Synthesis and application of magnetic beads for enriching low-abundance proteins

Abstract

The present invention discloses the synthesis and application of magnetic beads for enriching low-abundance proteins. The present invention combines X-type molecular sieves with MXenes for the first time to synthesize MXenes nanomagnetic beads Ti3C2Tx‑Fe3O4@NaX with surface-modified molecular sieves, which are used for the efficient enrichment of low-abundance proteins in blood, achieving highly specific enrichment of low-abundance proteins in blood and achieving a higher proteome coverage depth.

Description

Technical Field

[0001] The invention belongs to the field of protein enrichment biotechnology, and in particular relates to the synthesis and application of low-abundance protein enrichment magnetic beads. Background Art

[0002] Human blood is a common clinical sample used in diagnostic analysis, but proteomic studies in blood are extremely challenging because high-abundance proteins in blood can seriously interfere with the identification and quantification of lower-abundance proteins. The dynamic range of protein abundance in plasma is 12-13 orders of magnitude, and low-abundance proteins are very difficult to detect due to the inhibitory effect of high-abundance proteins in plasma and serum. Usually, plasma proteomic strategies based on tandem mass spectrometry can only detect the first few hundred proteins with higher concentrations. Although currently commonly used methods, such as immunoaffinity to remove high-abundance proteins and chromatographic fractionation to reduce sample complexity, can reduce the inhibitory effect of high-abundance proteins to a certain extent and increase the identification scale of the plasma proteome, they can still only achieve limited proteome coverage depth and cannot meet the sensitivity and throughput levels required for clinical sample analysis.

[0003] Sara Linse first proposed the concept of "Protein Corona" (PC) in 2007. Once nanoparticles (NPs) enter the biological environment, their surface is quickly covered by a layer of biomolecules (mainly proteins) to form a protein corona. Studies have shown that the protein corona may be used to diagnose the health status of patients. Therefore, the protein corona can be used as a valuable tool for plasma proteome analysis to discover potential biomarkers. The introduction of nanoparticles in the field of proteomics provides a new method for enriching low-abundance proteins in biological samples. Studies have shown that the composition of the protein corona is usually determined by the physicochemical properties of the nanoparticles. Therefore, the target protein corona can be obtained through the rational design of nanoparticles, and the subtle changes in the qualitative and quantitative properties of proteins can be revealed through the characterization of the protein corona.

[0004] MXenes are an emerging two-dimensional nanomaterial with a graphene-like structure that has received increasing attention in recent years. MXenes have excellent chemical diversity and hydrophilicity. In recent years, some achievements have been made in biosensors, therapeutics, cancer, etc., but there is still great development prospect in proteomics applications, which deserves further development and promotion. Molecular sieves are excellent adsorbents with unique and rich surface properties, and have obvious potential in reducing the "masking" effect caused by high-abundance proteins. Summary of the invention

[0005] In view of this, in order to overcome the above-mentioned technical problems existing in the art, the purpose of the present invention is to provide a low-abundance protein enrichment magnetic bead Ti3C2Tx-Fe3O4@NaX. The present invention combines X-type molecular sieves with MXenes for the first time to synthesize a surface-modified molecular sieve MXenes nanomagnetic bead (Ti3C2Tx-Fe3O4@NaX) for efficient enrichment of low-abundance proteins in blood. Based on the "protein corona" principle, high-specificity and high-throughput enrichment of low-abundance proteins in blood is achieved. The combination of high-specificity magnetic bead technology and mass spectrometry technology enables in-depth research on the blood proteome.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides a method for preparing low-abundance protein enrichment magnetic beads.

[0008] Further, the method comprises the following steps:

[0009] (1) adding Ti3AlC2MAX to LiF solution to react and prepare Ti3C2Tx Mxene;

[0010] (2) Dispersing Ti3C2Tx Mxene in ethylene glycol, adding FeCl3, trisodium citrate, and anhydrous sodium acetate to react, thereby preparing Ti3C2Tx-Fe3O4;

[0011] (3) preparing a solution A comprising a NaAlO2 solution and Ti3C2Tx-Fe3O4, preparing a solution B comprising a NaOH solution and Na2SiO3·9H2O, dropping the solution B into the solution A for reaction, and preparing low-abundance protein enrichment magnetic beads Ti3C2Tx-Fe3O4@NaX.

[0012] Furthermore, the LiF solution in step (1) is obtained by dissolving 1 g of LiF in 20 mL of 30% HCl;

[0013] Preferably, the amount of Ti3AlC2MAX in step (1) is 1 g;

[0014] Preferably, the reaction conditions in step (1) are 35° C. for 24 h;

[0015] Preferably, step (1) further comprises washing the product obtained by the reaction, removing the supernatant until the pH is ≥ 6, adding deionized water, shaking sufficiently to obtain a stratified and stable Ti3C2Tx Mxene hydrocolloid solution, centrifuging and discarding the supernatant, and freeze-drying to obtain Ti3C2Tx Mxene;

[0016] More preferably, the washing conditions are washing with deionized water, centrifuging at 5000 g for 5 min;

[0017] More preferably, the centrifugal conditions for discarding the supernatant are 2000g and centrifugation for 1 hour.

[0018] Further, the amount of Ti3C2Tx Mxene in step (2) is 30 mg;

[0019] Preferably, the amount of ethylene glycol used in step (2) is 30 mL;

[0020] Preferably, the amounts of FeCl3, trisodium citrate and anhydrous sodium acetate in step (2) are 160 mg, 30 mg and 700 mg respectively;

[0021] Preferably, in step (2), the Ti3C2TxMxene is dispersed in ethylene glycol, and after water bath sonication until the Ti3C2TxMxene is uniformly dispersed, the FeCl3 and trisodium citrate are added, and after continuing sonication until the Ti3C2TxMxene is completely dissolved, the anhydrous sodium acetate is added, and the mixture is stirred at room temperature, and the obtained uniform solution is transferred to a high-pressure reactor for reaction;

[0022] More preferably, the stirring time is 30 min;

[0023] More preferably, the autoclave is a Teflon-lined stainless steel autoclave;

[0024] More preferably, the reaction conditions are heating the reaction at 200° C. for 8 h;

[0025] More preferably, step (2) further comprises washing and drying the reaction product to obtain Ti3C2Tx-Fe3O4;

[0026] Most preferably, the cleaning condition is ethanol and pure water cleaning 3 times.

[0027] Furthermore, the NaAlO2 solution in step (3) is obtained by dissolving 500 mg NaAlO2 in 10 mL deionized water;

[0028] Preferably, the amount of Ti3C2Tx-Fe3O4 used in step (3) is 150 mg;

[0029] Preferably, the NaOH solution in step (3) is obtained by dissolving 4 g of NaOH in 50 mL of deionized water;

[0030] Preferably, the amount of Na2SiO3·9H2O used in step (3) is 2.5 g;

[0031] Preferably, the reaction conditions in step (3) are as follows: solution B is slowly dripped into solution A under stirring, stirred for 30 minutes, left to stand at room temperature for 45 hours, and reacted at 60° C. under stirring for 5 hours;

[0032] Preferably, step (3) further comprises washing the product obtained by the reaction until the solution is neutral, and drying to obtain low-abundance protein enrichment magnetic beads Ti3C2Tx-Fe3O4@NaX;

[0033] More preferably, the cleaning condition is cleaning with deionized water.

[0034] Furthermore, the Ti3C2Tx Mxene in step (1) is prepared by a minimum intensity layer delamination (MILD) method.

[0035] Furthermore, the Ti3C2Tx Mxene obtained by freeze-drying in step (1) is stored in a dark environment at 4°C.

[0036] Furthermore, the FeCl3 described in step (2) is FeCl3·6H2O.

[0037] Furthermore, the Teflon-lined stainless steel autoclave described in step (2) is a 50 mL Teflon-lined stainless steel autoclave.

[0038] Furthermore, after the reaction in step (2) is completed, the following steps are also included: taking out the reaction kettle, rinsing it with cold water and cooling it to room temperature, and then washing it with ethanol and pure water three times.

[0039] In some embodiments, the main principle of the low-abundance protein enrichment is to affinity adsorb the low-abundance proteins in the sample to be tested using the low-abundance protein enrichment magnetic beads Ti3C2Tx-Fe3O4@NaX prepared by the method described above to form a "protein corona", which can then be effectively removed by washing, thereby achieving highly specific enrichment of low-abundance proteins in the sample to be tested.

[0040] In the present invention, MXenes refers to titanium vacancy nitrogen-containing michelin (transition metal carbon / nitride), which is an emerging two-dimensional material with advantages such as high electrical conductivity, excellent processability, adjustable surface properties and excellent mechanical strength. MXenes is a new type of two-dimensional nanomaterial with a graphene-like structure. Its general chemical expression is M n+1 X nTx, where M is an early transition metal element, X represents a C or N element, Tx represents a surface end group (such as O, OH, F, etc.) formed during the synthesis process, and the subscript n = 1-4, which means that MXenes are two-dimensional nanosheets with a thickness of 3-9 atomic layers, and are the thickest two-dimensional materials to date. In a specific embodiment of the present invention, the Mxene is Ti3C2Tx Mxene.

[0041] In the present invention, the X-type molecular sieve (NaX-type molecular sieve, NaX) is an alkali metal aluminosilicate, has a certain alkalinity, belongs to a class of solid bases, has a pore size of 10A, adsorbs any molecule greater than 3.64A and less than 10A, and has good adsorption performance and ion exchange performance. In a specific embodiment of the present invention, the molecular sieve is an X-type molecular sieve.

[0042] The second aspect of the present invention provides low-abundance protein enrichment magnetic beads prepared by the method described in the first aspect of the present invention.

[0043] Furthermore, the low-abundance protein enrichment magnetic beads are Ti3C2Tx-Fe3O4@NaX.

[0044] The third aspect of the present invention provides a product of low-abundance protein enriched magnetic beads prepared by the method described in the first aspect of the present invention.

[0045] Furthermore, the product includes reagents for enriching low-abundance proteins, kits for enriching low-abundance proteins, instruments for enriching low-abundance proteins, equipment for enriching low-abundance proteins, devices for enriching low-abundance proteins, and systems for enriching low-abundance proteins.

[0046] In some embodiments, the reagent for low-abundance protein enrichment comprises low-abundance protein enrichment magnetic beads prepared by the method described in the first aspect of the present invention.

[0047] In some embodiments, the kit for low-abundance protein enrichment comprises low-abundance protein enrichment magnetic beads prepared by the method described in the first aspect of the present invention, magnetic bead cleaning solution, wash buffer, lysis solution, enzyme, stop solution and desalting column, etc.

[0048] In some embodiments, the kit for low-abundance protein enrichment also includes a liquid reaction container and a liquid collection container. The container refers to any type of reactor, receiver, tank or combination thereof, in which a biochemical reaction can occur. The container can be solid-walled (when the boundary of the container is solid, such as glass, plastic or polydimethylsiloxane (PDMS)), or liquid-walled (when the boundary of the container is liquid, such as oil). The solid-walled container can contain a solid support, which is a continuous solid connecting all containers. The container can contain a variety of polymerizable or gelable polymers and / or monomers. A variety of polymerizable or gelable polymers and / or monomers can form a hydrogel or a hardened matrix when polymerized or gelled, thereby forming hardened particles. The hardened particles can be beads. The hardened particles can be porous particles. The hardened particles can be hydrogel particles. Hydrogel particles can be made of gel-like polymers, such as cross-linked polyacrylamide, cross-linked PEG, agarose or alginate. The hardened particles can be melted after treatment or stimulation.

[0049] In some embodiments, the instrument for low-abundance protein enrichment, the equipment for low-abundance protein enrichment, the device for low-abundance protein enrichment, and the system for low-abundance protein enrichment comprise low-abundance protein enrichment magnetic beads prepared by the method described in the first aspect of the present invention.

[0050] The fourth aspect of the present invention provides the use of low-abundance protein enrichment magnetic beads prepared by the method described in the first aspect of the present invention or the product described in the third aspect of the present invention in enriching low-abundance proteins.

[0051] In some embodiments, after low-abundance proteins in the sample to be tested are enriched, high performance liquid chromatography combined with high-resolution mass spectrometry can be used to identify and quantitatively analyze the low-abundance proteins in the enriched sample to be tested.

[0052] Furthermore, the samples to be tested include: blood samples from subjects, serum samples from subjects, plasma samples from subjects, cerebrospinal fluid samples from subjects, lymph fluid samples from subjects, urine samples from subjects, saliva samples from subjects, etc.

[0053] The fifth aspect of the present invention provides a method for enriching low-abundance proteins in a sample to be tested.

[0054] Furthermore, the method comprises the following steps: treating the sample to be tested with the low-abundance protein-enriched magnetic beads prepared by the method described in the first aspect of the present invention.

[0055] Furthermore, the method comprises the following steps: washing the low-abundance protein-enriched magnetic beads Ti3C2Tx-Fe3O4@NaX prepared by the method described in the first aspect of the present invention with a cleaning solution, magnetically separating, removing the supernatant, resuspending with a cleaning solution and adding it to the sample to be tested, incubating, magnetically separating and removing the supernatant, washing the Ti3C2Tx-Fe3O4@NaX with a wash buffer, adding a lysate, sonicating, and magnetically separating to obtain a supernatant containing low-abundance proteins.

[0056] Further, the dosage of the Ti3C2Tx-Fe3O4@NaX is 100 μg;

[0057] Preferably, the cleaning solution is 10 mM Tris-HCl (pH 7.4), 150 mM KCl, 0.05% CHAPS;

[0058] Preferably, the amount of cleaning solution used during the cleaning is 100 μL;

[0059] Preferably, the amount of the washing solution used during the resuspension is 100 μL;

[0060] Preferably, the incubation condition is 37°C for 30 min;

[0061] Preferably, the washing condition with wash buffer is washing 3 times with 200 μL wash buffer;

[0062] Preferably, the lysate is 50 mM TEAB, 0.5% SDC, 10 mM TCEP, 40 mM CAA;

[0063] Preferably, the amount of the lysate is 30 μL;

[0064] Preferably, the sample to be tested is a blood sample from a subject.

[0065] Furthermore, the enriched supernatant containing low-abundance proteins is the supernatant containing the low-abundance proteins in the sample to be tested.

[0066] Furthermore, the method may further include the following steps: placing the supernatant containing low-abundance proteins obtained by the above enrichment in a metal bath at 95°C for 10 minutes, adding 50 μL of 50 mM TEAB and 0.5 μg of Trypsin enzyme, and incubating overnight at 37°C. Adding 10 μL of 20% TFA to the solution, centrifuging at 16000g for 10 minutes at 4°C, taking the supernatant, desalting with a C18 SPE filter, and heat drying for subsequent proteomics analysis.

[0067] In some embodiments, the sample to be tested includes, but is not limited to, a blood sample from a subject, and may also include other body fluid samples from a subject, for example, a serum sample, a plasma sample, a cerebrospinal fluid sample, a lymph sample, a urine sample, a saliva sample, a tear sample, a gastric juice sample, a mucus sample, a sweat sample, a milk sample, a liquid biopsy sample, or a vomitus sample, etc. The sample to be tested may be taken directly from the subject, or may be taken from a previously separated body fluid sample material from the subject. The present invention does not particularly limit the specific type and source of the sample to be tested, and any sample to be tested that can be used for enrichment of low-abundance proteins is within the scope of protection of the present invention.

[0068] In some embodiments, typical body fluids such as serum, plasma, cerebrospinal fluid, etc. as mentioned above are commonly used samples in the field of proteomics research based on mass spectrometry platforms. Such samples contain a rich variety of proteins, among which the proportion of high-abundant proteins (HAP) reaches 97%-99%, including albumin, IgG, IgA, fibrinogen, transferrin, haptoglobin, antitrypsin, etc. Low-abundant proteins (LAP) are often specific biomarkers of diseases or target protein molecules for drug action. Therefore, removing high-abundant proteins that interfere with detection and enriching as many low-abundant proteins as possible has become one of the key factors in the number of mass spectrometry identifications, and has important research value in early diagnosis of diseases, prognosis and treatment, and biomarker research.

[0069] In some embodiments, the high-abundance proteins include but are not limited to: any one or more of albumin, IgA, IgD, IgE, IgG, IgM, α1-acid glycoprotein, α1-antitrypsin, α2-macroglobulin, apolipoprotein A1, fibrinogen, haptoglobin, transferrin, complement C3, apolipoprotein A-II, α-2-HS-glycoprotein, apolipoprotein C-III, α-1-antichymotrypsin, vitamin D binding protein, ceruloplasmin, complement C4-A, complement C1q, hemagglutinin, kininogen-1, synaptotagmin 5, histidine-rich glycoprotein, vitronectin, complement factor H, plasma proteinase C1 inhibitor, C4b binding protein and fibronectin.

[0070] In some embodiments, the subject is an animal, preferably a mammal (human and non-human animals), including but not limited to: humans, non-human primates (particularly higher primates, such as macaques, crab-eating macaques, short-tailed macaques, bear monkeys, flat-topped monkeys, golden monkeys and tree shrews), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, any livestock or pets, etc. In a preferred embodiment, the subject is a human.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] The present invention prepares for the first time a new type of high-specificity low-abundance protein enriched magnetic bead material Ti3C2Tx-Fe3O4@NaX. The present invention applies MXenes to blood proteomics for the first time. MXenes are combined with X-type molecular sieves. Based on the excellent chemical diversity and hydrophilicity of MXenes and the specific adsorption effect of molecular sieves, high-specific enrichment of low-abundance proteins in blood is achieved, and a higher proteome coverage depth is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 :Synthesis flow chart of mikene-molecular sieve magnetic beads (Ti3C2Tx-Fe3O4@NaX).

[0074] Figure 2 : Transmission electron microscopy results, among which, Figure a: Ti3AlC2MAX, Figure b: Ti3C2Tx MXene, Figure c: Ti3C2Tx-Fe3O4, Figure d: Ti3C2Tx-Fe3O4@NaX.

[0075] Figure 3 : Energy dispersive X-ray spectroscopy results, among which, Figure a: Ti3AlC2MAX, Figure b: Ti3C2Tx MXene, Figure c: Ti3C2Tx-Fe3O4, Figure d: Ti3C2Tx-Fe3O4@NaX.

[0076] Figure 4 :The number of proteins and peptides was identified by enriching plasma proteins with mikrolene-molecular sieve magnetic beads (Ti3C2Tx-Fe3O4@NaX).

[0077] Figure 5 :The matching of plasma proteins enriched by mikropicene-molecular sieve magnetic beads (Ti3C2Tx-Fe3O4@NaX) to the absolute concentration of blood proteins in the human protein atlas. DETAILED DESCRIPTION

[0078] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0079] The reagents and raw materials used in the present invention are easily obtained by ordinary technicians in the field and can be obtained from commercial channels unless otherwise specified. The experimental methods in the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0080] Example Preparation of low-abundance protein enrichment magnetic beads Ti3C2Tx-Fe3O4@NaX and its application in low-abundance protein enrichment

[0081] 1. Experimental Materials

[0082] Ti3AlC2MAX was purchased from Eleven Technology Co., Ltd. (YY201701), LiF was purchased from Aladdin (L104230), ferric chloride hexahydrate (FeCl3·6H2O) was purchased from McLean (10025-77-1), and NaAlO2 was purchased from McLean (11138-49-1).

[0083] 2. Experimental methods

[0084] (1) Synthesis of Ti3C2Tx MXene

[0085] The minimum intensity layer delamination (MILD) method was used to synthesize Ti3C2Tx MXene. The specific experimental method was as follows: 1g lithium fluoride (LiF) was dissolved in 20mL 30% HCl, 1g Ti3AlC2 MAX was slowly added to the solution, and reacted at 35°C for 24h. Washed with deionized water several times, centrifuged at 5000g for 5min, and the supernatant was removed until the pH was ≥6. Deionized water was added and shaken thoroughly to obtain a layered and stable Ti3C2Tx MXene hydrocolloid solution, centrifuged at 2000g for 1h, the supernatant was discarded, freeze-dried, and stored in a dark environment at 4°C.

[0086] (2) Synthesis of Ti3C2Tx-Fe3O4

[0087] The hydrothermal method was used to synthesize ferroferric oxide nanoparticles (Fe3O4) on the surface of Ti3C2Tx MXene to form Ti3C2Tx-Fe3O4. The specific experimental method is as follows: 30 mg of Ti3C2Tx MXene was weighed and dispersed in 30 mL of ethylene glycol. The Ti3C2Tx MXene was ultrasonicated in a water bath until it was uniformly dispersed. 160 mg of ferric chloride hexahydrate (FeCl3·6H2O) and 30 mg of trisodium citrate were added, and the ultrasonication was continued until it was completely dissolved. 700 mg of anhydrous sodium acetate was added and mechanically stirred at room temperature for 30 min. The resulting uniform solution was transferred to a 50 mL Teflon-lined stainless steel autoclave and heated at 200 ° C for 8 h. After the reaction, the reactor was taken out, rinsed with cold water, cooled to room temperature, washed with ethanol and pure water 3 times, and dried to obtain Ti3C2Tx-Fe3O4.

[0088] (3) Synthesis of Ti3C2Tx-Fe3O4@NaX

[0089] X-type molecular sieve was synthesized on the surface of Ti3C2Tx-Fe3O4 to obtain Ti3C2Tx-Fe3O4@NaX. The specific experimental method is as follows: 500mg NaAlO2 was dissolved in 10mL deionized water and added to a 100mL three-necked flask. 150mg Ti3C2Tx-Fe3O4 was added under mechanical stirring and stirred to form solution A. 4g NaOH was dissolved in 50mL deionized water, and then 2.5g Na2SiO3·9H2O was added and completely dissolved to form solution B. Under mechanical stirring, solution B was slowly dripped into solution A, stirred for 30min, and then allowed to stand at room temperature for 45h. Under mechanical stirring, the reaction was carried out at 60℃ for 5h. After the reaction was completed, it was washed with deionized water several times until the solution was neutral, and dried to obtain Ti3C2Tx-Fe3O4@NaX.

[0090] (4) Enrichment of low-abundance proteins in blood and proteomic analysis

[0091] The prepared Ti3C2Tx-Fe3O4@NaX was used for enrichment of low-abundance proteins in blood and proteomic analysis. The specific experimental method is as follows: 100μg Ti3C2Tx-Fe3O4@NaX magnetic beads were washed with 100μL washing solution (10mM Tris-HCl (pH 7.4), 150mM KCl, 0.05% CHAPS), magnetically separated, the supernatant was removed, and resuspended with 100μL washing solution, added to 500μL plasma, and incubated at 37℃ for 30min. After the incubation, the supernatant was removed by magnetic separation, and the magnetic beads were washed 3 times with 200μL wash buffer. 30μL lysis solution (50mM TEAB, 0.5% SDC, 10mM TCEP, 40mM CAA) was added, ultrasonicated for 10min, magnetically separated, the supernatant was taken to a new centrifuge tube, and the supernatant was placed in a 95℃ metal bath for 10min. Add 50 μL 50 mM TEAB and 0.5 μg Trypsin enzyme, incubate overnight at 37°C. Add 10 μL 20% TFA to the solution, centrifuge at 16000 g for 10 min at 4°C, take the supernatant, desalt with C18 SPE filter, and heat dry.

[0092] (5) LC-MS / MS analysis

[0093] A timsTOF_HT mass spectrometer (Bruker, Germany) was connected to an UltiMate 3000 liquid phase mass spectrometer system (Thermo Fisher Scientific, USA). The sample dissolved in 0.1% FA aqueous solution was loaded with a sample amount of 400 ng. The liquid phase conditions were: C18 reverse phase analytical column (C18, 1.5 μm, 100 μm×15 cm). Mobile phase A was 0.1% FA; mobile phase B was 80% ACN, 0.1% FA. At a constant flow rate of 500 nL / min, within the dynamic range of the gradient length, the gradient was 0-17 min (3.5-32% B); 17-18 min (32-95% B); 18-20 min (95% B); 21-22 min (95%-1% B). The DIA acquisition mode was used, the mass spectrometer scanning range was m / z 300-1500, the primary mass spectrometer resolution was set to 60000 (1222 m / z), and the accumulation time was set to 50 ms in the TIMS tunnel. The capillary voltage was set to 1.5 kV, and the mobility was set to 0.70-1.30 cm 2 / (V). The total cycle time is 1.23s. The mass spectrometry raw data (.d) are generated.

[0094] (6) Data analysis

[0095] The .d file generated by the timsTOF_HT mass spectrometer was searched using the DIANN database search. The target database was uniprot.human. The search parameters were set to trypsin full digestion, 2 protease missed cleavage sites, carbamidomethyl (C) as a fixed protein modification, and oxidation (M) as a variable modification. The mass spectrometry primary mass error was 15 ppm, the secondary mass error was 15 ppm, and the false positive rate was set to 1%.

[0096] 3. Experimental results

[0097] Figure 1 The synthesis flow chart of MXene-molecular sieve magnetic beads (Ti3C2Tx-Fe3O4@NaX) and the transmission electron microscopy results of Ti3AlC2MAX and the synthesized Ti3C2Tx MXene, Ti3C2Tx-Fe3O4, and Ti3C2Tx-Fe3O4@NaX are shown in the figure. Figure 2 As shown in ad, it can be seen that Ti3AlC2 MAX and Ti3C2Tx MXene are lamellar structures. After the synthesis of magnetic Fe3O4, a large number of Fe3O4 particles can be observed on the surface of Ti3C2Tx MXene. After the molecular sieve is adsorbed on the surface of Fe3O4, cubic nanoparticles are found on the surface of Ti3AlC2 MAX. The above results show that Ti3C2Tx-Fe3O4@NaX is successfully synthesized.

[0098] Energy dispersive X-ray spectroscopy (EDS) results of Ti3AlC2MAX and synthesized Ti3C2Tx MXene, Ti3C2Tx-Fe3O4, and Ti3C2Tx-Fe3O4@NaX are shown in the figure below. Figure 3 As shown in ad, from the EDS analysis, it can be seen that Ti3AlC2 MAX is composed of C, O, Ti, and Al. After the synthesis of Ti3C2Tx MXene, the element composition is C, O, Ti, F, Al, and Cl. After the synthesis of magnetic Fe3O4, the element composition is C, O, Ti, F, Al, and Fe. After the synthesis of molecular sieves, the element composition is C, O, Ti, F, Al, Fe, Si, and Na. The above results once again prove that Ti3C2Tx-Fe3O4@NaX is successfully synthesized.

[0099] The results of plasma protein enrichment and identification of protein and peptide fragments by Michaelene-molecular sieve magnetic beads (Ti3C2Tx-Fe3O4@NaX) are shown in Figure 2. Figure 4 As shown in Figure 2, the matching results of plasma protein enriched by Michaelene-molecular sieve magnetic beads (Ti3C2Tx-Fe3O4@NaX) and the absolute concentration of blood proteins in the human protein atlas are as follows: Figure 5As shown, the above results indicate that the Ti3C2Tx-Fe3O4@NaX prepared by the present invention achieves highly specific enrichment of low-abundance proteins in blood and achieves a higher proteome coverage depth.

Claims

1. A method for preparing magnetic beads for enriching low-abundance proteins, characterized in that: The method comprises the following steps: (1) Add Ti3AlC2 MAX to LiF solution to react and prepare Ti3C2Tx Mxene; (2) Dispersing Ti3C2Tx Mxene in ethylene glycol, adding FeCl3, trisodium citrate and anhydrous sodium acetate to react, and preparing Ti3C2Tx-Fe3O4; (3) Solution A containing NaAlO2 solution and Ti3C2Tx-Fe3O4 was prepared, and solution B containing NaOH solution and Na2SiO3·9H2O was prepared. Solution B was dropped into solution A for reaction to prepare low-abundance protein enrichment magnetic beads Ti3C2Tx-Fe3O4@NaX.

2. The method according to claim 1, characterized in that The LiF solution described in step (1) is obtained by dissolving 1 g of LiF in 20 mL of 30% HCl.

3. The method according to claim 1, characterized in that The amount of Ti3AlC2 MAX used in step (1) is 1 g.

4. The method according to claim 1, characterized in that: The reaction conditions in step (1) are 35° C. for 24 h.

5. The method according to claim 1, characterized in that Step (1) further comprises washing the product obtained by the reaction, removing the supernatant until the pH is ≥ 6, adding deionized water, and fully shaking to obtain a stratified and stable Ti3C2Tx Mxene hydrocolloid solution, centrifuging and discarding the supernatant, and freeze-drying to obtain Ti3C2Tx Mxene.

6. The method according to claim 5, characterized in that The washing conditions are deionized water washing, 5000g, and centrifugation for 5 min.

7. The method according to claim 5, characterized in that The conditions for centrifuging and discarding the supernatant are 2000 g and centrifugation for 1 hour.

8. The method according to claim 1, characterized in that The amount of Ti3C2Tx Mxene used in step (2) is 30 mg.

9. The method according to claim 1, characterized in that: The amount of ethylene glycol used in step (2) is 30 mL.

10. The method according to claim 1, characterized in that The amounts of FeCl3, trisodium citrate and anhydrous sodium acetate used in step (2) are 160 mg, 30 mg and 700 mg respectively.

11. The method according to claim 1, characterized in that: In step (2), the Ti3C2Tx Mxene is dispersed in ethylene glycol, and after water bath sonication until the Ti3C2Tx Mxene is uniformly dispersed, the FeCl3 and trisodium citrate are added, and after continued sonication until the Ti3C2Tx Mxene is completely dissolved, the anhydrous sodium acetate is added, and the mixture is stirred at room temperature. The resulting uniform solution is transferred to a high-pressure reactor for reaction.

12. The method according to claim 11, characterized in that The stirring time is 30 min.

13. The method according to claim 11, characterized in that The high-pressure reactor is a Teflon-lined stainless steel high-pressure reactor.

14. The method according to claim 11, characterized in that The reaction conditions are heating at 200° C. for 8 h.

15. The method according to claim 1, characterized in that Step (2) also includes washing and drying the reaction product to obtain Ti3C2Tx-Fe3O4.

16. The method according to claim 15, characterized in that The cleaning conditions are ethanol and pure water cleaning 3 times.

17. The method according to claim 1, characterized in that The NaAlO2 solution in step (3) is obtained by dissolving 500 mg NaAlO2 in 10 mL deionized water.

18. The method according to claim 1, characterized in that The amount of Ti3C2Tx-Fe3O4 used in step (3) is 150 mg.

19. The method according to claim 1, characterized in that The NaOH solution in step (3) is obtained by dissolving 4 g of NaOH in 50 mL of deionized water.

20. The method according to claim 1, characterized in that The amount of Na2SiO3·9H2O used in step (3) is 2.5 g.

21. The method according to claim 1, characterized in that The reaction conditions in step (3) are as follows: solution B is slowly dripped into solution A under stirring, stirred for 30 min, left to stand at room temperature for 45 h, and reacted at 60° C. under stirring for 5 h.

22. The method according to claim 1, characterized in that Step (3) also includes washing the product obtained from the reaction until the solution is neutral, and drying to obtain low-abundance protein enrichment magnetic beads Ti3C2Tx-Fe3O4@NaX.

23. The method according to claim 22, characterized in that The cleaning condition is deionized water cleaning.

24. Low-abundance protein enrichment magnetic beads prepared by the method according to any one of claims 1 to 23.

25. A product comprising low-abundance protein enrichment magnetic beads prepared by the method of any one of claims 1-23.

26. Use of the low-abundance protein enrichment magnetic beads prepared by the method of any one of claims 1 to 23 or the product of claim 25 in enriching low-abundance proteins.

27. A method for enriching low-abundance proteins in a sample to be tested, characterized in that: The method comprises the following steps: treating a sample to be tested with low-abundance protein-enriched magnetic beads prepared by the method of any one of claims 1 to 23.

28. The method according to claim 27, characterized in that The method comprises the following steps: washing the low-abundance protein enrichment magnetic beads Ti3C2Tx-Fe3O4@NaX prepared by the method of any one of claims 1 to 23 with a washing solution, magnetically separating, removing the supernatant, resuspending with a washing solution and adding the beads to be tested, incubating, magnetically separating and removing the supernatant, washing the Ti3C2Tx-Fe3O4@NaX with a washbuffer, adding a lysis solution, ultrasonicating, and magnetically separating to obtain a supernatant containing low-abundance proteins.

29. The method according to claim 28, characterized in that The amount of Ti3C2Tx-Fe3O4@NaX used is 100 μg.

30. The method according to claim 28, characterized in that The cleaning solution is 10 mM Tris-HCl (pH 7.4), 150 mM KCl, and 0.05% CHAPS.

31. The method according to claim 28, characterized in that The amount of cleaning solution used during the cleaning is 100 μL.

32. The method according to claim 28, characterized in that The amount of the washing solution used during the resuspension is 100 μL.

33. The method according to claim 28, characterized in that The incubation condition is 37° C. for 30 min.

34. The method according to claim 28, characterized in that The washing condition with wash buffer is washing 3 times with 200 μL wash buffer.

35. The method according to claim 28, characterized in that The lysis solution contains 50 mM TEAB, 0.5% SDC, 10 mM TCEP, and 40 mM CAA.

36. The method according to claim 28, characterized in that The amount of the lysate used is 30 μL.

37. The method according to claim 28, characterized in that The sample to be tested is a blood sample from a subject.

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