Method for processing single-cell proteome sample and method for single-cell proteome analysis

Abstract

The application relates to a single-cell proteome sample processing method, which comprises the following steps: taking a single-cell sample, adding 0.01-0.2% non-ionic surfactant for ultrasonic lysis to obtain a pretreated sample; performing denaturation reduction alkylation treatment on the pretreated sample to obtain an alkylated sample; adding an immobilized enzyme reactor to the alkylated sample for enzymolysis for 0.5-4 hours, centrifuging to obtain supernatant, and obtaining a processed sample; and a preparation method of the immobilized enzyme reactor, which comprises the following steps: adding polyethyleneimine to amino polymer microspheres to obtain hydrophilic amino polymer microspheres after reaction; adding graphene oxide to the hydrophilic amino polymer microspheres to obtain graphene oxide-polymer microspheres after reaction; adding a protease solution containing benzalkonium to the graphene oxide-polymer microspheres to obtain the immobilized enzyme reactor after reaction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a single-cell proteome sample processing method and a single-cell proteome analysis method. BACKGROUND

[0002] The popularity of second-generation sequencing technology and the continuous development of third-generation sequencing technology have greatly promoted the research of single-cell genome and transcriptome, and scientists have greatly improved the resolution of cell understanding. However, the genomic and transcriptomic data of single cells are still far from enough to describe the phenotype and function of cells in the complex environment of an organism. Proteins, as the direct executors of all functions in cells, can perceive and respond to almost all external and internal stimuli through proteins and their post-translational modifications, thereby affecting the function and state of the entire organism. However, due to the lack of amplification characteristics, the large number of types, low abundance, and wide dynamic distribution range of proteins, compared with nucleic acids and other biological macromolecules, single-cell proteomics research is relatively lagging and extremely challenging. How to achieve high-coverage single-cell proteomics analysis has become one of the important scientific problems to be solved in proteomics research.

[0003] In recent years, with the development of ion mobility separation technology and a series of high-sensitivity high-resolution mass spectrometers such as Orbitrap Eclipse, Orbitrap Exploris480, timsTOF pro 2 and timsTOF SCP, proteomics analysis of single cells has gradually become a reality. However, the traditional proteomics sample processing process includes multiple steps of reagent transfer, desalting and drying, which can easily cause loss of samples. At the same time, due to the extremely low protein amount of single cells, the complex sample processing process can easily cause inconsistent loss of different types of proteins, thereby affecting the final quantitative accuracy. SUMMARY

[0004] Therefore, the present application provides a single-cell proteome sample processing method and a single-cell proteome analysis method with less sample loss and high efficiency.

[0005] The specific technical solutions are as follows:

[0006] In one aspect, the present application provides a single-cell proteome sample processing method, which comprises the following steps:

[0007] A single-cell sample is taken, and 0.01w%-0.2w% of a non-ionic surfactant is added for ultrasonic lysis to obtain a pretreated sample;

[0008] The pretreated sample is subjected to denaturation, reduction and alkylation treatment to obtain an alkylated sample;

[0009] adding an immobilized enzyme reactor to the alkylated sample for enzymolysis for 0.5h-4h, centrifuging to obtain supernatant, and obtaining a treated sample; a preparation method of the immobilized enzyme reactor, comprising the following steps:

[0010] adding polyethyleneimine to the amino polymer microspheres to obtain hydrophilic amino polymer microspheres after reaction;

[0011] adding graphene oxide to the hydrophilic amino polymer microspheres to obtain graphene oxide-polymer microspheres after reaction;

[0012] adding a protease solution containing benzamidine to the graphene oxide-polymer microspheres to obtain the immobilized enzyme reactor after reaction.

[0013] In one of the embodiments, the non-ionic surfactant satisfies at least one of conditions (1)-(2):

[0014] (1) the non-ionic surfactant is dodecyl-β-D-maltoside;

[0015] (2) the non-ionic surfactant is added in an amount of 5μL-15μL.

[0016] In one of the embodiments, the steps of denaturation reduction and alkylation of the pretreated protein sample comprise:

[0017] taking 14μL-16μL of the pretreated sample, adding 0.1μL-1μL of a reducing agent with a concentration of 25mM-100mM, and incubating at 56℃-100℃ for 20min-60min to obtain a denatured and reduced protein sample;

[0018] adding 0.1μL-1μL of an alkylation reagent with a concentration of 25mM-100mM to the denatured and reduced protein sample for alkylation treatment to obtain the alkylated protein sample.

[0019] In one of the embodiments, the reducing agent comprises at least one of dithiothreitol or tris-carboxyethyl phosphine.

[0020] In one of the embodiments, the alkylation reagent comprises at least one of iodoacetamide, chloroacetamide, iodoacetic acid and N-ethylmaleimide.

[0021] In one of the embodiments, the amino polymer microspheres satisfy at least one of conditions (3)-(4):

[0022] (3) the particle size of the amino polymer microspheres is 1μm-10μm;

[0023] (4) the pore size of the amino polymer microspheres is

[0024] In one of the embodiments, the polyethyleneimine satisfies at least one of (5) to (6):

[0025] (5) the concentration of the polyethyleneimine is 3 mg / mL to 5 mg / mL;

[0026] (6) the polyethyleneimine is added in an amount of 1 mL to 2 mL per 150 mg of the amino polymer microspheres.

[0027] In one of the embodiments, the graphene oxide satisfies at least one of (7) to (8):

[0028] (7) the concentration of the graphene oxide is 1 mg / mL to 3 mg / mL;

[0029] (8) the graphene oxide is added in an amount of 1 mL to 2 mL per 150 mg of the amino polymer microspheres.

[0030] In one of the embodiments, the protease includes at least one of trypsin, pepsin, chymotrypsin, carboxypeptidase or glycosidase.

[0031] The application also provides a single-cell proteome analysis method, which obtains the treated sample by the above method and then performs chromatography-mass spectrometry analysis.

[0032] The single-cell proteome sample processing method uses a non-ionic surfactant to reduce protein adsorption on the inner wall of the test tube, further uses an immobilized enzyme reactor to perform enzymolysis on the single-cell protein sample, and easily separates the enzyme from the sample solution after the enzymolysis, thereby avoiding the influence of the enzyme on subsequent mass spectrometry analysis of the single-cell proteome sample. At the same time, due to the extremely low protein amount of the single cell, the use of graphene oxide-polyethyleneimine-amino polymer microspheres improves the enzyme loading amount compared with the traditional amino polymer microspheres, improves the enzymolysis efficiency, and reduces non-specific adsorption. The protein enzymolysis product can be directly subjected to liquid chromatography-mass spectrometry analysis, thereby avoiding the loss of the single-cell proteome sample caused by desalting, evaporation and transfer processes. The single-cell proteome sample processing method provides technical support for efficient single-cell proteome analysis. Research proves that the single-cell proteome sample processing method can reduce sample loss and improve the number of single-cell protein identifications. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 A single human oocyte sample proteome analysis graph in Example 1;

[0034] Figure 2 A proteome analysis result graph of samples treated under different DDM concentrations in Examples 1 to 6;

[0035] Figure 3 Figure for proteomic analysis results of samples in Example 1, Example 7-9 under different enzymatic hydrolysis time treatment;

[0036] Figure 4 Figure for proteomic analysis results of samples in Example 1, Comparative Example 1-5 under different enzyme treatment. DETAILED DESCRIPTION

[0037] In order to make the above objectives, features and advantages of the present application more apparent, the specific embodiments of the present application are described in detail below, and preferred embodiments of the present application are given. In the following description, a large number of specific details are set forth in order to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. The reagents or instruments used herein are not specified by the manufacturer, and are conventional products that can be purchased.

[0039] In the conventional technology, the single-cell proteomic sample pretreatment technology based on microfluidic chip has great potential, but it needs to use a microfluidic chip designed in a clever way and a high-precision mechanical platform, which has a high technical and engineering threshold and is difficult to popularize and apply in most proteomic laboratories. At present, the single-cell proteomic sample pretreatment method in the test tube has the following problems: on the one hand, most of such methods need to desalt and evaporate the sample before mass spectrometric analysis, which is easy to cause loss of the sample; on the other hand, in order to improve the enzymatic hydrolysis efficiency, several times or even tens of times of the amount of single-cell protein is used for enzyme, so that such a high amount of enzyme is more likely to be self-degraded, and after entering the mass spectrometer, it may also affect the identification of the original protein in the single cell.

[0040] The applicant has found through a large number of creative experimental researches that: using a specific immobilized enzyme reactor for single-cell protein sample enzymatic hydrolysis can easily separate the enzyme from the sample solution, improve the enzymatic hydrolysis efficiency, and at the same time avoid the influence of the enzyme on the subsequent mass spectrometric analysis of the single-cell sample, so that the protein enzymatic hydrolysis product can be directly subjected to liquid chromatography-mass spectrometry analysis, thereby providing technical support for realizing efficient single-cell proteomic analysis.

[0041] An embodiment of the present application provides a single-cell proteomic sample processing method, which comprises steps S10-S30.

[0042] Step S10: taking a single cell sample, adding a non-ionic surfactant for ultrasonic lysis to obtain a pretreated sample.

[0043] In some embodiments, the single cell is a mammalian oocyte.

[0044] In a specific example, the single cell is a single human oocyte.

[0045] In some embodiments, the non-ionic surfactant is dodecyl-β-D-maltoside (DDM).

[0046] In some embodiments, the mass fraction of the non-ionic surfactant is 0.01% to 0.2%.

[0047] In a specific example, the mass fraction of the non-ionic surfactant is 0.1%.

[0048] In some embodiments, the amount of the non-ionic surfactant added is 5 μL to 15 μL.

[0049] In a specific example, the amount of the non-ionic surfactant added is 10 μL.

[0050] Step S20: denaturing reduction and alkylation of the pretreated sample in step S10 to obtain an alkylated sample.

[0051] Specifically, the step of denaturing reduction and alkylation of the pretreated sample in step S10 includes steps S21 to S22.

[0052] Step S21: taking 14 μL to 16 μL of the pretreated sample in step S10, adding 0.1 μL to 1 μL of a reducing agent with a concentration of 25 mM to 100 mM, and incubating at 56°C to 100°C for 20 min to 60 min to obtain a denatured and reduced protein sample.

[0053] In some embodiments, the reducing agent includes at least one of dithiothreitol or tricarboxyethyl phosphine.

[0054] In a specific example, the reducing agent is dithiothreitol (DTT).

[0055] It can be understood that DTT can reduce disulfide bonds in proteins, open disulfide bonds, and make protein molecules change from spherical to chain as much as possible, increase the solubility of proteins, and expose as many enzyme cutting sites as possible.

[0056] In a specific example, the temperature of the incubation treatment is 75°C.

[0057] In a specific example, the incubation time is 40 min.

[0058] Step S22: 0.1 μL to 1 μL of alkylating agent with a concentration of 25 mM to 100 mM is added to the denatured and reduced protein sample for alkylating treatment, and an alkylated protein sample is obtained.

[0059] In some embodiments, the alkylating agent includes at least one of iodoacetamide (IAA), chloroacetamide (ClAM), iodoacetic acid, and N-ethylmaleimide.

[0060] Step S30: The immobilized enzyme reactor is added to the alkylated sample obtained in step S20 for enzymatic hydrolysis for 0.5 h to 4 h, and the supernatant is obtained by centrifugation, and a treated sample is obtained.

[0061] Optionally, the step of preparing the immobilized enzyme reactor includes steps a, b, and c.

[0062] Step a: After adding polyethyleneimine to the amino polymer microspheres, hydrophilic amino polymer microspheres are obtained.

[0063] It can be understood that the polyethyleneimine is a water-soluble polymer produced by polymerization of ethyleneimine, and after reacting with the amino polymer microspheres, the hydrophilicity of the microspheres is increased, and non-specific adsorption is reduced.

[0064] In some embodiments, step a further includes a step of activating the amino polymer microspheres.

[0065] In some embodiments, the step of activating the amino polymer microspheres is to add the amino polymer microspheres to a glutaraldehyde-containing phosphate buffer with a volume fraction of 5% for reaction, and then to obtain coupled amino polymer microspheres.

[0066] In some embodiments, the particle size of the amino polymer microspheres is 1 μm to 10 μm.

[0067] In a specific example, the particle size of the amino polymer microspheres is 10 μm.

[0068] In some embodiments, the pore size of the amino polymer microspheres is

[0069] In a specific example, the pore size of the amino polymer microspheres is

[0070] In some embodiments, the concentration of the polyethyleneimine is 3 mg / mL to 5 mg / mL.

[0071] In a specific example, the concentration of polyethyleneimine is 5 mg / mL.

[0072] In some embodiments, the amount of polyethyleneimine added is 1 mL to 2 mL per 150 mg of the amino polymer microspheres.

[0073] In a specific example, the amount of polyethyleneimine added is 1 mL per 150 mg of the amino polymer microspheres.

[0074] In some embodiments, the reaction time is 2 h to 4 h.

[0075] In a specific example, the reaction time is 3 h.

[0076] In some embodiments, after the addition of polyethyleneimine to the amino polymer microspheres in step a, the method further comprises a step of washing the unreacted polyethyleneimine with ethanol and water.

[0077] Step b: After the addition of graphene oxide to the hydrophilic amino polymer microspheres, graphene oxide-polymer microspheres are obtained.

[0078] It can be understood that the graphene oxide is adsorbed on the amino polymer porous microspheres to form graphene oxide-polymer microspheres.

[0079] In some embodiments, the concentration of graphene oxide is 1 mg / mL to 3 mg / mL.

[0080] In a specific example, the concentration of graphene oxide is 1 mg / mL.

[0081] In some embodiments, the amount of graphene oxide added is 1 mL to 2 mL per 150 mg of the amino polymer microspheres.

[0082] In a specific example, the amount of graphene oxide added is 1.5 mL per 150 mg of the amino polymer microspheres.

[0083] In some embodiments, the reaction time is 5 h to 8 h.

[0084] In a specific example, the reaction time is 6 h.

[0085] In some embodiments, after the addition of graphene oxide to the hydrophilic amino polymer microspheres in step b, the method further comprises a step of washing the unreacted graphene oxide with ethanol and water.

[0086] Step c: After the addition of the benzyiamide-containing protease solution to the graphene oxide-polymer microspheres, an immobilized enzyme reactor is obtained.

[0087] It can be understood that the protease is immobilized by adding the protease solution and the electrostatic interaction between the protease and the graphene oxide in the graphene oxide-polyethyleneimine-aminopolymer microspheres.

[0088] In some embodiments, the concentration of the benzenecarboxamidine is 50 mM.

[0089] In some embodiments, the concentration of the protease solution is 1 mg / mL-5 mg / mL.

[0090] In a specific example, the concentration of the protease solution is 2 mg / mL.

[0091] In some embodiments, the protease includes at least one of trypsin, pepsin, chymosin, carboxypeptidase or glycosidase.

[0092] In some embodiments, the reaction temperature in step c is 4°C.

[0093] In some embodiments, the reaction time in step c is 24 h.

[0094] In some embodiments, after the reaction in step c, a step of washing away the unreacted trypsin solution by using an ammonium bicarbonate solution with a concentration of 50 mM is further included.

[0095] An embodiment of the present application further provides a single-cell proteome analysis method, which uses the above-mentioned processing method to obtain a processed sample, and then performs liquid chromatography-mass spectrometry analysis.

[0096] The above-mentioned single-cell proteome sample processing method uses a non-ionic surfactant to reduce protein adsorption on the inner wall of a test tube, and further uses an immobilized enzyme reactor to perform enzymolysis on the single-cell protein sample. After the enzymolysis, the enzyme is easily separated from the sample solution, thereby avoiding the influence of the enzyme on subsequent mass spectrometry analysis of the single-cell proteome sample. At the same time, due to the extremely low protein amount of the single cell itself, the use of graphene oxide-polyethyleneimine-aminopolymer microspheres improves the immobilization amount of the enzyme compared with traditional aminopolymer microspheres, improves the enzymolysis efficiency, and reduces non-specific adsorption. The protein enzymolysis product can be directly subjected to liquid chromatography-mass spectrometry analysis, thereby avoiding the loss of the single-cell proteome sample caused by desalting, evaporation and transfer processes. The above-mentioned single-cell proteome sample processing method provides technical support for efficient single-cell proteome analysis. Research proves that the above-mentioned single-cell proteome sample processing method can reduce sample loss and improve the number of single-cell protein identifications.

[0097] The following detailed description is made with reference to the accompanying drawings, in which the following embodiments are described. The following examples are not intended to be limiting, unless otherwise specified, and include only those components which are essential for a proper understanding of the application, as well as those that are desirable for its practice. In the examples, reagents and instruments are used as are conventional in the art, unless otherwise specified. Methods of experimentation not specifically noted in the examples are performed according to conventional conditions, such as those described in the literature, in books, or according to the manufacturer's recommendations.

[0098] Example 1

[0099] 1. Preparation of single-cell protein sample

[0100] (1) Collection of single human oocyte: immature oocytes discarded in the process of clinical diagnosis and treatment were collected with informed consent, and after removing granulosa cells, the oocytes were washed 3 times with PBS buffer, and collected in a 200 uL low adsorption PCR tube soaked with 0.1% DDM in advance.

[0101] (2) Protein extraction: 10 uL of 0.1% DDM was added to the PCR tube in step (1), and cell disruption was performed in a non-contact ultrasonic disruptor. After 5 minutes, centrifugation was performed, and the supernatant was taken to obtain a pretreated sample.

[0102] (3) Protein reduction: 0.5 uL of 100 mM DTT was added to the pretreated sample obtained in step (2), and incubated at 75°C for 40 min to obtain denatured and reduced protein sample.

[0103] (4) Protein alkylation: 0.5 uL of 100 mM IAA was added to the denatured and reduced protein sample obtained in step (3) for alkylation treatment, and reacted at room temperature for 30 min in the dark to obtain an alkylated sample.

[0104] (5) Synthesis of enzyme reactor: 150 mg of amino polymer microspheres (particle size 10 pm, pore size 100 nm) were added to a phosphate buffer containing 5% (v / v) glutaraldehyde, and reacted at room temperature for 3 hours. After centrifugation, excess glutaraldehyde was washed away with ethanol and water; 1 mL (5 mg / mL) of polyethyleneimine was added and reacted for another 3 hours. After centrifugation, unreacted polyethyleneimine was washed away with ethanol and water; 1.5 mL (1 mg / mL) of graphene oxide was added and shaken at room temperature for 6 hours. After centrifugation, unreacted graphene oxide solution was washed away with ethanol and water; finally, 1 mL of 2 mg / mL trypsin solution containing 50 mM benzamidine was added, and reacted at 4°C for 24 hours. Unreacted trypsin solution was washed away with 50 mM ammonium bicarbonate solution, and the prepared enzyme reactor was stored at 4°C for standby use.

[0105] ​(6) Proteolysis: the alkylated sample obtained in step (4) was subjected to proteolysis by adding the immobilized enzyme reactor prepared in step (5), and after proteolysis for 0.5 h, the supernatant was obtained by centrifugation to obtain a treated sample.

[0106] 2. Single-cell proteome analysis

[0107] Mass spectrometry analysis: the treated sample was transferred to a sample bottle of a liquid chromatograph-mass spectrometer for direct injection, liquid chromatograph-mass spectrometry determination was performed, and proteomic analysis was performed on the determination results.

[0108] The results are shown in Figure 1 , wherein Figure 1 A is a single human oocyte sample pretreatment process based on an immobilized enzyme reactor; Figure 1 B is the number of identified proteins of GV stage and MI stage single human oocytes. A number of GV stage and MI stage single human oocytes were collected for whole proteome analysis, and the results are shown in Figure 1 B: for GV stage single human oocytes, an average of 3697 proteins (n=6) can be identified; for MI stage single human oocytes, an average of 3816 proteins (n=13) can be identified.

[0109] Examples 2-6

[0110] Examples 2-6 and the single-cell proteome sample treatment method in Example 1 are basically the same, except that different DDM concentrations are used to obtain the pretreated sample. The obtained treated sample was transferred to a sample bottle of a liquid chromatograph-mass spectrometer for direct injection, liquid chromatograph-mass spectrometry determination was performed, and proteomic analysis was performed on the determination results. The results are shown in Figure 2 , wherein Figure 2 A is the number of identified proteins (n=3) under each DDM concentration; Figure 2 B is the number of identified peptides (n=3) under each DDM concentration; Figure 2 C is the quantitative CV value distribution between 3 repeats under each DDM concentration.

[0111] The experimental results show that under the concentration of 0.1% DDM, better protein Figure 2 A), peptide Figure 2 B) identification depth can be obtained, indicating that it has better extraction effect on the whole proteome; at the same time, under this concentration, the quantitative reproducibility Figure 2 C) between the 3 technical repeats is also better.

[0112] Examples 7-9

[0113] Examples 7-9 were substantially identical to the sample processing method of Example 1, except that the immobilized enzyme reactor was added to the previously processed sample for different lengths of time, and the resulting processed sample was transferred to a sample vial for liquid chromatography-mass spectrometry and proteomic analysis of the results. The results are shown in Table 1. Figure 3 Figure 3 Table 1: Proteomic analysis of single-cell protein samples Figure 3 Table 1: Proteomic analysis of single-cell protein samples Figure 3 Table 1: Proteomic analysis of single-cell protein samples Figure 3 Table 1: Proteomic analysis of single-cell protein samples Figure 3 Table 1: Proteomic analysis of single-cell protein samples Figure 3 Table 1: Proteomic analysis of single-cell protein samples

[0114] Comparative Example 1

[0115] Comparative Example 1 was substantially identical to the sample processing method of Example 1, except that the protease added to the previously processed sample was different, specifically, the enzyme added was Sigma enzyme (Cat# T6567), and the enzyme was allowed to digest overnight. After centrifugation, the supernatant was obtained as the processed sample, and the resulting processed sample was transferred to a sample vial for liquid chromatography-mass spectrometry and proteomic analysis of the results.

[0116] Comparative Example 2

[0117] Comparative Example 2 was substantially identical to the sample processing method of Example 1, except that the protease added to the previously processed sample was different, specifically, the enzyme added was HLS (Cat# TRY001C), and the enzyme was allowed to digest overnight. After centrifugation, the supernatant was obtained as the processed sample, and the resulting processed sample was transferred to a sample vial for liquid chromatography-mass spectrometry and proteomic analysis of the results.

[0118] Comparative Example 3

[0119] ​The treatment method of the single cell proteome sample in Comparative Example 3 is basically the same as that in Example 1, except that the method of adding protease to the pre-treatment sample for enzymolysis is different. Specifically, the added enzyme is Promega (product model number Cat#V5280), and overnight enzymolysis is performed. After centrifugation, the supernatant is taken to obtain the treated sample. The obtained treated sample is transferred to the sample bottle of the liquid chromatograph-mass spectrometer for direct injection, liquid chromatograph-mass spectrometry is performed, and proteomic analysis is performed on the determination results.

[0120] Comparative Example 4

[0121] The treatment method of the single cell proteome sample in Comparative Example 4 is basically the same as that in Example 1, except that the method of adding protease to the pre-treatment sample for enzymolysis is different. Specifically, the added enzyme is Thermo (product model number Cat#90057), and overnight enzymolysis is performed. After centrifugation, the supernatant is taken to obtain the treated sample. The obtained treated sample is transferred to the sample bottle of the liquid chromatograph-mass spectrometer for direct injection, liquid chromatograph-mass spectrometry is performed, and proteomic analysis is performed on the determination results.

[0122] Comparative Example 5

[0123] The treatment method of the single cell proteome sample in Comparative Example 5 is basically the same as that in Example 1, except that the immobilized enzyme reactor is an immobilized enzyme reactor that is not modified with polyethyleneimine and graphene oxide. The obtained treated sample is transferred to the sample bottle of the liquid chromatograph-mass spectrometer for direct injection, liquid chromatograph-mass spectrometry is performed, and proteomic analysis is performed on the determination results.

[0124] The results of Example 1 and Comparative Examples 2-5 are shown in Table Figure 4 Figure 4 Table A is the number of protein identifications (n=3) obtained using different enzymes; Figure 4 Table B is the number of peptide segment identifications (n=3) obtained using different enzymes; Figure 4 Table C is the protein quantification CV value distribution between 3 repeated uses of different enzymes; Figure 4 ​The proportion of peptides without missed cleavage (0), one missed cleavage (1), and two missed cleavages (2) in the identification results using different enzymes is shown in Table 4. The same sample was used to compare the results of the 0.5 h enzymatic reaction of the immobilized enzyme reactor in Example 1 with the results of the overnight enzymatic reaction of four commercial enzymes in solution in Comparative Examples 2-4 and the results of the 0.5 h enzymatic reaction of the immobilized enzyme reactor without polyethyleneimine and graphene oxide modification in Comparative Example 5. The results show that the number of proteins and peptides identified in the 0.5 h enzymatic reaction of the immobilized enzyme reactor modified with polyethyleneimine and graphene oxide is basically the same as that of the overnight enzymatic reaction of commercial enzymes in solution, and is better than that of the 0.5 h enzymatic reaction of the immobilized enzyme reactor without polyethyleneimine and graphene oxide modification Figure 4 (A and B in Table 4), indicating that the hydrophilic modification of polyethyleneimine reduces the non-specific adsorption of proteins or peptides; in addition, the results of the enzymatic reaction of the immobilized enzyme reactor modified with polyethyleneimine and graphene oxide have better quantitative reproducibility, indicating that the consistency and stability of the enzymatic reaction of the enzyme reactor are better Figure 4 (C); at the same time, the modification of graphene oxide improves the enzyme loading capacity, ensuring the completeness of the enzymatic reaction, and the proportion of peptides without missed cleavage is better than that of the overnight enzymatic reaction of four commercial enzymes in solution and the enzymatic reaction of the immobilized enzyme reactor without polyethyleneimine and graphene oxide modification for the same length of time, as shown in Figure 4 (D).

[0125] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present disclosure.

[0126] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope of the application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the protection scope of the present application. Therefore, the patent protection scope of the present application should be subject to the appended claims.

Claims

1. A method of processing a single-cell proteome sample, characterized by, The method comprises the following steps: a single cell sample is taken, 0.01w%-0.2w% non-ionic surfactant is added for ultrasonic lysis to obtain a pretreated sample; the pretreated sample is subjected to denaturation reduction and alkylation treatment to obtain an alkylated sample; an immobilized enzyme reactor is added to the alkylated sample for enzymolysis for 0.5h-4h, the supernatant is taken by centrifugation to obtain a treated sample; the preparation method of the immobilized enzyme reactor comprises the following steps: hydrophilic amino polymer microspheres are obtained by adding polyethyleneimine to the amino polymer microspheres after reaction; graphene oxide-polymer microspheres are obtained by adding graphene oxide to the hydrophilic amino polymer microspheres after reaction; a protease solution containing benzamidine is added to the graphene oxide-polymer microspheres, and the immobilized enzyme reactor is obtained after reaction; the electrostatic interaction between the protease and the graphene oxide in the graphene oxide-polyethyleneimine-amino polymer microspheres realizes the immobilization of the protease; the concentration of the polyethyleneimine is 3mg / mL-5mg / mL; the pore size of the amino polymer microspheres is 200Å-1000Å.

2. The treatment method according to claim 1, characterized in that, The non-ionic surfactant satisfies at least one of conditions (1)-(2): (1) the non-ionic surfactant is dodecyl-β-D-maltoside; (2) the addition amount of the non-ionic surfactant is 5μL-15μL.

3. The treatment method according to any one of claims 1 to 2, characterized in that, The step of subjecting the pretreated sample to denaturation reduction and alkylation comprises: 14μL-16μL of the pretreated sample is taken, 0.1μL-1μL of a reducing agent with a concentration of 25mM-100mM is added, and incubation is carried out at 56°C-100°C for 20min-60min to obtain a denatured and reduced protein sample; 0.1μL-1μL of an alkylation reagent with a concentration of 25mM-100mM is added to the denatured and reduced protein sample for alkylation treatment to obtain the alkylated sample.

4. The treatment method according to claim 3, characterized in that, The reducing agent comprises at least one of dithiothreitol or tricarboxyethyl phosphine.

5. The treatment method of claim 3, wherein The alkylation reagent comprises at least one of iodoacetamide, chloroacetamide, iodoacetic acid and N-ethylmaleimide.

6. The treatment method according to any one of claims 1 to 2, characterized in that, The particle size of the amino polymer microspheres is 1μm-10μm.

7. The treatment method according to any one of claims 1 to 2, characterized in that, The addition amount of the polyethyleneimine is 1mL-2mL per 150mg of the amino polymer microspheres.

8. The treatment method according to any one of claims 1 to 2, characterized in that, The graphene oxide satisfies at least one of conditions (3)-(4): (3) the concentration of the graphene oxide is 1mg / mL-3mg / mL; (4) the addition amount of the graphene oxide is 1mL-2mL per 150mg of the amino polymer microspheres.

9. The treatment method according to any one of claims 1 to 2, characterized in that, The protease comprises at least one of trypsin, pepsin, chymosin, carboxypeptidase or glycosidase.

10. A method of single-cell proteome analysis, characterized by, The treated sample is obtained by using the treatment method in any one of claims 1-9, and then chromatography-mass spectrometry analysis is carried out.

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