Cell lysis buffer and protein pretreatment method based on digital microfluidic system

Abstract

The invention relates to a cell lysis solution, which is prepared from the following active components: 0.08 to 0.2 percent of DDM (Dichloromethane), 35 to 45 mM of CAA (Ceamic Acid Acetic Acid), 5 to 15 mM of TCEP (Trichloroethylene Phosphate), 85 to 100 mM of TEAB (Tetraethyl Ammonium Bromide), 0 to 0.25 percent of Protease MAX and 0 to 0.25 percent of RapiGest SF (SF). The invention further provides a single-cell protein pretreatment method based on the digital microfluidic system, the cell lysis solution is used for lysis, the number of detectable proteins and the number of detectable peptide fragments can be high, and the method can be well used for single-cell proteomics analysis.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a cell lysate and protein pretreatment method based on a digital microfluidic system. Background Art

[0002] Cells are the basic units of structure and function of organisms. Due to factors such as the random expression of genetic information and changes in the microenvironment, heterogeneity between cells is ubiquitous. However, since the analysis based on population cells reflects the average value and cannot truly reflect the differences between each cell, it is necessary to analyze heterogeneity at the single-cell level. As the direct executor of biological functions in cells, proteins can sense and respond to external and internal stimuli through the dynamic changes of proteins and their post-translational modifications, thereby affecting the function and state of the entire organism. Therefore, single-cell proteomics has received more and more attention from researchers, especially in research directions such as cancer, immunity, development, and evolution. Revealing changes in cell heterogeneity has become a hot topic and opportunity. However, since the protein content of a single cell is very low and there are many types of proteins in the same cell, in order to discover the cell heterogeneity and functional characteristics in a complex population, it is necessary to analyze different types of proteins in a large number of cells. Therefore, single-cell proteomics has very high requirements for sensitivity and throughput.

[0003] At present, among the various methods for analyzing single-cell proteomics, the method based on liquid chromatography and mass spectrometry can obtain more comprehensive information about proteins in a single cell. Protein quantification is divided into relative quantification and absolute quantification. Although absolute quantification seems to be more ideal, it is more complicated and expensive, while relative quantification can often meet scientific research needs. Therefore, the non-targeted relative quantification method is more commonly used by comparing the chromatographic peaks of the same peptide with the same ionization efficiency in different samples. Non-targeted relative quantification is further divided into non-labeled quantification and labeled quantification technology. Non-labeled quantification refers to the direct delivery of cell enzymes into peptides to mass spectrometry detection. Compared with the labeling method, it does not require the use of expensive labeling reagents and the operation process is simple. The commonly used labeling reagent for labeling quantitative technology is Thermo Fisher's TMT label. The TMT labeling method is to react peptides with labeling reagents on the basis of non-standard samples and then mix them into mass spectrometry analysis. The system error is small, the repeatability is good, and the sensitivity is high.

[0004] The single-cell non-labeled quantitative sample pre-processing process on the microfluidic chip includes single-cell separation, cell lysis, protease cleavage, and sample output.

[0005] The single-cell TMT labeling quantitative sample pre-processing process includes single-cell separation, cell lysis, protease cleavage, TMT labeling, termination labeling, and sample output.

[0006] Both label-free quantitative and labeled quantitative techniques require single-cell isolation to obtain suitable samples. However, there are still huge challenges in how to process trace single-cell samples to obtain peptide solutions before mass spectrometry analysis, avoid introducing mass spectrometry-incompatible reagents, reduce sample volume, and effectively transfer peptides to the mass spectrometry detector.

[0007] The pre-treatment of single-cell proteomics samples generally includes cell lysis, reduction, alkylation, enzymatic hydrolysis, TMT labeling, termination and other processes. Currently, the most commonly used processing method is to use a cell sorter to sort single cells into a 96-well plate, and then manually add and transfer reagents to achieve pre-treatment for single-cell proteomics.

[0008] Commonly used methods for single-cell sorting technology are as follows: infinite dilution method, precision-controlled manual picking method, laser acquisition microdissection (LCM) technology, which are manually operated and have low efficiency; although technologies such as fluorescent activated cell sorting (FACS) and magnetic separation technology (MACS) can improve sorting efficiency, they are relatively expensive.

[0009] The single-cell protein sample pretreatment system currently has a product called CellenONE, which uses droplet imaging technology to sort single cells. The ultra-micro nanoliter reagent distribution system distributes the mixed solution to a standard well plate or chip (including lysate and enzyme reagent), incubates the reaction at a temperature control, and connects to the liquid phase injection system for direct injection. The final reaction volume of the system is about 200nL, and the throughput is 12 groups of 16 labels, 192 per chip.

[0010] Traditional single-cell sorting technology cannot achieve the high-throughput requirements of single-cell proteomics; due to its reliance on manual operations, it may cause errors and interference in the analysis results and cannot achieve the high-sensitivity requirements of single-cell proteomics; moreover, more reaction reagents are required, which will make the experimental cost higher, the reaction efficiency lower, and it is impossible to achieve parallel processing when processing the same batch of samples. There is a time difference between the addition of the first sample reagent and the last sample reagent, and the reaction time cannot be parallel and consistent.

[0011] The digital microfluidic system can directly manipulate cell suspensions, separate large droplets into small droplets, and thus separate single cells. It can also operate multiple droplets simultaneously, achieving the full process of high-throughput single-cell protein sample non-labeled quantification and TMT labeled quantification pre-treatment, with the advantages of full automation, process-based, and controllable operation. The digital microfluidic system technology is relatively new and has its own system technology requirements. In particular, how to obtain a higher number of detectable proteins and detectable peptides is one of the important requirements for the digital microfluidic system. Summary of the invention

[0012] Based on this, the purpose of the present invention is to provide a cell lysis solution based on a digital microfluidics system and a single-cell protein pretreatment method using the cell lysis solution. Through such a treatment method, the time required for single-cell protein pretreatment is greatly shortened, and a higher number of detectable proteins and detectable peptides can be obtained.

[0013] The above-mentioned objectives are achieved by means of the following technical solutions.

[0014] The first aspect of the present invention is to provide a cell lysis solution based on a digital microfluidic system, which includes the following active components: 0.08% to 0.2% DDM, 35 to 45 mM CAA, 5 to 15 mM TCEP, 85 to 100 mM TEAB, 0 to 0.25% ProteaseMAX, and 0 to 0.25% RapiGest SF.

[0015] In some embodiments thereof, it comprises the following active ingredients: 0.08%-0.15% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB, and 0.05-0.2% ProteaseMAX; or it comprises the following active ingredients: 0.08%-0.15% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB and 0.05-0.2% RapiGest SF.

[0016] In some preferred embodiments, it includes the following active ingredients: 0.09%-0.12% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB, 0.12-0.18% ProteaseMAX; or it includes the following active ingredients: 0.09%-0.12% DDM, 38-42mM CAA, 8-12mM TCEP, 90-100mM TEAB, and 0.12-0.18% RapiGest SF.

[0017] The second aspect of the present invention provides a single cell protein pretreatment method based on a digital microfluidics system, the method comprising the following steps:

[0018] S1. Prepare cell suspension;

[0019] S2. Inject the cell suspension into the digital microfluidic chip and tear it into single cell droplets;

[0020] S3. Inject any of the above lysates onto the digital microfluidic chip, tear the lysate into single droplets of lysate, merge with the single cell droplets, perform cell lysis, and obtain protein solution droplets;

[0021] S4. Injecting the enzymatic hydrolysate onto the digital microfluidic chip, tearing the enzymatic hydrolysate into single droplets, merging the droplets with the protein solution, and reacting to obtain peptide solution droplets;

[0022] S5. Mark or not mark as needed;

[0023] S6. If labeling is not required, the protein sample is directly obtained; if labeling is required, after the labeling reaction is completed, the labeling is terminated to obtain the protein sample.

[0024] In some of the embodiments, the cell solubility in the cell suspension injected in step S2 is 5×105 / mL to 8×105 / mL, and further preferably, the cell solubility in the injected cell suspension is 5.5×105 / mL to 6.5×105 / mL, and most preferably, the cell solubility in the injected cell suspension is 6x 105 / mL, at which time the single cell acquisition efficiency is the highest.

[0025] In some embodiments, the enzymatic hydrolysis solution includes 0.05 ug / uL to 0.1 ug / uL Trypsin / Lys-CMix and 90 mM to 110 mM TEAB. In this case, a larger number of detectable proteins and peptides are obtained.

[0026] In some of the embodiments, in step S6, the protein sample can be obtained by dipping with a gun tip or by capillary aspiration, and the capillary aspiration can obtain a higher number of detectable proteins and peptides.

[0027] In some of the embodiments, the tearing method is to tear the droplet into half with equal volume. By using this method to separate single droplets, single cell droplets or reaction liquid droplets can be formed quickly and controllably.

[0028] In some embodiments, the temperature for cell lysis in step S3 is 52° C.-60° C., preferably 54° C.-58° C.; and / or the lysis time is 13-17 min.

[0029] In some embodiments, the reaction temperature in step S4 is 35° C. to 39° C., and the reaction time is 1.5 h to 2.5 h.

[0030] The present invention has found in the research that in a digital microfluidic system, when cells are lysed by a suitable lysis solution (0.08% to 0.2% DDM + 35 to 45mM CAA + 5 to 15mM TCEP + 85 to 100mM TEAB + 0 to 0.25% ProteaseMAX + 0 to 0.25% RapiGest SF), the lysis is most complete and a higher number of detectable proteins and peptides can be obtained. The single-cell protein pretreatment method of the present invention greatly shortens the time required for single-cell protein pretreatment through the above-mentioned treatment method, and can realize the simultaneous reaction of all parallel samples, and obtain a high number of detectable proteins and peptides.

[0031] Furthermore, using 0.05ug / uL-0.1ug / uL Trypsin / Lys-C Mix and 90mM-110mM TEAB as the enzymatic solution can well complete the enzymatic cleavage reaction, thereby obtaining a higher number of detectable proteins and peptides, which can meet the high requirements of single-cell proteomics for sensitivity and throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the microfluidic core machine in the digital microfluidic system.

[0033] Figure 2 Schematic diagram of the imaging system in the digital microfluidic system.

[0034] Figure 3 This is the internal structure diagram of the temperature control system in the digital microfluidic system.

[0035] Figure 4 Schematic diagram of the structure of the digital microfluidic chip.

[0036] Figure 5 Schematic diagram of the digital microfluidic chip partitioning during the experiment.

[0037] Figure 6 The present invention is an experimental flow chart of a single cell protein pre-treatment method according to an embodiment of the present invention.

[0038] 7A to 7I Schematic diagram of each step of the single cell protein pretreatment method in Example 1.

[0039] Figure 8 Schematic diagram of the droplet being torn in half. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0042] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0043] Below are some definitions of terms.

[0044] PBS Phosphate buffer saline: phosphate buffer saline.

[0045] TMT Tandem mass tags: Tandem mass tags.

[0046] DDM n-Dodecyl-β-D-maltoside: n-Dodecyl β-D-maltoside, a non-ionic detergent, destroys membrane glycoproteins and phospholipid bilayers, releasing proteins from the cell membrane. At the same time, DDM can also stabilize membrane proteins and dissolve them in the solution.

[0047] CAA Chloracetamide: Chloroacetamide, a thiol protecting agent, can prevent repeated oxidation of disulfide bonds and the formation of new disulfide bonds.

[0048] TCEP triethylcarboxyurea: Reducing agent, reducing disulfide bonds, making the reduced protein easier to cleave and dissociate. TEAB triethylammonium bicarbonate buffer: pH 8.5±0.1, is a highly efficient protein cleavage agent under alkaline conditions, which can promote protein cleavage in an anhydrous environment and release ions in the ion trap, thereby increasing the ion signal intensity. In addition, the buffering properties of TEAB can help stabilize the structure of proteins, making it easier to maintain them in a high-quality state.

[0049] The single-cell protein pretreatment method of the present invention is based on the design of a chip using digital microfluidics technology, a fully automated process, almost no manual intervention is required, the experimental cycle is greatly shortened, samples can be quantitatively distributed, modular integration, flexible structure, and diverse functions can be adjusted according to demand; by using digital microfluidics technology to control samples and reagents, react and transfer to the upper cover detection point, the experimental environment is relatively independent and the risk of contamination is low. After process optimization, the method for single-cell proteomics pretreatment based on digital microfluidics technology, wherein the TMT-labeled single-cell proteomics sample pretreatment scheme includes the following experimental process: single cell separation, cell lysis, protease cleavage, TMT labeling, termination labeling, sample output, including the following steps:

[0050] (1) Preparation of cell suspension: HeLa cells were cultured in a 37°C incubator with a CO2 concentration of 5%. The concentrations of fetal bovine serum and penicillin-streptomycin in the culture medium were 10% and 1%, respectively. When the cell density increased to 80% of the entire culture dish, the cells were collected into a centrifuge tube and centrifuged at 100×g to discard the supernatant. The cells were washed three times with 1×PBS and then resuspended and counted.

[0051] (2) Single cell separation: inject the chip with the medium hexadecane, load the chip into the core machine, set the corresponding parameters, inject the cell suspension, introduce the tearing path to run, tear into single cell droplets, confirm the single cell in the single droplet, and calculate the single cell yield.

[0052] (3) Cell lysis: The lysis solution path is introduced, the cell lysis solution of the present invention is injected, and the lysis solution is run to split the lysis solution into single droplets in half, and the single cell droplets are merged to obtain single cell lysis droplets, and the temperature is controlled at 56° C. and the lysis is performed for 10 minutes. Through this step, the cell lysis, alkylation, and reduction reactions are completed together.

[0053] (4) Protease cleavage: introduce the enzymatic solution path, inject the enzymatic solution of the present invention, run, tear the enzymatic solution in half into single droplets, merge with the single cell lysis droplet, control the temperature to 37° C., and maintain for 2 h.

[0054] (5) TMT labeling: inject TMT reagent, mix with peptide solution, and incubate at room temperature for 1 h;

[0055] (6) Stop labeling: inject the stop reagent, mix with the reaction solution, and incubate at room temperature for 15 min;

[0056] (7) Sample output: Move the sample to the sample outlet, lower the temperature to 15°C, and solidify the medium hexadecane. Pipette the sample solution into a liquid phase injection vial and analyze it by mass spectrometry.

[0057] In the following embodiments, the digital microfluidic chip used is: a digital microfluidic A3B4 chip, on which a certain voltage is applied to perform droplet control and tearing.

[0058] In the following embodiments, the tearing method is tearing the droplet volume in half: by controlling the power on and power off of the electrodes on the chip, the droplet is controlled; the droplet will move toward the adjacent powered electrode and away from the powered off electrode; in this way, the droplets of equal volume are controlled to move in the opposite direction and tear in half, and each group obtains 2 droplets after tearing in half; according to the above method, the droplets after tearing in half are again subjected to a second round of tearing in half to obtain 4 droplets, and then the 4 droplets are subjected to a third round of tearing in half to obtain 8 droplets. Figure 8 , where the normal framed square represents the unpowered electrode, the bold black rounded frame represents the droplet, and the oblique shadow square represents the powered electrode. By controlling the power on and off of the electrode, the droplet can be controlled and torn in half to meet the experimental needs.

[0059] In the following examples, the experimental reagents used are as follows:

[0060]

[0061]

[0062] Take one of the lysis buffer configurations as an example:

[0063] First prepare 10× stock solution:

[0064] 1% DDM: weigh 100 mg, dissolve in 1 mL 100 mM TEAB, and mix thoroughly;

[0065] 100mM TCEP: weigh 28.6mg, dissolve in 1mL 100mM TEAB, mix thoroughly;

[0066] 400mM CAA: weigh 37.4mg, dissolve in 1mL 100mM TEAB, mix thoroughly;

[0067] 1% RapiGest SF: 1 mg of lyophilized powder, dissolved in 100 uL 100 mM TEAB, mix thoroughly;

[0068] 1mL lysis buffer 0.1% DDM + 40mM CAA + 10mM TCEP + 100mM TEAB + 0.15% Rap iGest SF preparation: 100uL 1% DDM + 100uL 100mM TCEP + 100uL 400mM CAA + 150uL 1% Rap iGest SF + 550uL 100mM TEAB, mix thoroughly to obtain the result.

[0069] The present invention is further described in detail below with reference to specific embodiments.

[0070] Example 1

[0071] Since the processing steps of non-labeled samples are the same as the first half of the TMT labeled sample preparation, the TMT labeled sample is shown on half of the chip as an example. Figure 6 , the specific operations are as follows:

[0072] 1. Collect the normally cultured cells in a 15 mL centrifuge tube and centrifuge at 100 × g for 5 minutes to remove the culture medium; wash the cell pellet three times with 1 × PBS buffer, and then count to obtain a cell suspension of a certain concentration.

[0073] 2. Use a 200μL pipette to tilt 45° and inject hexadecane into the oil filling hole on the upper cover corresponding to the digital microfluidic chip to provide an environment for the movement of single-cell droplets, and load the chip into the core machine.

[0074] 3. Turn on the core machine and open the microfluidic system interface of the host computer. The internal structure of the system is as follows: Figure 1 As shown, set the corresponding parameters, such as the corresponding row and column voltages, row hold time, frame delay time, etc.; open the camera interface, the internal structure of the imaging system is as follows Figure 2 As shown, it includes high-power and low-power microscopes and a light source, and the position calibration of the chip is performed under the high-power microscope.

[0075] 4. Select stage 1 in the microfluidic system, inject 512nL of cell suspension and cell lysate (0.08%~0.2% DDM+35~45mM CAA+5~15mM TCEP+85~100mM TEAB+0~0.25% ProteaseMAX+0~0.25% RapiGest SF) into different injection holes respectively, tear them into single droplets respectively, and run. The system moves to the specified position according to the edited path and completes the tearing. The movement of droplets and cell lysate can be observed under a low-power microscope.

[0076] 5. Execute stage 2, screen single-cell droplets, and perform single-cell identification. The system automatically switches to the high-power microscope, identifies single-cell droplets, and automatically moves to the bottom of the successfully torn lysate according to the preset path. Non-single-cell droplets and excess lysate are automatically moved to the waste liquid area.

[0077] 6. Open the temperature control interface. The internal structure of the temperature control system is as follows: Figure 3As shown. Set the temperature to 56°C, the time to 15 min, perform the merging function, and mix at the same time, that is, merge the single droplet of lysate with the single cell droplet to obtain a single cell lysate mixed droplet, perform cell lysis, and cell membrane rupture can be seen under the microscope, and cell lysis, alkylation and reduction reactions are completed at the same time. Figure 5 and Fig. 7A .like Figure 5 As shown, the lysis agent is in the middle area of ​​the chip, and the cells and other reagents are torn into single droplets and then go to the lysis liquid area to mix and complete each step of the reaction.

[0078] 7. After the cleavage reaction is completed, inject 512nL of enzyme digestion solution (0.05-0.15ug / uL Trypsin / Lys-C Mix + 100mM TEAB) from the injection hole with a 10μL pipette, run the automatic path to tear the enzyme digestion solution into a single droplet, perform automatic identification, screen the successfully torn single droplet of enzyme digestion solution, and move it to the bottom of the protein solution droplet formed after the cell lysis of the above single cell lysis solution mixed droplet until all enzyme digestion solutions are run. Figure 7B .

[0079] 8. Open the temperature control interface, reset the temperature to 37°C, maintain it for 2 hours, then combine the corresponding protein droplets with the enzyme digestion solution, mix them at the same time, and react to obtain the peptide solution. Figure 7C .

[0080] 9. After the enzyme digestion reaction is completed, use the automatic sample injection device to inject 16 TMT reagents into the sample injection hole, perform droplet injection and tearing, and move the successfully torn TMT droplets to the bottom of the peptide solution after the system recognizes them until all TMT reagents are run. Fig.7D .

[0081] 10. The TMT reagent droplets and the single-cell polypeptide solution droplets were fused and mixed, and incubated at room temperature for 1 hour. Fig. 7E .

[0082] 11. After the TMT reaction is completed, 0.45-0.55% hydroxylamine solution is injected, and liquid drop injection and tearing are performed. After the system recognizes that the successfully torn hydroxylamine solution is moved to the bottom of the above liquid, until all hydroxylamine reagents are run.

[0083] The TMT labeling termination reaction experimental conditions are room temperature for 15 minutes, and the hydroxylamine solution is combined with the above reaction solution and mixed. Figure 7F and Figure 7G .

[0084] 12 After the termination reaction is completed, the automatic mixing output path is executed, and each set of samples contains 16 TMT-labeled single cell peptide solutions. Figure 7H .

[0085] Move the sample to the sample outlet, open the temperature control interface, set the temperature to 15°C, and solidify the medium hexadecane. Use the automatic sample outlet device to take out the sample into the liquid phase vial, dilute it with 0.1% formic acid to the minimum injection volume, and then analyze it on the machine. Fig.7I .

[0086] The above experimental process only takes half of the area as an example, and 256 single-cell reactions can be performed simultaneously.

[0087] Each example was tested in triplicate.

[0088] Experimental group 1: Lysis solution was 0.1% DDM + 40mM CAA + 10mM TCEP + 100mM TEAB + 0.15% ProteaseMAX, enzymatic agent was 0.1ug / uL Trypsin / Lys-C Mix + 100mM TEAB, capillary aspiration method, according to the above method, different concentrations of cell suspension were loaded on the slice, and the single cell yield was calculated. The results are shown in Table 1.

[0089] Table 1.

[0090]

[0091]

[0092] The results showed that when the concentration of the cell suspension on the slide was 6×10^5 cells / mL, the single cell yield was higher. When it was less than 4×10^5 or greater than 8×10^5 cells / mL, the single cell yield was lower. After multiple experimental analyses, the optimal value was 4-8×10^5 cells / mL.

[0093] Experimental Group 2: Investigate the lysing agents with different concentrations (the preparation method is to weigh each component according to the concentration and dissolve it in TEAB), with the cell suspension concentration on the slice being 6×10^5 cells / mL, 0.05ug / uL Trypsin / Lys-C Mix+100mM TEAB as the enzymatic agent, and output the sample through the capillary sampling device to detect the number of proteins and peptides. The results are shown in Table 2.

[0094] Table 2.

[0095]

[0096]

[0097] The results showed that the lysis reagent 0.1% DDM + 40mM CAA + 10mM TCEP + 0 / 0.15% RapiGest SF / 0.15% RapiGest SF had no significant difference in sample pretreatment, but lowering the DDM concentration to 0.01%, increasing the CAA concentration to 200mM or increasing the TCEP concentration to 80mM may affect the number of protein and peptide identifications due to incomplete cell lysis. Although increasing the RapiGest SF concentration had no effect on the number of protein identifications, high concentrations are not recommended considering the reagent cost and the impact on the mass spectrometry end ion source.

[0098] Experimental group 3: different concentrations of enzymatic hydrolysates were investigated, with the cell suspension concentration of 6×10^5 cells / mL, 0.1% DDM+40mM CAA+10mM TCEP+100mM TEAB+0.15% RapiGest SF as the lysing agent, and the sample was output through the capillary sampling device to detect the number of proteins and peptides. The results are shown in Table 3.

[0099]

[0100] The experimental results showed that the enzymatic hydrolysate 0.05 / 0.1ug / uL Trypsin / Lys-C Mix had no significant difference in sample pretreatment; however, when the concentration of the enzymatic hydrolysate was reduced to 0.005ug / uL, the number of protein and peptide identifications may be affected due to insufficient enzymatic hydrolysis.

[0101] Experimental group 4: In the pre-treatment step, the cell suspension concentration was 6×10^5 cells / mL, 0.1% DDM+40mMCAA+10mM TCEP+100mM TEAB+0.15% RapiGest SF was used as the lysis solution, and 0.05ug / uL Trypsin / Lys-CMix+100mM TEAB was used as the enzymatic agent. The effects of different sampling methods on the number of detected proteins and peptides were investigated. The results are shown in Table 4.

[0102] Example Sampling method Detectable protein quantity Number of detectable peptides 18 Tip of gun 970±6 4450±101 19 Capillary aspiration 1293±79 8974±648

[0103] The experimental results show that capillary sampling is better than pipette sampling.

[0104] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A cell lysis solution for a digital microfluidic system, characterized in that: It includes the following active ingredients: 0.08%-0.2% DDM, 35-45mM CAA, 5-15mM TCEP, 85-100mM TEAB, 0-0.25% ProteaseMAX, and 0-0.25% RapiGest SF.

2. The cell lysis solution according to claim 1, characterized in that It includes the following active ingredients: 0.08% to 0.15% DDM, 38 to 42 mM CAA, 8 to 12 mM TCEP, 90 to 100 mM TEAB, and 0.05 to 0.2% ProteaseMAX; or it includes the following active ingredients: 0.08%-0.15% DDM, 38-42 mM CAA, 8-12 mM TCEP, 90-100 mM TEAB and 0.05-0.2% RapiGest SF.

3. The cell lysis solution according to claim 1, characterized in that It includes the following active components: 0.09% to 0.12% DDM, 38 to 42 mM CAA, 8 to 12 mM TCEP, 90 to 100 mM TEAB, and 0.12 to 0.18% ProteaseMAX; or it includes the following active components: 0.09% to 0.12% DDM, 38 to 42 mM CAA, 8 to 12 mM TCEP, 90 to 100 mM TEAB, and 0.12 to 0.18% RapiGest SF.

4. A single cell protein pretreatment method based on a digital microfluidics system, characterized in that: The following steps are involved: S1. Prepare cell suspension; S2. Inject the cell suspension into the digital microfluidic chip and tear it into single cell droplets; S3. injecting the lysate according to any one of claims 1 to 3 onto the digital microfluidic chip, tearing the lysate into single droplets of the lysate, merging the single droplets with the single cell droplets, performing cell lysis, and obtaining protein solution droplets; S4. Injecting the enzymatic hydrolysate onto the digital microfluidic chip, tearing the enzymatic hydrolysate into single droplets, merging the droplets with the protein solution, and reacting to obtain a peptide solution; S5. Mark or not mark as needed; S6. Obtain protein samples.

5. The single cell protein pretreatment method based on the digital microfluidic system according to claim 4, characterized in that: The cell concentration of the cell suspension injected in step S2 is 5×10 5 / mL~8×10 5 Pieces / mL.

6. The single cell protein pretreatment method based on the digital microfluidic system according to claim 5, characterized in that: The cell concentration of the cell suspension injected in step S2 is 5.5×10 5 / mL~6.5×10 5 Pieces / mL.

7. The single cell protein pretreatment method based on the digital microfluidic system according to claim 4, characterized in that: The enzymatic hydrolysis solution includes 0.05ug / uL-0.1ug / uL Trypsin / Lys-C Mix and 90mM-110mM TEAB.

8. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 4 to 7, characterized in that: In step S6, a protein sample is obtained by capillary aspiration.

9. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 4 to 7, characterized in that: The tearing method is to tear the droplet into half with equal volume.

10. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 4 to 7, characterized in that: The temperature for cell lysis in step S3 is 52° C. to 60° C., preferably 54° C. to 58° C.; and / or the lysis time is 13 to 17 min.

11. The single cell protein pretreatment method based on a digital microfluidic system according to any one of claims 4 to 7, characterized in that: The reaction temperature in step S4 is 35° C. to 39° C., and the reaction time is 1.5 h to 2.5 h.

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