A Method for Pretreatment of Cell Lysate and Protein Based on Digital Microfluidic System

Abstract

This invention relates to cell lysis buffers comprising the following active components: 0.08%–0.2% DDM, 35–45 mM CAA, 5–15 mM TCEP, 85–100 mM TEAB, 0–0.25% ProteaseMAX, and 0–0.25% RapiGest SF. This invention also provides a single-cell protein pretreatment method based on a digital microfluidic system. Using the aforementioned cell lysis buffer for lysis yields high numbers of detectable proteins and peptides, making it well-suited for single-cell proteomics analysis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for pretreatment of cell lysate and protein based on a digital microfluidic system. Background Technology

[0002] Cells are the basic structural and functional units of organisms. Due to the randomness of genetic information expression and changes in the microenvironment, heterogeneity is prevalent among cells. However, since population-based cell analysis reflects average values ​​and cannot truly reflect the differences between individual cells, it is necessary to analyze heterogeneity at the single-cell level. Proteins, as the direct executors of intracellular biological functions, can sense and respond to external and internal stimuli through dynamic changes in proteins and their post-translational modifications, thereby influencing the function and state of the entire organism. Therefore, single-cell proteomics has received increasing attention from researchers, especially in areas such as cancer, immunity, development, and evolution, where revealing changes in cellular heterogeneity has become a current hot topic and opportunity. However, because the protein content of a single cell is very low, and there are many different types of proteins within the same cell, discovering cellular heterogeneity and functional characteristics in complex populations requires analyzing different types of proteins in a large number of cells. Therefore, single-cell proteomics has very high requirements for sensitivity and throughput.

[0003] Currently, among various methods for analyzing single-cell proteomics, methods based on liquid chromatography-mass spectrometry (LC-MS) can provide more comprehensive information on proteins within a single cell. Protein quantification is divided into relative quantification and absolute quantification. Although absolute quantification seems more ideal, it is more complex and expensive, while relative quantification often meets research needs. Therefore, non-targeted relative quantification methods, which compare chromatographic peaks of the same peptide with the same ionization efficiency in different samples, are more commonly used. Non-targeted relative quantification is further divided into label-free quantification and label-based quantification techniques. Label-free quantification involves directly transmitting peptides from cell enzyme digestion to mass spectrometry for detection. Compared to labeling methods, it eliminates the need for expensive labeling reagents and simplifies the process. The commonly used labeling reagent for labeling quantification is Thermo Fisher Scientific's TMT label. The TMT labeling method involves reacting peptides with a labeling reagent in a non-labeled sample before mixing them into the mass spectrometer for analysis. This method exhibits smaller systematic errors, better repeatability, and higher sensitivity.

[0004] The preprocessing flow for single-cell label-free quantitative samples on a microfluidic chip includes single-cell isolation, cell lysis, protease digestion, and sample output.

[0005] The preprocessing steps for single-cell TMT labeling quantitative samples include single-cell isolation, cell lysis, protease digestion, TMT labeling, stop labeling, and sample output.

[0006] Both label-free and label-based quantitative techniques require single-cell isolation to obtain suitable samples. However, there are still significant challenges in processing micro-single-cell samples to obtain peptide solutions before mass spectrometry analysis, without introducing mass spectrometry-incompatible reagents, reducing sample volume, and effectively transferring peptides to the mass spectrometry detector.

[0007] Pretreatment of single-cell proteomics samples generally includes cell lysis, reduction, alkylation, enzymatic digestion, TMT labeling, and termination. The most common method is to use a cell sorter to sort single cells into 96-well plates, and then manually add and transfer reagents to achieve pretreatment for single-cell proteomics.

[0008] The commonly used methods for single-cell sorting include: infinite dilution method, precision control manual pick-up method, and laser harvesting microdissection (LCM) technology. These are manual operations with low efficiency. Fluorescent cell activation sorting (FACS) and magnetic separation technology (MACS) can improve sorting efficiency, but they are more expensive.

[0009] CellenONE, a single-cell protein sample pretreatment system, is currently available. It uses droplet imaging technology for single-cell sorting, and an ultra-micro nanoliter reagent dispensing system to distribute the mixture (including lysis buffer and enzyme reagent) onto standard well plates or chips. The reaction is then carried out under temperature control and linked to a liquid chromatography-mass spectrometry (LC-MS) system for direct sample injection. The final reaction volume of this system is approximately 200 nL, with a throughput of 12 groups of 16 standards, or 192 cells / chip.

[0010] Traditional single-cell sorting techniques cannot meet the high-throughput requirements of single-cell proteomics. Due to their reliance on manual operation, they may cause errors and interference in the analysis results, failing to meet the high sensitivity requirements of single-cell proteomics. Moreover, they require more reaction reagents, resulting in higher experimental costs, lower reaction efficiency, and the inability to achieve parallel processing when processing the same batch of samples. There is a time difference between the addition and mixing of the reagents for the first sample and the last sample, and the reaction times cannot be parallel and consistent.

[0011] Digital microfluidic systems can directly manipulate cell suspensions, separating large droplets into smaller ones for single-cell isolation. Simultaneously, multiple droplets can be manipulated, enabling a complete pretreatment process for high-throughput label-free quantification and TMT-labeled quantification of single-cell protein samples. This offers advantages such as full automation, streamlined processes, and controllable operation. However, digital microfluidic technology is relatively new and has its own system requirements, particularly in achieving high levels of detectable proteins and peptides. Summary of the Invention

[0012] Based on this, the purpose of this invention is to provide a cell lysis buffer based on a digital microfluidic system and a method for pre-treating single-cell proteins using the cell lysis buffer. This method greatly shortens the time required for single-cell protein pre-treatment and can obtain a high number of detectable proteins and peptides.

[0013] The following technical solutions are used to achieve the above objectives.

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

[0015] In some embodiments, it comprises 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% ProteaseMAX; or comprises 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.

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

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

[0018] S1. Prepare cell suspension;

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

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

[0021] S4. Inject the enzymatic hydrolysate into the digital microfluidic chip, tear the enzymatic hydrolysate into single droplets, combine them with the protein solution droplets, and react to obtain peptide solution droplets;

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

[0023] S6. If no labeling is required, obtain the protein sample directly; if labeling is required, stop the labeling reaction after it is completed and obtain the protein sample.

[0024] In some embodiments, the cell concentration in the injected cell suspension in step S2 is 5×10⁵ cells / mL to -8×10⁵ cells / mL. More preferably, the cell concentration in the injected cell suspension is 5.5×10⁵ cells / mL to 6.5×10⁵ cells / mL. Most preferably, the cell concentration in the injected cell suspension is 6×10⁵ cells / mL, at which point the single-cell acquisition efficiency is the highest.

[0025] In some of these embodiments, the enzymatic hydrolysate comprises 0.05 ug / uL to 0.1 ug / uL Trypsin / Lys-CMix and 90 mM to 110 mM TEAB, in which case a larger number of detectable proteins and peptides are obtained.

[0026] In some embodiments, protein samples can be obtained in step S6 by dipping the tip of a pipette or by aspirating through a capillary tube, with capillary aspiration yielding a higher number of detectable proteins and peptides.

[0027] In some embodiments, the tearing is performed by tearing the droplet in half of equal volume. Using this method for single droplet separation, single-cell droplets or reaction liquid droplets can be formed quickly and controllably.

[0028] In some of these embodiments, the cell lysis temperature 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 of these embodiments, the reaction in step S4 is carried out at a temperature of 35°C to 39°C for a time of 1.5h to 2.5h.

[0030] This invention discovered that in digital microfluidic systems, cell lysis is most complete and yields a high number of detectable proteins and peptides when cells are lysed using a suitable lysis buffer (0.08%–0.2% DDM + 35–45 mM CAA + 5–15 mM TCEP + 85–100 mM TEAB + 0–0.25% ProteaseMAX + 0–0.25% RapiGest SF). The single-cell protein pretreatment method described in this invention significantly reduces the time required for single-cell protein pretreatment, allows for simultaneous reaction of all parallel samples, and achieves 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 digestion solution can effectively complete the enzymatic digestion reaction, thereby obtaining a higher number of detectable proteins and peptides, which can meet the high sensitivity and throughput requirements of single-cell proteomics. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the microfluidic core machine in a digital microfluidic system.

[0033] Figure 2 This is a schematic diagram of the imaging system in a digital microfluidic system.

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

[0035] Figure 4 This is a schematic diagram of the structure of a digital microfluidic chip.

[0036] Figure 5 This is a schematic diagram of the partitioning of the digital microfluidic chip during the experiment.

[0037] Figure 6 This is an experimental flowchart of a single-cell protein pretreatment method according to an embodiment of the present invention.

[0038] Figures 7A to 7I This is a schematic diagram of each step in the single-cell protein pretreatment method in Example 1.

[0039] Figure 8 This is a schematic diagram of the tearing mechanism of a droplet in half. Detailed Implementation

[0040] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0041] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0042] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0043] The following are some terminology definitions.

[0044] PBS Phosphate buffer saline: phosphate buffer.

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

[0046] DDM n-Dodecyl-β-D-maltoside: a nonionic detergent that disrupts the membrane glycoprotein and phospholipid bilayer, releasing proteins from the cell membrane. Simultaneously, DDM also stabilizes membrane proteins and dissolves them in solution.

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

[0048] TCEP (triethylcarboxyurea): A reducing agent that reduces disulfide bonds, making the reduced protein easier to cleave and dissociate. TEAB (triethylammonium bicarbonate buffer): pH 8.5±0.1, a highly efficient protein cleavage agent under alkaline conditions. It promotes protein cleavage in an anhydrous environment and releases ions in an ion trap, thereby increasing the ion signal intensity. Additionally, the buffering properties of TEAB help stabilize protein structure, making it easier to maintain a high-quality protein state.

[0049] The single-cell protein pretreatment method described in this invention is based on a chip design using digital microfluidic technology. It features a fully automated process with almost no manual intervention, significantly shortening the experimental cycle. It allows for quantitative sample allocation, modular integration, flexible structure, and diverse functions, and can be adjusted according to needs. By using digital microfluidic technology to control samples and reagents, the reaction process, and transfer to the detection point on the cover, the experimental environment is relatively independent, resulting in a low risk of contamination. After process optimization, the single-cell proteomics pretreatment method based on digital microfluidic technology includes the following experimental procedure: single-cell isolation, cell lysis, protease digestion, TMT labeling, termination labeling, and sample output, comprising the following steps:

[0050] (1) Preparation of cell suspension: HeLa cells were cultured in a 37°C incubator with 5% CO2. The concentrations of fetal bovine serum and penicillin-streptomycin antibiotics 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 centrifuge tubes, centrifuged at 100×g and the supernatant was discarded. The cells were washed three times with 1×PBS and then resuspended and counted.

[0051] (2) Single-cell separation: The chip is injected with hexadecane, loaded into the core machine, and the corresponding parameters are set. Cell suspension is injected, and the tearing path is introduced to run. After tearing into single-cell droplets, single cells are identified in the single droplets, and the single-cell yield is calculated.

[0052] (3) Cell lysis: The cell lysis buffer described in this invention is injected into the lysis buffer pathway and run. The lysis buffer is torn in half into single droplets, which are then combined with single-cell droplets to obtain single-cell lysis droplets. The temperature is controlled at 56°C, and lysis is carried out for 10 minutes. Through this step, cell lysis, alkylation, and reduction reactions are completed simultaneously.

[0053] (4) Protein digestion: The enzyme digestion solution is introduced into the pathway, and the enzyme digestion solution described in this invention is injected. The process is run to tear the enzyme digestion solution in half into single droplets, which are then combined with the single cell lysis droplets. The temperature is controlled at 37°C and maintained for 2 hours.

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

[0055] (6) Termination marking: Inject the termination reagent, mix it 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 allow the hexadecane medium to solidify. Aspirate the sample solution into a liquid chromatography vial for mass spectrometry analysis.

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

[0058] In the following embodiments, the tearing method is droplet volume halving: the droplets are manipulated by controlling the energization and de-energization of electrodes on the control chip; the droplets move towards the adjacent energized electrode and away from the de-energized electrode; in this way, equal-volume droplets are controlled to move in the opposite direction to perform equal-volume halving, resulting in 2 droplets after halving in each group; following the above method, the halved droplets are subjected to a second round of halving to obtain 4 droplets, and then these 4 droplets are subjected to a third round of halving to obtain 8 droplets. See also Figure 8 In this diagram, a square with a plain border represents an electrode without power, a thick black rounded-corner border represents a droplet, and a shaded square with a diagonal line represents an electrode with power. By controlling the power on and off of the electrodes, droplet control and halving are achieved to meet experimental requirements.

[0059] The experimental reagents used in the following examples are as follows:

[0060]

[0061]

[0062] Taking the preparation of one type of lysis buffer as an example:

[0063] First, prepare 10× stock solution:

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

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

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

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

[0068] Prepare 1 mL of lysis buffer by mixing 0.1% DDM + 40 mM CAA + 10 mM TCEP + 100 mM TEAB + 0.15% Rap iGest SF. Then mix 100 μL of 1% DDM + 100 μL of 100 mM TCEP + 100 μL of 400 mM CAA + 150 μL of 1% Rap iGest SF + 550 μL of 100 mM TEAB thoroughly.

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

[0070] Example 1

[0071] Since the processing steps for non-standard samples are the same as the first half of the processing steps for preparing TMT labeled samples, we will use half of the chip to illustrate the TMT labeled sample as an example. (See also...) Figure 6 The specific steps are as follows:

[0072] 1. Collect normally cultured cells into 15 mL centrifuge tubes 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 the cells to obtain a cell suspension of a certain concentration.

[0073] 2. Using a 200μL pipette tilted at 45°, inject hexadecane into the oil injection hole corresponding to the top cover of the digital microfluidic chip to provide an environment for the movement of single-cell droplets, and then load the chip into the core unit.

[0074] 3. Power on the core machine and open the microfluidic system interface on 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, and the internal structure of the imaging system is as follows. Figure 2 As shown, it includes high- and low-magnification microscopes and a light source, while chip positioning is calibrated under the high-magnification microscope.

[0075] 4. In the microfluidic system, select stage 1 and inject 512 nL of cell suspension and cell lysis buffer (0.08%–0.2% DDM + 35–45 mM CAA + 5–15 mM TCEP + 85–100 mM TEAB + 0–0.25% ProteaseMAX + 0–0.25% RapiGest SF) into different injection wells to tear them into single droplets. Run the system, and the system will move to the designated position according to the edited path and complete the tearing. The movement of the droplets and cell lysis buffer can be observed under low magnification.

[0076] 5. Execution Phase 2: Screening single-cell droplets and performing single-cell identification. The system automatically switches to high magnification, identifies single-cell droplets, and automatically moves them along a preset path to the area below the successfully ruptured lysate. 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. The temperature was set to 56℃, the time to 15 minutes, and the merging function was executed, simultaneously mixing the single droplets of lysis buffer with the single-cell droplets to obtain a mixed droplet of single-cell lysis buffer. Cell lysis was then performed, and under a microscope, cell membrane rupture was observed. Simultaneously, cell lysis, alkylation, and reduction reactions were completed. (See also...) Figure 5 and Figure 7A .like Figure 5 As shown, the lysis agent is located in the middle region of the chip. After the cells and other reagents are torn into single droplets, they move to the lysis buffer region to mix and complete each step of the reaction.

[0078] 7. After the lysis reaction is complete, inject 512 nL of enzyme digestion buffer (0.05–0.15 μg / μL Trypsin / Lys-C Mix + 100 mM TEAB) into the injection well using a 10 μL pipette. Run the automatic path to tear the enzyme digestion buffer into single droplets, automatically identify and select successfully torn single droplets, and move them below the protein solution droplets formed after cell lysis of the single-cell lysis buffer mixture. Repeat this process until all enzyme digestion buffers have been processed. See also Figure 7B .

[0079] 8. Open the temperature control interface, reset the temperature to 37℃, and maintain the temperature for 2 hours. Then, simultaneously combine and mix the corresponding protein droplets with the enzyme digestion solution to obtain the peptide solution. See [link to relevant documentation]. Figure 7C .

[0080] 9. After the enzyme digestion reaction is complete, inject 16 standard TMT reagents into each well using an automated injection device. Perform droplet injection and tearing. The system identifies successfully torn TMT droplets and moves them below the peptide solution until all TMT reagents have been processed. See also Figure 7D .

[0081] 10. Fuse and mix the TMT reagent droplets with the single-cell polypeptide solution droplets, then incubate at room temperature for 1 hour. See also Figure 7E .

[0082] 11. After the TMT reaction is completed, inject 0.45-0.55% hydroxylamine solution, perform droplet injection and tearing, and the system will identify the successfully torn hydroxylamine solution and move it to the bottom of the above liquid until all hydroxylamine reagents have been processed.

[0083] The experimental conditions for stopping the TMT-labeled reaction were: room temperature for 15 minutes, and the hydroxylamine solution was mixed with the above reaction solution. (See reference...) Figure 7F and Figure 7G .

[0084] 12. After the reaction is terminated, the automatic mixing output path is executed, with each sample containing 16 TMT-labeled single-cell peptide solutions. See also Figure 7H .

[0085] Transfer the sample to the sample outlet, open the temperature control interface, set the temperature to 15℃, and allow the hexadecane medium to solidify. Using the automatic sample dispensing device, remove the sample into a liquid chromatography vial, dilute it to the minimum injection volume with 0.1% formic acid, and then analyze it. See [link to relevant documentation]. Figure 7I .

[0086] The above experimental procedure uses only half of the region as an example, which can simultaneously perform 256 single-cell reactions.

[0087] Each embodiment was tested in three parallel replicates.

[0088] Experimental Group 1: The lysis buffer was 0.1% DDM + 40mM CAA + 10mM TCEP + 100mM TEAB + 0.15% ProteaseMAX; the enzyme digestion agent was 0.1ug / uL Trypsin / Lys-C Mix + 100mM TEAB. Cells were aspirated using capillary aspiration and loaded onto slides with different concentrations of cell suspension according to the above method. 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 cell suspension on the slide was 6×10^5 cells / mL, the single cell yield was high. When the concentration was less than 4×10^5 or greater than 8×10^5 cells / mL, the single cell yield was even lower. After multiple experimental analyses, the optimal concentration was 4-8×10^5 cells / mL.

[0093] Experimental Group 2: Different concentrations of lysis agents were investigated (prepared by weighing each component according to its concentration and dissolving it in TEAB). The cell suspension was loaded onto slides at a concentration of 6 × 10^5 cells / mL, and 0.05 μg / μL Trypsin / Lys-C Mix + 100 mM TEAB was used as the enzymatic digestion agent. Samples were output via a capillary sampling device for protein and peptide quantity analysis. Results are shown in Table 2.

[0094] Table 2.

[0095]

[0096]

[0097] The results showed that the lysis reagents 0.1% DDM + 40mM CAA + 10mM TCEP + 0 / 0.15% RapiGest SF / 0.15% RapiGest SF had no significant difference in sample pretreatment. However, decreasing the DDM concentration to 0.01%, increasing the CAA concentration to 200mM, or increasing the TCEP concentration to 80mM may result in incomplete cell lysis, thus affecting the number of proteins and peptides identified. Although increasing the RapiGest SF concentration did not affect the number of proteins identified, high concentrations are not recommended considering reagent costs and the impact on the ion source at the mass spectrometer end.

[0098] Experimental Group 3: Different concentrations of enzymatic digestion agents were investigated. Cell suspension was loaded onto slides at a concentration of 6 × 10^5 cells / mL. The lysis agents were 0.1% DDM + 40 mM CAA + 10 mM TCEP + 100 mM TEAB + 0.15% RapiGest SF. Samples were extracted using a capillary sampling device for protein and peptide quantity analysis. Results are shown in Table 3.

[0099]

[0100] Experimental results showed that the enzyme digestion agent 0.05 / 0.1ug / uL Trypsin / Lys-C Mix had no significant difference in sample pretreatment; however, when the concentration of the enzyme digestion solution was reduced to 0.005ug / uL, the number of proteins and peptides identified may be affected due to insufficient enzyme digestion.

[0101] Experimental Group 4: In the pretreatment step, the cell suspension concentration was 6 × 10^5 cells / mL. The lysis buffer was 0.1% DDM + 40 mM MCA + 10 mM TCEP + 100 mM TEAB + 0.15% RapiGest SF, and the enzyme digestion agent was 0.05 μg / μL Trypsin / Lys-CMix + 100 mM TEAB. 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 Dipping the tip of the gun 970±6 4450±101 19 Capillary absorption 1293±79 8974±648

[0103] Experimental results show that capillary sampling is superior to pipette sampling.

[0104] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A single-cell protein pretreatment method based on a digital microfluidic system, characterized in that, Includes the following steps: S1. Prepare cell suspension; S2. The cell suspension is injected into a digital microfluidic chip and torn into single-cell droplets; the cell concentration in the injected cell suspension is 5 × 10⁻⁶. 5 8 × 10⁸ cells / mL ~ 8 × 10⁻⁸ 5 cells / mL; S3. Inject the lysis buffer onto the digital microfluidic chip, tear the lysis buffer into single droplets, merge them with single cell droplets, perform cell lysis, and obtain protein solution droplets; S4. Inject the enzymatic hydrolysate into the digital microfluidic chip, tear the enzymatic hydrolysate into single droplets, combine them with the protein solution droplets, and react to obtain a peptide solution; S5. Mark or not mark as needed; S6. Obtain protein samples; The cell lysis buffer comprises the following active components: 0.08%~0.15% DDM, 38~42mM CAA, 8~12mM TCEP, 90~100mM TEAB, and 0.05~0.2% ProteaseMAX; or comprising the following active ingredients: 0.08%~0.15% DDM, 38~42mM CAA, 8~12mM TCEP, 90~100mM TEAB, and 0.05~0.2% RapiGestSF; The enzymatic hydrolysate comprises 0.05 ug / uL - 0.1 ug / uL Trypsin / Lys-C Mix and 90 mM - 110 mM TEAB.

2. The single-cell protein pretreatment method based on a digital microfluidic system according to claim 1, characterized in that, The cell lysis buffer comprises the following active components: 0.09%~0.12% DDM, 38~42mM CAA, 8~12mM TCEP, 90~100mM TEAB, 0.12~0.18% ProteaseMAX; or comprising 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.

3. The single-cell protein pretreatment method based on a digital microfluidic system according to claim 1, characterized in that, The cell concentration in the cell suspension injected in step S2 was 5.5 × 10⁻⁶. 5 ~6.5×10⁻⁶ cells / mL 5 per mL.

4. The single-cell protein pretreatment method based on a digital microfluidic system according to claim 1, characterized in that, The enzymatic hydrolysate comprises 0.05 ug / uL - 0.1 ug / uL Trypsin / Lys-C Mix and 90 mM - 110 mM TEAB.

5. The single-cell protein pretreatment method based on a digital microfluidic system according to any one of claims 1-4, characterized in that, In step S6, a protein sample is obtained by capillary aspiration.

6. The single-cell protein pretreatment method based on a digital microfluidic system according to any one of claims 1-4, characterized in that, The tearing method is that the droplet is torn in half with equal volume.

7. The single-cell protein pretreatment method based on a digital microfluidic system according to any one of claims 1-4, characterized in that, In step S3, the cell lysis temperature is 52℃~60℃; and / or the lysis time is 13~17min.

8. The single-cell protein pretreatment method based on a digital microfluidic system according to claim 7, characterized in that... The cell lysis temperature in step S3 is 54℃~58℃.

9. The single-cell protein pretreatment method based on a digital microfluidic system according to any one of claims 1-4, characterized in that, The reaction in step S4 is carried out at a temperature of 35℃~39℃ for 1.5h~2.5h.

10. The application of a cell lysis buffer in a single-cell protein pretreatment method based on a digital microfluidic system, wherein the cell lysis buffer comprises the following active components: 0.08%~0.15% DDM, 38~42mM CAA, 8~12mM TCEP, 90~100mM TEAB, and 0.05~0.2% ProteaseMAX; or comprising the following active ingredients: 0.08%~0.15% DDM, 38~42mM CAA, 8~12mM TCEP, 90~100mM TEAB and 0.05~0.2% RapiGestSF.

11. The application according to claim 10, characterized in that, The cell lysis buffer comprises the following active components: 0.09%~0.12% DDM, 38~42mM CAA, 8~12mM TCEP, 90~100mM TEAB, 0.12~0.18% ProteaseMAX; or comprising 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.

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