3-sulfopropyltetradecyldimethyl betaine (MSB), etc.).

[0033] Nano-ferroferric oxide: particle size is 20-1000nm, spherical or amorphous nano-ferroferric oxide particles.

[0034] 1.1. Preparation of surfactant solution: Take an appropriate amount of surfactant and mix it with ultrapure water to make a 0.1%-5% (W / V) solution;

[0035] 1.2 Surface modification of nano-ferroferric oxide: 30 mg of nano-ferroferric oxide was weighed and placed in a 15 mL centrifuge tube. 10 mL of the above-mentioned surfactant solution was added and vortexed to mix. The suspension was then sonicated in an ultrasonic disruptor at 20% power for 15 minutes and incubated at room temperature for 45 minutes. The suspension was then adsorbed on a magnetic rack for 3 minutes, and the liquid portion was removed. The solid portion was washed twice with 10 mL of deionized water by rotation and oscillation for 5 minutes each. The modified ferroferric oxide nanoparticles were stored in deionized water. Before use, they were prepared into a 3 mg / mL suspension in deionized water and sonicated again at 20% power for 5 minutes.

[0036] Step 2. Enrichment of low-abundance plasma proteins

[0037] 2.1. Plasma samples were taken out of the -80°C freezer and thawed on ice. Vortex-mixed and centrifuged at 16,000 × g at 4°C for 15 min.

[0038] 2.2. Place 50 μL of the intermediate clear layer of the centrifuged plasma sample into a new 1.5 mL centrifuge tube. Add 200 μL of dilution buffer (containing 1 mM EDTA, 10 mM TRIS, 0.05% CHAPS, and 150 mM KCl in water), vortex to mix, and then rapidly centrifuge for 2 seconds.

[0039] Wherein, EDTA: ethylenediaminetetraacetic acid; TRIS: tris(hydroxymethyl)aminomethane; CHAPS: 3-((3-cholamidopropyl)dimethylaminopropyl)-1-propanesulfonic acid; KCl: potassium chloride.

[0040] 2.3. Take 100 μL of the modified nano-ferroferric oxide particles with a concentration of 3 mg / mL and add them to the plasma solution diluted in step 2. Then, place the sample on a shaker with the settings set at 24°C and 900 rpm and incubate for 30 minutes.

[0041] 2.4 After incubation, centrifuge rapidly for 2 seconds, then place the sample on a magnetic rack for adsorption for 5 minutes and remove the liquid portion. Add 200 μL of 150 mM KCl solution to the solid portion and wash it on a magnetic rack for 5 minutes using the same parameters as in step 3. Then adsorb it on a magnetic rack for another 5 minutes, remove the wash solution, and repeat the wash three times. After the final wash and removal of the wash solution, the enrichment process is complete. Different eluents may be used to elute the adsorbed protein in accordance with different subsequent analytical detection methods.

[0042] Step 3. Detection and analysis of enriched proteins

[0043] 3.1. For Western blotting analysis and SDS-PAGE silver staining, add 50 μL of 3% (w / v) SDS solution to the enriched sample. Sonicate in a water bath for 5 minutes to dissociate the adsorbed proteins into the solution. Then, adsorb on a magnetic rack for 5 minutes, and aspirate the solution for subsequent sample processing and analysis.

[0044] 3.2 For proteomic analysis, 50 μL of an aqueous solution containing 10 mM TCEP, 40 mM CAA, and 50 mM TEAB was added and sonicated for 5 minutes, followed by heating at 95°C for 10 minutes. The sample tube was then placed on ice to cool. 60 μL of chromatography-grade acetonitrile was added to the sample solution, and the sample tube was shaken at 900 rpm at 24°C for 5 minutes. The tube was removed and centrifuged at 16,000 × g at 4°C for 10 minutes. The supernatant was discarded, and 50 μL of a 50 mM TEAB solution containing 0.01% DDM (w / v) was added. The nanoparticles were then sonicated in a water bath for 2 minutes to disperse. Then, 1 μg of mass spectrometry-grade trypsin was added to the sample solution, and enzymatic digestion was performed on a shaker at 37°C and 1500 rpm for 4-16 hours. After digestion, the sample was desalted and heat-dried to produce a dry peptide powder. Finally, the dry powder was stored in a -80°C or -20°C refrigerator. Before testing, it was redissolved with 0.1% (V / V) formic acid aqueous solution and then detected and analyzed using a liquid chromatography-mass spectrometer.

[0045] Wherein, TCEP: tris(2-carboxyethyl)phosphine; CAA: chloroacetamide; TEAB: tetraethylammonium bromide; DDM: dodecyl-β-D-maltoside.

[0046] Example 2

[0047] like Figure 1 The figure shows a comparison of SDS-PAGE silver staining of proteins enriched by the method of the present invention and commercial antibody depletion kits (Neat: unenriched original plasma, HMNP: protein enriched by the method of the present invention, TOP-14: protein enriched after depletion of high abundance by Thermo Fisher Scientific TOP-14 depletion kits);

[0048] Neat method: directly take plasma with the same amount of protein as that obtained by the method of the present invention and perform SDS-PAGE silver staining experiment;

[0049] TOP-14 method: According to the manufacturer's instructions, 10 μl of plasma was added to a high-abundance protein removal column and incubated at room temperature for 10 minutes. The filtrate was then collected by centrifugation. The protein concentration in the filtrate was determined and an amount of the filtrate equal to the protein obtained by the method of the present invention was subjected to SDS-PAGE silver staining.

[0050] Figure 1 Conclusion: The method of the present invention (HMNP) removes more high-abundance proteins than the TOP-14 high-abundance protein removal kit.

[0051] like Figure 2 The figure shows the comparison of the number of proteins enriched in the nano-iron tetroxide particles modified by four surfactants (CTAC: hexadecyltrimethylammonium chloride modified iron tetroxide nanoparticles, HPS: 3-sulfopropyl hexadecyldimethylbetaine modified iron tetroxide nanoparticles, SDS: sodium dodecyl sulfate modified iron tetroxide nanoparticles, SDBS: sodium dodecylbenzenesulfonate modified iron tetroxide nanoparticles);

[0052] Figure 2 Conclusion: Compared with CTAC modification, HPS, SDS and SDBS modified nanoferroferric oxide particles have better enrichment effect of low-abundance plasma proteins.

[0053] like Figure 3 As shown, the comparison of the number of proteins enriched by the method of the present invention and commercial antibody depletion kits was performed for proteomic analysis (Neat: unenriched original plasma, HMNP: proteins enriched by the method of the present invention, TOP-14: proteins enriched after depletion of high abundance by Thermo Fisher Scientific TOP-14 depletion kits);

[0054] Neat method: 10 μL of plasma is added to 190 μL of 50 mM TEAB solution, 10 mM TCEP, and 40 mM CAA solution, mixed, and heated at 95°C for 10 minutes. After cooling to room temperature, the sample solution is transferred to a 10 kD ultrafiltration tube. The solution is replaced three times with 200 μL of 50 mM TEAB. Trypsin is then added at a ratio of 50:1 protein to trypsin and digested overnight at 37°C. The reaction is then terminated by adding trifluoroacetic acid to a final concentration of 0.1%, and the filtrate is collected by centrifugation. The filtrate is then dried and stored in a refrigerator at -80°C or -20°C. Prior to analysis, it is reconstituted with 0.1% (v / v) formic acid in water and analyzed by liquid chromatography-mass spectrometry.

[0055] TOP-14 Method: Following the manufacturer's instructions, 10 μl of plasma was added to a high-abundance protein removal column, incubated at room temperature for 10 minutes, and the filtrate was collected by centrifugation. After determining the protein concentration in the filtrate, 10 mM TCEP and 40 mM CAA solutions were added, mixed, and heated at 95°C for 10 minutes. After cooling to room temperature, the sample solution was transferred to a 10 kD ultrafiltration tube. The solution was replaced three times with 200 μl of 50 mM TEAB. Trypsin was then added at a ratio of 50:1 protein to trypsin and digested overnight at 37°C. The reaction was then terminated by adding trifluoroacetic acid to a final concentration of 0.1%, and the filtrate was collected by centrifugation. The filtrate was dried and stored at -80°C or -20°C. Prior to analysis, it was reconstituted with 0.1% (v / v) formic acid in water and analyzed by liquid chromatography-mass spectrometry.

[0056] Figure 3 Conclusion: The number of proteins identified by the method of the present invention for plasma proteomic analysis far exceeds that of the TOP-14 high-abundance protein removal kit.

[0057] like Figure 4 As shown, the number of low-abundance proteins identified by proteomic analysis of proteins enriched by the method of the present invention and commercial antibody depletion kits is compared (Neat: unenriched original plasma, HMNP: proteins enriched by the method of the present invention, TOP-14: proteins enriched after depletion of high abundance by Thermo Fisher Scientific's TOP-14 depletion kits).

[0058] Figure 4 Conclusion: Compared with the TOP-14 high-abundance protein removal kit, the method of the present invention has more obvious advantages in the identification of low-abundance plasma proteome.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for enriching low-abundance proteins in plasma / serum, comprising the following steps: Step 1. Surface modification of nano-ferroferric oxide particles using surfactants 1.

1. Preparation of surfactant solution: Take an appropriate amount of surfactant and mix it with ultrapure water to make a 0.1%-5% (W / V) solution; 1.2 Surface modification of nano-ferroferric oxide: 30 mg of nano-ferroferric oxide was weighed and placed in a 15 mL centrifuge tube. 10 mL of the above-mentioned surfactant solution was added and vortexed to mix. The suspension was then sonicated in an ultrasonic disruptor at 20% power for 15 minutes and incubated at room temperature for 45 minutes. The suspension was then adsorbed on a magnetic rack for 3 minutes, and the liquid portion was removed. The solid portion was washed twice with 10 mL of deionized water by rotation and oscillation for 5 minutes each. The modified ferroferric oxide nanoparticles were stored in deionized water. Before use, they were prepared into a 3 mg / mL suspension in deionized water and sonicated again at 20% power for 5 minutes. Step 2. Enrichment of low-abundance plasma proteins 2.

1. Plasma samples were thawed on ice, vortexed, and centrifuged at 16,000 × g, 4°C for 15 min. 2.

2. Take 50 μl of the clear intermediate layer of the centrifuged plasma sample and place it in a new 1.5 ml centrifuge tube. Add 200 μl of dilution buffer, vortex to mix, and then quickly centrifuge for 2 seconds. 2.

3. Take 100 μL of the modified nano-ferroferric oxide particles with a concentration of 3 mg / mL and add them to the plasma solution diluted in step 2. Then, place the sample on a shaker with the settings set at 24°C and 900 rpm and incubate for 30 minutes. 2.4 After incubation, centrifuge quickly for 2 seconds, then place the sample on a magnetic rack for adsorption for 5 minutes and remove the liquid portion; add 200 μL of 150 mM KCl solution to the solid portion and wash it on a magnetic rack for 5 minutes with the same parameters as step 3, then adsorb it on a magnetic rack for 5 minutes, remove the washing solution, and repeat the washing three times; after the final washing and removal of the washing solution, the enrichment process is complete.

2. The method for enriching low-abundance proteins in plasma / serum according to claim 1, wherein: Also includes step 3. Enriched protein detection and analysis 3.

1. For protein immunoblotting analysis and polyacrylamide gel electrophoresis silver staining, add 50 μL of 3% (w / v) SDS solution to the enriched sample. Sonicate in a water bath for 5 minutes to dissociate the adsorbed proteins into the solution. Then, adsorb on a magnetic rack for 5 minutes, and then aspirate the solution for subsequent sample processing and analysis. 3.2 For proteomic analysis, 50 μL of an aqueous solution containing 10 mM TCEP, 40 mM CAA, and 50 mM TEAB was added and sonicated for 5 minutes, then heated at 95°C for 10 minutes. The centrifuge tube containing the sample was then placed on ice to cool. 60 μL of chromatographic grade acetonitrile was then added to the sample solution. The centrifuge tube containing the sample was placed on a shaker at 24°C and 900 rpm for 5 minutes. The centrifuge tube was removed and centrifuged at 16,000 × g, 4°C for 10 minutes. The supernatant was discarded and 50 μL of 50 mM TEAB containing 0.01% DDM (W / V) was added. TEAB solution, and then water bath sonication for 2 minutes to disperse the nanoparticles; then add 1 microgram of mass spectrometry-grade trypsin to the sample solution, set the temperature to 37°C and the speed to 1500 rpm on the oscillating mixer for enzymatic hydrolysis for 4-16 hours; after the enzymatic hydrolysis is completed, desalination and heat drying are carried out to prepare peptide dry powder; finally, the dry powder is stored in a -80°C or -20°C refrigerator and redissolved with 0.1% (V / V) formic acid aqueous solution before testing and analyzed using a liquid chromatography-mass spectrometer.

3. The method for enriching low-abundance proteins in plasma / serum according to claim 1, wherein: In the step 1, the surfactant includes: anionic surfactants (sodium dodecyl sulfate (SDS), sodium dodecyl sulfonate (SDSO), sodium dodecylbenzene sulfonate (SDBS) etc.), cationic surfactants (cetyltrimethylammonium chloride (CTAC), dodecyltrimethylammonium bromide (DTAB) etc.), zwitterionic surfactants (3-(3-cholaminylpropyl)dimethylamino-1-propanesulfonic acid (CHAPS), 3-sulfopropyl hexadecyldimethyl betaine (HPS), 3-sulfopropyl tetradecyldimethyl betaine (MSB) etc.).

4. The method for enriching low-abundance proteins in plasma / serum according to claim 1, characterized in that: The nanometer ferroferric oxide particles have a size of 20-1000 nm and are spherical or amorphous.

5. The method for enriching low-abundance proteins in plasma / serum according to claim 1, characterized in that: In step 2.2, 200 μl of dilution buffer: an aqueous solution containing 1 mM EDTA, 10 mM TRIS, 0.05% CHAPS and 150 mM KCl.

6. The method for enriching low-abundance proteins in plasma / serum according to claim 1, characterized in that: In step 2.4, after the enrichment process is completed, different eluents need to be used to elute the adsorbed protein in accordance with different subsequent analytical and detection methods.

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