Efficient enzymolysis method for trace protein sample based on combination of immobilized enzyme and digital microfluidic technology

Abstract

The invention discloses an efficient enzymolysis method for a trace protein sample based on combination of immobilized enzyme and a digital microfluidic technology. The method comprises the following steps: S1, immobilizing trypsin on magnetic beads to obtain immobilized trypsin magnetic beads; s2, moving the protein sample solution and the reducing agent solution in the digital micro-fluidic chip until the solution and the reducing agent solution are uniformly mixed, and performing incubation for the first time; s3, moving the alkylating agent solution and the mixed solution after the first incubation in the digital micro-fluidic chip until the mixed solution is uniformly mixed, and performing second incubation; s4, moving the immobilized trypsin magnetic beads and a mixed solution after the second incubation in a digital micro-fluidic chip until the mixed solution is mixed, carrying out enzyme digestion reaction, and after the reaction is finished, collecting digestive juice through magnetic separation. According to the method, the enzymolysis time can be greatly shortened, automatic analysis is achieved, the proteomics sample preparation process is remarkably simplified, sample loss is remarkably reduced, and efficient enzymolysis of trace protein is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of proteomics, and in particular to a method for realizing efficient enzymatic hydrolysis of trace protein samples by combining immobilized enzyme with digital microfluidics. Background Art

[0002] As the main executor of cellular physiological functions, proteins play a vital physiological role in organisms. Proteomics research is to comprehensively elucidate biological processes by systematically analyzing the expression of proteins in cells or tissues. Bottom-up mass spectrometry analysis is usually used in classical proteomics research. This strategy achieves accurate identification of proteins by enzymatically hydrolyzing complex protein molecules into smaller polypeptide fragments, and then through ionization, chromatographic separation and mass spectrometry detection. The identification effect of this process is highly dependent on the efficiency of the previous sample processing. Therefore, rapid, efficient and complete protein enzymatic hydrolysis has become an extremely important link in accurate, high-throughput identification and quantitative research of the proteome.

[0003] The traditional method is to use free trypsin to hydrolyze proteins. Trypsin itself hydrolyzes proteins, and the free protease used usually requires more than 12 hours of catalytic reaction time and is difficult to separate from the product. This not only prolongs the entire experimental cycle, but may also cause sample degradation and denaturation, posing a challenge to the accuracy and reproducibility of experimental results. In addition, the traditional proteomics sample preparation process mainly includes cell lysis, protein reduction alkylation, and enzymatic protein hydrolysis. These steps all need to be performed in test tubes, making the entire preparation process relatively complex and time-consuming.

[0004] Therefore, it is particularly important to develop a fast, efficient and automated sample pretreatment method. Compared with the traditional method of free trypsin hydrolysis, immobilized enzyme hydrolysis can significantly reduce the time required for hydrolysis, so it has received widespread attention in recent years. However, most traditional immobilized enzyme strategies only stay at the stage of hydrolysis of standard proteins and have not been used in the efficient preparation of proteomics samples.

[0005] In 2020, Liu Zhaosheng's research group fixed trypsin to the surface of polytrimethylolpropane trimethacrylate (TRIM) monomers through sulfhydryl-ene click chemistry, achieving rapid and efficient online digestion of proteins, significantly simplifying the manual operation steps in proteomics analysis, reducing sample consumption, and increasing the speed of analysis. However, this method has disadvantages such as low flexibility, high sample consumption, low throughput, and difficulty in preparation.

[0006] It can be seen that although the current enzymatic hydrolysis method speeds up the protein enzymatic hydrolysis process, it is still impossible to achieve efficient digestion of trace proteins in a short time. On the one hand, this is undoubtedly affected by the relatively complicated preparation process of the existing immobilized enzyme reactor, which limits the widespread application of this technology. On the other hand, the traditional technology of fixing proteases to the surface of biological magnetic beads still completes the enzymatic hydrolysis process in a test tube, which is difficult to quickly hydrolyze trace protein samples. At the same time, the contact area between the sample and the test tube wall is large, which causes the risk of sample loss. Therefore, it is urgent to develop a new method that can achieve efficient digestion of trace proteins.

[0007] Digital microfluidics is a technology that precisely manipulates liquids at a microscale. It achieves functions such as separation and mixing of liquids on microfluidic chips by manipulating discrete microdroplets. Among them, digital microfluidics based on dielectric wetting is a technology that uses the dielectric wetting effect to control the position, merging, separation and movement of microdroplets. The principle is to change the contact angle between the droplet and the surface of the dielectric layer by applying an alternating electric field to the electrode at the bottom of the droplet, thereby achieving precise manipulation of the microdroplet. Due to its unique advantages (such as high precision, programmability, fast response and real-time control), digital microfluidics based on dielectric wetting has been widely used in fields such as biology and chemical analysis. It can be used for various experimental operations such as sample mixing, reaction control, cell manipulation, enzyme reaction, gene analysis, etc. At present, there is no report on combining digital microfluidics with magnetic bead-immobilized enzymes to achieve efficient enzymatic hydrolysis of trace protein samples. Summary of the invention

[0008] The purpose of the present invention is to provide an efficient enzymatic hydrolysis method for trace protein samples based on the combination of immobilized enzyme and digital microfluidics technology. The method achieves efficient enzymatic hydrolysis of trace samples and efficient preparation of proteomics samples by fixing trypsin to the surface of N-hydroxysuccinimide magnetic beads and then combining digital microfluidics technology, thereby significantly reducing sample loss and simplifying the proteomics sample preparation process.

[0009] To achieve the above object, the present invention adopts the following technical solution: In a first aspect, the present invention provides an efficient enzymatic hydrolysis method for trace protein samples, comprising the following steps: S1, immobilizing trypsin on magnetic beads to obtain immobilized trypsin magnetic beads; S2, moving the protein sample solution and the reducing agent solution in the digital microfluidic chip until they are evenly mixed and performing the first incubation; S3, moving the alkylating agent solution and the mixed solution after the first incubation in the digital microfluidic chip until they are evenly mixed, and performing a second incubation; S4, moving the immobilized trypsin magnetic beads and the mixed solution after the second incubation in a digital microfluidic chip to mix, performing an enzyme digestion reaction, and collecting the digestion solution by magnetic separation after the reaction is completed.

[0010] In the above-mentioned efficient enzymatic hydrolysis method for trace protein samples, preferably, the fixing step in step S1 is as follows: N-hydroxysuccinimide magnetic beads are mixed with trypsin solution and then incubated, and after the incubation is completed, the magnetic beads are collected by magnetic separation.

[0011] Further, each 1 mg of N-hydroxysuccinimide magnetic beads was mixed with 15 μL of a 5 mg / mL trypsin solution; The NaCl concentration in the trypsin solution is 0.15 M and the pH is 5.5; The incubation temperature is 15-30° C. and the incubation time is 8-10 hours.

[0012] In the above-mentioned efficient enzymatic hydrolysis method for trace protein samples, the reducing agent may be dithiothreitol or tris(2-carboxyethyl)phosphine; As an example, the first incubation temperature is 56° C. and the time is 30 minutes.

[0013] In the above-mentioned efficient enzymatic hydrolysis method for trace protein samples, the alkylating agent may be iodoacetamide; As an example, the second incubation temperature is 37° C. and the time is 30 minutes.

[0014] In the above-mentioned high-efficiency enzymatic hydrolysis method for trace protein samples, as an example, the temperature of the enzymatic reaction is 37° C. and the time is 15 minutes.

[0015] In the above-mentioned efficient enzymatic hydrolysis method for trace protein samples, the method further includes the following steps: mixing the magnetic beads and ultrapure water after the enzymatic cleavage reaction in a digital microfluidic chip to wash the magnetic beads, and combining the washing solution with the digestion solution after washing.

[0016] In the above-mentioned high-efficiency enzymatic hydrolysis method for trace protein samples, the protein in the protein sample is bovine serum albumin, casein and / or transferrin.

[0017] In a second aspect, the present invention provides a proteomics analysis method, comprising any of the above-mentioned efficient enzymatic hydrolysis methods for trace protein samples.

[0018] In the above-mentioned proteomics analysis method, as an example, the protein sample is a HeLa cell protein extract.

[0019] The present invention has the following beneficial effects: (1) The present invention combines immobilized trypsin magnetic beads with digital microfluidics technology to achieve efficient enzymatic hydrolysis of trace protein samples. The enzymatic hydrolysis method only takes 15 minutes, which is significantly shorter than the method of using free trypsin to hydrolyze protein solutions in traditional centrifuge tubes (10 hours). It can also reduce manual operations, achieve automated analysis, and significantly simplify the proteomics sample preparation process. In addition, due to the smaller enzymatic hydrolysis environment on the digital microfluidics chip, it can significantly reduce sample loss caused by nonspecific adsorption, thereby achieving efficient enzymatic hydrolysis of trace proteins. Compared with the method of using free trypsin to hydrolyze protein solutions in traditional centrifuge tubes, it can cover more sequences and have a higher protein identification rate, thereby achieving rapid preparation of proteomics samples and in-depth proteomics analysis.

[0020] (2) The present invention fixes trypsin on N-hydroxysuccinimide magnetic beads through click chemistry reaction, which effectively improves the thermal stability and resistance to pH changes of trypsin.

[0021] (3) The present invention optimizes the cross-linking conditions and finds that when the NaCl concentration in the trypsin solution is 0.15 M and the pH is 5.5, the trypsin loading capacity is the largest. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flow chart of the preparation of immobilized trypsin magnetic beads in an embodiment of the present invention.

[0023] Figure 2 1 is a flowchart of the process of preparing samples using immobilized trypsin on digital microfluidics in an embodiment of the present invention.

[0024] Figure 3 is a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) spectrum of each sample in the characterization of immobilized trypsin magnetic beads in Example 1 of the present invention, wherein, Figure 3 (a) is the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) spectrum of N-benzoyl-L-arginine ethyl ester solution; Figure 3 (b) is a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) spectrum of a mixed solution of N-benzoyl-L-arginine ethyl ester and trypsin; Figure 3 (c) is a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) spectrum of the supernatant after N-benzoyl-L-arginine ethyl ester was mixed with immobilized trypsin magnetic beads; Figure 3 (d) is the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) spectrum of the supernatant after N-benzoyl-L-arginine ethyl ester was mixed with untreated magnetic beads.

[0025] Figure 4is the cross-linking reaction condition optimization curve in Example 1 of the present invention, wherein: Figure 4 (a) is the Bradford method calibration curve; Figure 4 (b) The effect of NaCl solubility on the immobilized amount of trypsin; Figure 4 (c) Effect of pH on the immobilized amount of trypsin.

[0026] Figure 5 is the stability analysis curve of immobilized trypsin in Example 1 of the present invention, wherein, Figure 5 (a) Effect of temperature on the stability of free and immobilized trypsin; Figure 5 (b) Effect of pH on the stability of free and immobilized trypsin.

[0027] Figure 6 This is the enzymatic kinetic analysis curve of immobilized trypsin in Example 1 of the present invention.

[0028] Figure 7 The comparison of the protein standard product cleaved by the immobilized enzyme on the digital microfluidic chip and the free trypsin in the test tube in Example 1 of the present invention is shown in FIG. Figure 7 (a) Sequence coverage corresponding to on-chip digestion with immobilized trypsin for 15 minutes and in vitro digestion with free trypsin for 10 hours; Figure 7 (b) is the corresponding peptide matching number.

[0029] Figure 8 The identification results of the proteomics samples prepared by using the immobilized enzyme on the digital microfluidic chip and the free trypsin in the test tube in Example 2 of the present invention, wherein: Figure 8 (a) is the number of proteomic identifications corresponding to 0.2 μg of HeLa cell protein extract; Figure 8 (b) is the corresponding peptide identification number; Figure 8 (c) Venn diagram of the number of proteomic identifications; Figure 8 (d) Pearson correlation between proteome quantifications.

[0030] Fig. 9 This is a heat map of the protein quantification of each sample prepared using immobilized enzyme on the digital microfluidic chip. DETAILED DESCRIPTION

[0031] As described in the background technology, although the current enzymatic hydrolysis method speeds up the protein enzymatic hydrolysis process, it still cannot achieve efficient digestion of trace proteins in a short time. Based on this, the present invention provides an efficient enzymatic hydrolysis method for trace protein samples, comprising the following steps: S1, fix trypsin on magnetic beads to obtain immobilized trypsin magnetic beads; S2, move the protein sample solution and the reducing agent solution in the digital microfluidic chip to mix, and perform the first incubation; S3, move the alkylating agent solution and the mixed solution after the first incubation in the digital microfluidic chip to mix, and perform the second incubation; S4, move the immobilized trypsin magnetic beads and the mixed solution after the second incubation in the digital microfluidic chip to mix, perform enzyme cutting reaction, and collect the digestion solution by magnetic separation after the reaction is completed. Based on this method, enzymatic hydrolysis only takes 15 minutes, which greatly shortens the enzymatic hydrolysis time, reduces manual operations, realizes automated analysis, and can significantly simplify the proteomics sample preparation process. In addition, due to the smaller enzymatic hydrolysis environment on the digital microfluidic chip, it can significantly reduce sample loss caused by nonspecific adsorption and achieve efficient enzymatic hydrolysis of trace proteins.

[0032] According to the present invention, preferably, the fixing step described in step S1 is as follows: N-hydroxysuccinimide magnetic beads are mixed with trypsin solution and incubated, and the magnetic beads are collected by magnetic separation after incubation. Based on the above fixing method, trypsin can not only be successfully fixed to the surface of N-hydroxysuccinimide magnetic beads, but also maintain a certain biological activity. Further stability tests show that the immobilized enzyme of the present invention exhibits a wide range of stable "activity platforms" (activity maintained at more than 75%) in the temperature range of 30-45 ° C, highlighting strong high temperature adaptability; and under extreme acid and alkali conditions, the activity of immobilized trypsin remains good, reflecting its significant stability and resistance to pH changes. Further preferably, each 1 mg of N-hydroxysuccinimide magnetic beads is mixed with 15 μL of a trypsin solution with a concentration of 5 mg / mL; the NaCl concentration in the trypsin solution is 0.15 M and the pH is 5.5; the incubation temperature is 15 to 30 ° C, including but not limited to 20 to 25 ° C, and the time is 8 to 10 hours, including but not limited to 8 hours. The present invention optimizes the conditions of the cross-linking reaction and finds that when the NaCl concentration in the trypsin solution is 0.15 M and the pH is 5.5, the amount of trypsin immobilized on the surface of the N-hydroxysuccinimide magnetic beads reaches a maximum value. Further enzyme kinetic analysis shows that the catalytic potential of the system can be significantly enhanced by regulating the amount of enzyme without significantly changing the substrate affinity.

[0033] In some embodiments of the present invention, the fixation step further includes the following steps after collecting the magnetic beads by magnetic separation: 1) washing the magnetic beads with a washing buffer to remove free trypsin that is not bound to the magnetic beads; 2) incubating the magnetic beads with a blocking buffer (such as 1 M ethanolamine, pH 9.0) to block the remaining active binding sites on the magnetic beads; 3) washing again and resuspending the magnetic beads in a PBS buffer containing 0.1% sodium azide (w / v) to maintain their stability.

[0034] According to the present invention, the reducing agent may be dithiothreitol or tris(2-carboxyethyl)phosphine (TECP). The addition of the reducing agent can open (break) the disulfide bonds of the protein in the trace protein sample, thereby opening the structure of the protein, which is more conducive to enzymatic hydrolysis. As an example, the temperature of the first incubation is 56°C and the time is 30 minutes.

[0035] According to the present invention, the alkylating agent may be iodoacetamide; the addition of iodoacetamide can prevent the disulfide bond from reforming, ensuring that the protein remains in a melted state during the subsequent enzymatic hydrolysis process. As an example, the second incubation temperature is 37° C. and the time is 30 minutes.

[0036] According to the present invention, the temperature of the enzyme cleavage reaction can be 37° C. and the time can be 15 minutes.

[0037] According to the present invention, the method further comprises the following steps: mixing the magnetic beads after the enzyme cleavage reaction and ultrapure water in a digital microfluidic chip to wash the magnetic beads, and combining the washing solution with the digestion solution after washing.

[0038] According to the present invention, as an example, the protein in the protein sample is bovine serum albumin, casein and / or transferrin. Taking the above proteins as an example for testing, it is found that the sequence coverage and peptide matching number obtained by digestion with immobilized trypsin for 15 minutes on the digital microfluidic chip are significantly higher than the sequence coverage obtained by digestion with free trypsin for 10 hours in a test tube.

[0039] The present invention further provides a proteomics analysis method, including any of the above-mentioned efficient enzymatic hydrolysis methods for trace protein samples. The above-mentioned efficient enzymatic hydrolysis method can realize rapid preparation of proteomics samples and in-depth proteomics analysis.

[0040] In some embodiments of the present invention, the protein sample is a HeLa cell protein extract. The results show that the method of immobilizing trypsin on the chip can identify far more proteomes and peptides than free trypsin in a test tube in a significantly shortened time.

[0041] Therefore, the advantages of the present invention are not only that the enzymatic hydrolysis time is greatly shortened, automated analysis is achieved, and the proteomics sample preparation process is simplified, but more importantly, due to the smaller enzymatic hydrolysis environment on the digital microfluidic chip, the sample loss caused by nonspecific adsorption can be significantly reduced, and efficient enzymatic hydrolysis of trace proteins can be achieved. Compared with the method of using free trypsin to hydrolyze the protein solution in a traditional centrifuge tube, more sequences can be covered and the protein identification amount is higher, which can realize the rapid preparation of proteomics samples and in-depth proteomics analysis.

[0042] According to the present invention, the digital microfluidic chip can adopt any digital microfluidic chip available on the market or disclosed in the prior art, and cooperate with the micro-droplet driving platform when used. For example, in a specific embodiment of the present invention, the micro-droplet manipulation chip disclosed in CN114778422 A can be used. Specifically, in the present invention, the digital microfluidic chip includes an upper plate, a lower plate and a conductive gasket therebetween, the upper plate includes an upper substrate and a conductive layer arranged on the lower surface of the upper substrate, the lower surface of the conductive layer is provided with an upper hydrophobic layer, the lower plate includes a lower substrate and a dielectric layer on the upper surface of the lower substrate, an electrode array is arranged between the lower substrate and the dielectric layer, and a lower hydrophobic layer is provided on the upper surface of the dielectric layer. Among them, the upper substrate can be a transparent material glass or acrylic plate; the lower substrate can be one of glass, silicon, paper, polyester film and printed circuit board. When in use, the droplet sample is between the upper hydrophobic layer and the lower hydrophobic layer, and the position, merging, separation and movement of the micro-droplets are controlled under the action of the electrodes. The electrode array includes a liquid reservoir electrode, a driving electrode, a ground electrode and a contact electrode; eight liquid reservoir electrodes are arranged into a 4×2 two-dimensional array to form a two-dimensional liquid reservoir electrode array, there are gaps in the two-dimensional liquid reservoir electrode array, a plurality of driving electrodes are arranged in the gaps of the two-dimensional liquid reservoir electrode array, and the edge of each liquid reservoir electrode is connected to the driving electrode located in the gap of the two-dimensional liquid reservoir electrode array through a corresponding driving electrode, each liquid reservoir electrode, driving electrode and ground electrode is respectively connected to a contact electrode, and connected to a micro-droplet driving platform through the contact electrode; the ground electrode is grounded; the conductive layer in the upper plate is connected to the ground electrode through a conductive gasket. The sample loading position can be set on the liquid reservoir electrode. The liquid reservoir electrode, driving electrode, ground electrode and contact electrode can be made of polysilicon, metal and its oxide.

[0043] like Figure 2As shown, when used, the reagent, sample solution and immobilized trypsin magnetic beads are placed on different reservoir electrodes respectively, first the micro-droplet driving platform is controlled to energize the reservoir electrode containing the solution, the reservoir electrode generates a dielectric wetting force, and then the driving electrode connected to one side of the reservoir electrode containing the solution is energized at the same time, the driving electrode generates a dielectric wetting force to pull the solution, so that the solution forms a long strip, and then the reservoir electrode is powered off, at this time the solution is not affected by the dielectric wetting force of the reservoir electrode, but only by the dielectric wetting force of the driving electrode on one side, so as to achieve sampling. When it is necessary to control the movement of the droplets, according to their respective moving paths, the micro-droplet driving platform is controlled to energize the driving electrodes located on the moving path of the droplets in turn, and the droplets are driven to move according to the set moving path. For immobilized trypsin magnetic beads, the mixing of the magnetic beads and the mixed solution after the second incubation can be specifically controlled by the following steps: 1) injecting the immobilized trypsin magnetic bead suspension into the chip, and then driving the immobilized trypsin magnetic bead suspension to move in the same way as the driving droplet principle, and then controlling the separation of the magnetic beads and the solution by magnetic separation (e.g., using a magnetic bar to enrich the immobilized trypsin magnetic beads, and then driving the droplets to separate the magnetic beads from the solution); 2) driving the mixed solution after the second incubation to mix with the immobilized trypsin magnetic beads. After the incubation is completed, magnetic separation is also used to control the separation of the magnetic beads and the digestion solution, such as using a magnetic bar to enrich the immobilized trypsin magnetic beads, and then driving the digestion solution to separate the digestion solution from the magnetic beads.

[0044] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0045] The methods used in the following examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0046] The N-hydroxysuccinimide magnetic beads in the following examples are products of PuriMag, the product name is NHS magnetic beads, and the product model is PuriMag G Series.

[0047] The trypsin in the following examples was purchased from Promega, the product name is Sequencing Grade Modified Trypsin, and the product number is V5113.

[0048] Example 1: Efficient enzymatic hydrolysis of trace protein samples 1. Preparation, characterization, cross-linking condition optimization and performance testing of immobilized trypsin magnetic beads (1) Preparation of immobilized trypsin magnetic beads in accordance with Figure 1 Immobilized trypsin magnetic beads were prepared according to the process shown. First, 1 mg of N-hydroxysuccinimide magnetic beads were placed in a centrifuge tube and washed three times with ultrapure water to completely remove the residual magnetic bead storage solution. Then, 15 μL of trypsin solution (5 mg / mL) (NaCl concentration was 0.15 M, pH was 5.5) was added and incubated at room temperature for 8 hours with continuous rotation to ensure that trypsin fully reacted with the N-hydroxysuccinimide ester on the surface of the magnetic beads. After the incubation, the magnetic beads were separated from the solution by magnetic separation technology, and the magnetic beads were washed three times with washing buffer to remove free trypsin that was not bound to the magnetic beads. Next, 100 μL of blocking buffer (1 M ethanolamine, pH 9.0) was added and incubated at room temperature for 1 hour to block the remaining active binding sites on the magnetic beads. Finally, the magnetic beads were washed three times again and resuspended in PBS buffer containing 0.1% sodium azide (w / v) to maintain their stability and stored at 4 ℃ for subsequent experiments.

[0049] (2) Characterization of immobilized trypsin magnetic beads Trypsin was used to characterize the specific hydrolysis of N-benzoyl-L-arginine ethyl ester. First, four samples were prepared, namely: N-benzoyl-L-arginine ethyl ester solution (10 mM), a mixed solution of 10 μL N-benzoyl-L-arginine ethyl ester (10 mM) and 5 μg trypsin incubated for 30 minutes, the supernatant of 10 μL N-benzoyl-L-arginine ethyl ester (10 mM) and 100 μg immobilized trypsin magnetic beads incubated for 5 minutes, and the supernatant of 10 μL N-benzoyl-L-arginine ethyl ester (10 mM) and 100 μg untreated N-hydroxysuccinimide magnetic beads incubated for 5 minutes. Then, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was performed on these four samples to detect the product components.

[0050] The results are as follows Figure 3As shown. The mass spectrum of the N-benzoyl-L-arginine ethyl ester solution showed a single clear peak with m / z 306.2 (a), which is the characteristic peak of the substrate N-benzoyl-L-arginine ethyl ester. When N-benzoyl-L-arginine ethyl ester was mixed with trypsin and incubated for 30 minutes, a new peak m / z 279.1 appeared on the mass spectrum (b), indicating that N-benzoyl-L-arginine ethyl ester had been hydrolyzed by trypsin to produce the product N-benzoyl-L-arginine. In further experiments, we found that after incubating N-benzoyl-L-arginine ethyl ester with immobilized enzyme magnetic beads for 5 minutes, the characteristic peak with m / z value of 279.1 in the supernatant still existed significantly (c), proving that trypsin immobilized on the surface of magnetic beads effectively hydrolyzed N-benzoyl-L-arginine ethyl ester. By comparing the supernatant (d) of untreated N-hydroxysuccinimide magnetic beads incubated with N-benzoyl-L-arginine ethyl ester for 5 minutes, it was found that the m / z 279.1 peak did not appear, which ruled out the interference of nonspecific hydrolysis. The above results show that trypsin was not only successfully immobilized on the surface of N-hydroxysuccinimide magnetic beads, but also maintained a certain biological activity.

[0051] (3) Optimization of cross-linking reaction conditions 5 mg / mL trypsin solution (MES buffer) with different NaCl concentrations and different pH values ​​was mixed and incubated with 1 mg N-hydroxysuccinimide magnetic beads. After the incubation, the supernatant was extracted and washed, and the supernatant and washing solution were combined in the same centrifuge tube. Then, the concentration of trypsin in the mixed solution was determined by the Bradford method. By calculating the mass difference between the total amount of trypsin involved in the reaction and the unreacted trypsin, the amount of trypsin immobilized on the surface of N-hydroxysuccinimide magnetic beads under different experimental conditions was determined, thereby determining the optimal reaction conditions for obtaining the maximum trypsin loading amount.

[0052] The results are as follows Figure 4 As shown. With the increase of NaCl concentration in the solution, the amount of trypsin fixed on the surface of magnetic beads increased significantly until the NaCl concentration reached 0.15 M, after which the amount of trypsin slowly decreased. We speculate that in the low concentration range, NaCl ions effectively shielded the surface charge of trypsin molecules, weakened the electrostatic repulsion between enzyme molecules, and made it easier for enzyme molecules to approach and bind to the surface of N-hydroxysuccinimide magnetic beads. However, when the NaCl concentration is too high, "salting out" will occur, causing trypsin to precipitate, thereby reducing its fixed amount. Therefore, the optimized NaCl concentration is 0.15 M. At this concentration, the charge shielding effect is most significant, while salting out has not yet appeared, which is conducive to maximizing the fixation of trypsin on the surface of magnetic beads. In addition, Figure 4(c) shows that when the pH is lower than 5.5, the immobilized amount of trypsin is relatively stable. When the solution pH is 5.5, the immobilized amount of trypsin reaches the maximum value. Thereafter, as the pH increases, the immobilized amount decreases significantly. This is because the ester bonds on the surface of N-hydroxysuccinimide magnetic beads are easily hydrolyzed in a weak acid environment, and as the pH increases, the degree of hydrolysis increases, thus affecting the immobilized amount of trypsin.

[0053] (4) Stability test of immobilized trypsin The trypsin activity detection kit was used to determine the stability differences of immobilized trypsin and free trypsin under different temperature and pH conditions. Trypsin can catalyze the hydrolysis of the ester bond in N-benzoyl-L-arginine ethyl ester to generate N-benzoyl-L-arginine, which has a characteristic absorption peak at 253 nm. Therefore, the activity of trypsin can be characterized by measuring the change in absorbance.

[0054] The results are as follows Figure 5 As shown in the figure, the optimal activity temperature of free trypsin is 37 °C, while the optimal temperature of immobilized trypsin is increased to 40 °C, and it shows a broad and stable "activity platform" (activity remains above 75%) in the temperature range of 30-45 °C, highlighting its strong high temperature adaptability. Free trypsin does not show this characteristic. This phenomenon is attributed to the covalent bond formed between the enzyme molecule and the carrier, which constrains the conformational changes of trypsin, thereby enhancing its overall stability.

[0055] In addition, the present invention also investigates the effect of different pH (5-10) on the activity of immobilized trypsin. Figure 5 As shown in (b), the optimum pH of trypsin is 8.0, whether in free or immobilized form. Although the activity of free trypsin is slightly higher than that of immobilized enzyme in the pH range of 7-8, the pH activity distribution of immobilized trypsin is wider, not only expanding in both alkaline and acidic regions, but also maintaining its activity above 60% in a wider pH range (6-10). The high activity of immobilized trypsin is due to the amide bond formed between the carrier and the trypsin molecules, which not only enhances the rigidity of trypsin, but also provides a protective microenvironment, effectively alleviating the adverse effects of pH fluctuations. Especially under extreme acid-base conditions, the activity of immobilized trypsin remains good, reflecting its remarkable stability and resistance to pH changes.

[0056] (5) Enzymatic kinetic analysis of immobilized trypsin In order to evaluate the enzymatic kinetics of immobilized trypsin, the present invention designed the following experiment. First, a series of N-benzoyl-L-arginine ethyl ester solutions of different concentrations (0.3125, 0.4, 0.625, 1.25, 2.5 mM) were prepared, and then mixed with 1 μg of free trypsin, 1 μg of immobilized trypsin, and 2 μg of immobilized trypsin (0.15M / L NaCl concentration, pH 5.5), respectively. Next, the rate of increase of the absorbance value at 253 nm was measured by a microplate reader. According to the Lambert-Beer law, when the thickness of the absorption layer is constant, the absorbance of the substance is proportional to the concentration, so the rate of increase of the absorbance value can be used as the initial rate of the enzyme reaction. Finally, a double reciprocal graph was drawn, and the Michaelis constant (Km) and the maximum reaction rate (Vmax) were calculated according to formula 1.

[0057]

[0058] The results are as follows Figure 6 As shown in the figure, compared with the free trypsin Km = 0.294 and Vmax = 0.117, the same amount of immobilized trypsin showed a higher Michaelis constant Km = 0.517 and a slightly lower maximum reaction rate Vmax = 0.106, indicating that the immobilization process may lead to a weakening of the affinity between the enzyme and the substrate and a slight decrease in the reaction rate, which may be due to the carrier interface limiting the direct exposure of the enzyme active site. Interestingly, when the immobilized trypsin loading amount was increased to two times, although the Km value did not change much (0.485), the Vmax increased significantly to 0.172, indicating that by loading more immobilized enzymes, the effect of reduced affinity after immobilization can be effectively compensated and the overall catalytic efficiency can be improved. This finding also emphasizes the advantages of immobilized enzyme technology, that is, by regulating the amount of enzyme, the catalytic potential of the system can be significantly enhanced without significantly changing the substrate affinity, highlighting the feasibility of the strategy of using excess immobilized enzymes to optimize reaction efficiency in specific applications.

[0059] 2. Digestion of protein standards using immobilized enzymes on digital microfluidic chips Bovine serum albumin, casein, and transferrin were used as model proteins, and a 0.5 mg / mL standard protein solution was prepared with 25 mM ammonium bicarbonate solution. The protein solution was denatured at 95 °C for 15 minutes. Figure 2As shown in the flow chart, the following steps are performed in the digital microfluidic chip in sequence: First, 2 µL of protein solution and 2 µL of 30 mM dithiothreitol solution are injected, and the two are fully mixed through the circulation path (indicated by the arrow), and incubated at 56 °C for 30 minutes, and then 2 µL of 30 mM iodoacetamide solution is added, mixed evenly, and incubated at 37 °C for another 30 minutes. Subsequently, 1 mg of immobilized trypsin magnetic beads are introduced, and the magnetic beads and the solution are fully mixed at 37 °C for 15 minutes to perform the enzyme digestion reaction. The magnetic beads and the digested solution are quickly separated by an external magnetic field, and the magnetic beads are washed three times with ultrapure water, and finally the washing solution is combined with the extracted digestion solution. All digestion products are analyzed by mass spectrometry to evaluate the enzyme digestion effect.

[0060] As a control group, free trypsin (enzyme: protein = 1:20 w / w) was used to digest the protein solution in a traditional centrifuge tube under the same conditions for 10 hours, and the digestion products were also analyzed by mass spectrometry. The steps for digesting the protein solution in a traditional centrifuge tube using free trypsin are as follows: first, 2 uL of standard protein solution was injected into the test tube, then 2 uL of 30 mM dithiothreitol solution was added and mixed and incubated at 56 °C for 30 minutes, then 2 uL of 30 mM iodoacetamide solution was added and mixed and incubated at 37 °C for 15 minutes, then 5 uL of trypsin solution (10 ng / uL) was added and mixed and incubated at 37 °C for 10 hours, and then 2 uL of 0.1% formic acid solution was added to terminate the digestion.

[0061] The results are as follows Figure 7 As shown in the figure, the sequence coverages obtained by digestion with immobilized trypsin for 15 minutes on the digital microfluidic chip were 93%, 71% and 52.8%, and the numbers of peptide matches were 92, 84 and 14, respectively, which were significantly higher than the sequence coverages (38.6%, 29.8%, 25.8%) and peptide matches (31, 24, 5) obtained by digestion with free trypsin for 10 hours in vitro.

[0062] Example 2: Efficient enzymatic hydrolysis and proteomic analysis of HeLa cell protein extract (1) Preparation of HeLa cell protein extract HeLa cells were cultured under controlled conditions in a cell culture incubator with 5% CO2, saturated humidity, and 37°C. The growth medium of HeLa cells was MEM supplemented with 10% fetal bovine serum (FBS), 100 μg / mL penicillin, and 100 μg / mL streptomycin (Eallbio, China).

[0063] The cultured HeLa cell suspension was centrifuged at 800 rpm for 8 minutes at 4 °C, and the supernatant was discarded. Then, 2 mL of urea buffer was added and lysed on ice for 15 minutes, during which time a pipette was used to repeatedly blow and beat to promote lysis. Subsequently, the lysate was centrifuged at 16000 g for 30 minutes at 4 °C to separate the clear liquid between the precipitate and the upper lipid layer, which was the protein extract of HeLa cells for subsequent use.

[0064] (2) HeLa cell protein extract The operation was the same as in Example 1. 0.2 μg of HeLa cell protein extract was treated by the method of enzymatic cleavage using an immobilized enzyme on a digital microfluidic chip of the present invention and the method of enzymatic cleavage of a protein solution in a traditional centrifuge tube using free trypsin under the same conditions (the operation was the same as the above model protein), and finally the protein identification amount was compared.

[0065] The results are as follows Figure 8 As shown in Figure 2, in a significantly shorter time, the immobilized trypsin on-chip method was able to identify 6,322 proteomes and 43,618 peptides, far more than the identification of free trypsin in the test tube (5,610, 32,255). Figure (c) shows that the on-chip method can identify 342 additional proteomes. In addition, correlation analysis of the quantitative status of proteins identified by both methods revealed a significant positive correlation (r = 0.82; P < 0.0001; Figure d).

[0066] Fig. 9 This is a heat map of the protein quantification of each sample prepared using immobilized enzyme on the digital microfluidic chip. The four samples are four proteomics samples prepared using the immobilized enzyme strategy on the digital microfluidic chip. The protein expression levels of any two samples were correlated. It can be seen from the figure that this method is highly reproducible.

[0067] From the comparison results of the method of the present invention and the method of free trypsin in the traditional centrifuge tube for enzymatic cleavage of protein solution in the above embodiment, it can be seen that the method of the present invention greatly shortens the enzymatic cleavage time, and can realize automated analysis, and can significantly reduce the sample loss caused by non-specific adsorption, and realizes efficient enzymatic cleavage of trace proteins, which can cover more sequences and have a higher protein identification amount, and can realize rapid preparation of proteomics samples and deep proteomics analysis. This may be because the traditional method uses free trypsin (enzyme: protein = 1: 20), and since the enzyme is a small amount, it takes a long time to consume, so that there are low efficiency, enzyme autolysis and non-specific enzyme cleavage phenomena; and the method of the present invention can effectively inhibit the autolysis of the enzyme while greatly increasing the amount of trypsin by fixing trypsin to the surface of biological magnetic beads, and can greatly shorten the enzyme cleavage time, reduce non-specific enzyme cleavage and adsorption. The above results all show the superiority of immobilized trypsin magnetic beads combined with digital microfluidics in enzyme cleavage protein and proteomics sample preparation.

[0068] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, can implement the present invention in a wider range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. An efficient enzymatic hydrolysis method for trace protein samples, characterized in that: The steps include: S1, immobilizing trypsin on magnetic beads to obtain immobilized trypsin magnetic beads; S2, moving the protein sample solution and the reducing agent solution in the digital microfluidic chip until they are evenly mixed and performing the first incubation; S3, moving the alkylating agent solution and the mixed solution after the first incubation in the digital microfluidic chip until they are evenly mixed, and performing a second incubation; S4, moving the immobilized trypsin magnetic beads and the mixed solution after the second incubation in a digital microfluidic chip to mix, performing an enzyme digestion reaction, and collecting the digestion solution by magnetic separation after the reaction is completed.

2. The efficient enzymatic hydrolysis method for trace protein samples according to claim 1, characterized in that: The fixing steps in step S1 are as follows: N-hydroxysuccinimide magnetic beads are mixed with trypsin solution and then incubated, and after the incubation is completed, the magnetic beads are collected by magnetic separation.

3. The efficient enzymatic hydrolysis method for trace protein samples according to claim 2, characterized in that: Each 1 mg of N-hydroxysuccinimide magnetic beads was mixed with 15 μL of 5 mg / mL trypsin solution; The NaCl concentration in the trypsin solution is 0.15 M and the pH is 5.5; The incubation temperature is 15-30° C. and the incubation time is 8-10 hours.

4. The efficient enzymatic hydrolysis method for trace protein samples according to any one of claims 1 to 3, characterized in that: The reducing agent is dithiothreitol or tris(2-carboxyethyl)phosphine; The first incubation was carried out at a temperature of 56° C. and for 30 minutes.

5. The efficient enzymatic hydrolysis method for trace protein samples according to any one of claims 1 to 4, characterized in that: The alkylating agent is iodoacetamide; The second incubation temperature was 37° C. and the time was 30 minutes.

6. The efficient enzymatic hydrolysis method for trace protein samples according to any one of claims 1 to 5, characterized in that: The temperature of the enzyme digestion reaction was 37° C. and the time was 15 minutes.

7. The efficient enzymatic hydrolysis method for trace protein samples according to any one of claims 1 to 6, characterized in that: The method further comprises the following steps: mixing the magnetic beads after the enzyme cleavage reaction with ultrapure water in a digital microfluidic chip to wash the magnetic beads, and combining the washing solution with the digestion solution after the washing is completed.

8. The efficient enzymatic hydrolysis method for trace protein samples according to any one of claims 1 to 7, characterized in that: The protein in the protein sample is bovine serum albumin, casein and / or transferrin.

9. A proteomics analysis method, characterized in that: A highly efficient enzymatic hydrolysis method for trace protein samples comprising any one of claims 1-8.

10. The proteomics analysis method according to claim 9, characterized in that: The protein sample is a HeLa cell protein extract.

Images