Method for rapidly preparing polypeptide

Abstract

The invention discloses a method for rapidly preparing polypeptide. The method comprises the following steps: 1) preparing a mixed reagent; and 2) mixing the mixed reagent obtained in the step 1) with protein for reaction to prepare the polypeptide. According to the method for rapidly preparing the polypeptide, the mixed reagent is firstly prepared and then mixed with the protein, reduction, sulfydryl alkylation and enzyme digestion reaction are completed in one step, the polypeptide is prepared from the protein through one-pot one-step reaction, the reaction time can be shortened to 60 min or below, the reaction can be carried out at room temperature, the preparation time and instrument cost can be remarkably reduced, and the method is suitable for industrial production. The speed of preparing polypeptide from protein is greatly increased, energy consumption is reduced, and related basic research and industrial production are accelerated.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide preparation, and particularly relates to a method for rapidly preparing a polypeptide. Background Art

[0002] Preparation of peptides from biological samples (such as tissues and serum) is widely used in protein-related scientific research and industrial production. In scientific research, protein disulfide bond reduction, alkylation of reduced sulfhydryl groups, and trypsin-based enzymatic digestion are currently widely used sample pretreatment steps in mass spectrometry-based protein qualitative and quantitative analysis. Currently, these three reactions are usually performed in three steps: first, dithiothreitol (DTT, incubation at 37°C for 1 hour) is used to reduce disulfide bonds, then iodoacetamide (IAA, 10mM, room temperature in the dark, 45 minutes) is used to alkylate the reduced sulfhydryl groups (and free sulfhydryl groups), and finally, trypsin (enzyme / protein mass ratio 1:50, 37°C, 16 hours) is used to enzymatically digest the reduced and alkylated proteins into peptides. For tissue proteins, studies have reported using a high-pressure Barocycler (Pressure Biosciences, USA) with a lysis buffer containing 6M urea, 2M thiourea, 100mM NH₄CO₃, 20mM tris(2-chloroethyl) phosphate (TCEP), and 40mM IAA. Pressure cycling (45,000 psi for 30 seconds followed by 90 cycles of 10 seconds at 30°C) was performed to achieve a one-pot combined reaction of tissue lysis and reduction-alkylation. This was followed by centrifugation in 30kDa ultrafiltration tubes for 1 hour and buffer exchange, followed by overnight trypsin digestion at 37°C. For plasma proteins, a 300-fold dilution with PBS followed by the addition of 1% SDS, 5mM TCEP, and 10mM CAA was reported. The lysis-reduction-alkylation reaction was completed within 15 minutes at 37°C, followed by enzymatic digestion using 50mM triethylammonium bicarbonate buffer at room temperature for 2-4 hours at a 1:25 enzyme-to-protein ratio. The recently reported single-tube solid-phase enhanced sample preparation (SP3) technique utilizes hydrophilic magnetic beads for non-selective protein binding and cleanup, shortening protein purification and enzymatic digestion times to 30 minutes and 18 hours, respectively. While the recently proposed dispersed droplet oscillation reaction technique reduces protein reduction, alkylation, and enzymatic digestion times to 1 minute, 1 minute, and 5 minutes, respectively, it still requires a three-step process. To address these issues, the present invention discloses a method for rapid peptide preparation. Summary of the Invention

[0003] In order to solve the technical problems existing in the prior art, the object of the present invention is to provide a method for rapidly preparing polypeptides.

[0004] In order to achieve the above-mentioned purpose and the above-mentioned technical effect, the technical solution adopted by the present invention is:

[0005] A method for rapidly preparing a polypeptide comprises the following steps:

[0006] 1) Prepare mixed reagents;

[0007] 2) The mixed reagent obtained in step 1) is mixed with the protein to react to produce a polypeptide.

[0008] Furthermore, in step 1), the mixed reagent includes at least one of the following components: pH buffer, protein denaturant, stabilizer, protein solubilizing agent I, protein solubilizing agent II, anti-protein adsorption agent by tube wall, enzyme structure stabilizing reagent, protein disulfide bond reducing agent, protein thiol alkylation reagent, protein hydrolase.

[0009] Furthermore, the pH buffer is at least one of NH4HCO3 buffer, tris-HCl buffer, and HEPES buffer, with a concentration of 0-5M.

[0010] Furthermore, the protein denaturant is at least one of urea, thiourea, and guanidine hydrochloride, with a concentration of 0-10M.

[0011] Furthermore, the stabilizer is at least one of glycerol and EDTA-free protease inhibitor.

[0012] Furthermore, the protein solubilizing agent I is at least one of sodium dodecyl sulfate, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, and 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate.

[0013] Furthermore, the enzyme structure stabilizing reagent is at least one of CaCl2 and MgCl2, with a concentration of 0-100 mM.

[0014] Furthermore, the protein disulfide bond reducing agent is at least one of DTT, TCEP, and β-mercaptoethanol, with a concentration of 0-1000 mM.

[0015] Furthermore, the protein thiol alkylation reagent is at least one of IAA and iodoacetamide CAA, with a concentration of 0-1000 mM; the protein hydrolase is at least one of trypsin and Lys-C.

[0016] Furthermore, in step 2), the reaction time is 1-60 min, and the reaction temperature is room temperature.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention discloses a method for rapidly preparing polypeptides. The method comprises the following steps: first preparing a mixed reagent, then mixing the mixed reagent with a protein, and completing reduction, thiol alkylation, and enzymatic cleavage reactions in one step, thereby realizing the preparation of polypeptides from proteins through a one-pot, one-step reaction. The reaction time can be shortened to less than 60 minutes and can be carried out at room temperature. This method can significantly reduce preparation time and instrument costs, greatly improve the speed of preparing polypeptides from proteins, reduce energy consumption, and accelerate related basic research and industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the base peak chromatogram of Comparative Example 1, Figure 1 a, b, and c are three test images;

[0020] Figure 2 is the base peak chromatogram of Example 1, Figure 2 a, b, and c are three test images;

[0021] Figure 3 1 is a diagram showing the length distribution of the amino acid sequences of the polypeptides of Example 1 and Comparative Example 1;

[0022] Figure 4 This is a hyperscore distribution diagram of the shared polypeptide identification credibility scores of Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0023] The present invention is described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more precise definition of the protection scope of the present invention.

[0024] The following is a brief summary of one or more aspects to provide a basic understanding of these aspects. This summary is not an exhaustive overview of all conceivable aspects and is neither intended to identify key or critical elements of all aspects nor to define the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form as a prelude to the more detailed description that will be provided later.

[0025] like Figure 2-4 As shown, a method for rapidly preparing a polypeptide comprises the following steps:

[0026] 1) Prepare mixed reagents;

[0027] The mixed reagent includes:

[0028] pH buffer, such as NH4HCO3, tris-HCl, HEPES, etc.; concentration 0-5M;

[0029] Protein denaturants, such as urea, thiourea, guanidine hydrochloride, etc.; concentration 0-10M;

[0030] Stabilizers, such as glycerol, EDTA-free protease inhibitors, etc.; volume ratio 0-30%;

[0031] Protein solubilizing agent I, such as sodium dodecyl sulfate (SDS), 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100), 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS), etc.; volume ratio 0-0.1%;

[0032] Protein dissolving aid II, such as acetonitrile, etc.; volume ratio 0-20%;

[0033] Anti-protein adsorption aids, such as bovine serum albumin (BSA), concentration 0-10 mg / mL;

[0034] Enzyme structure stabilizing reagents, such as CaCl2, MgCl2, etc.; concentration 0-100mM;

[0035] Protein disulfide bond reducing agents, such as DTT, TCEP, β-mercaptoethanol, etc.; concentration 0-1000mM;

[0036] Protein thiol alkylation reagents, such as IAA, iodoacetamide CAA, etc.; concentration 0-1000mM;

[0037] Protein hydrolases, such as trypsin, Lys-C, etc.; protein / enzyme mass ratio 10000:1-10:1.

[0038] 2) The mixed reagent obtained in step 1) is mixed with the protein, and reacted for a period of time (1-60 minutes) under certain conditions (such as droplet reaction, etc.).

[0039] The technical effect of the present invention is embodied in that the traditional three-step method is shortened to a one-step method, which greatly shortens the preparation time, and the entire process can be completed at room temperature without heating, with lower energy consumption.

[0040] The present invention is also applicable to any form of reaction conditions, including droplets (water-in-oil, oil-in-water), pressurized or normal pressure, heated or room temperature, etc.

[0041] The present invention is also applicable to all pH ranges, which are determined by the type and concentration of the buffer solution, and mainly meet the reaction requirements of the hydrolase.

[0042] The present invention is also applicable to all denaturation conditions, including heating, adding protein denaturants (such as urea, SDS), etc.

[0043] The present invention is also applicable to all protein disulfide bond reducing agents, such as DTT, β-mercaptoethanol, TCEP, etc.

[0044] The present invention is also applicable to all blocking reagents for protein sulfhydryl groups, such as alkylating reagents IAA and CAA.

[0045] The present invention is also applicable to all enzyme structure and activity stabilizing reagents, such as CaCl2, MgCl2, glycerol, EDTA-free protease inhibitors, etc.

[0046] The present invention is also applicable to all reagents for preventing proteins from being adsorbed by tube walls, such as bovine serum albumin (BSA).

[0047] The present invention is also applicable to all protein solubilizing reagents, such as sodium dodecyl sulfate SDS, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol Triton X-100, 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate CHAPS, acetonitrile, and the like.

[0048] The present invention is also applicable to all protein hydrolases and their variants, such as trypsin, Glu-C, Asp-N, chymotrypsin, etc.

[0049] The present invention is also applicable to all reagents that are beneficial to protein disulfide bond reduction reaction, sulfhydryl blocking reaction, and / or hydrolysis reaction.

[0050] Example 1

[0051] A method for rapidly preparing a polypeptide comprises the following steps:

[0052] 1) Prepare mixed reagents;

[0053] Prepare a mixed reagent by adding the following reagents in sequence to a 1.5 mL centrifuge tube:

[0054] 13 μL 8 M urea, 5 μL 5 M tris-HCl buffer, 5 μL 400 mM thiourea, 5 μL 40 mM TCEP, 2 μL 200 mM IAA, 20 μL 0.2 μg / L trypsin, 1 μL 100 mM MgCl2, 1 μL 100 mM CaCl2, 5 μL glycerol, 1 μL 1 μg / L BSA, 40 μL H2O, 1 mL toluene;

[0055] 2) Add 2 μL of serum (at a concentration of 50 μg / μL) to the centrifuge tube containing the mixed reagent, seal and shake (manually or mechanically) for 5 minutes, and complete the reduction (reduction of protein disulfide bonds to sulfhydryl groups), alkylation (sulfhydryl alkylation), and enzymatic cleavage (enzymatic cleavage of protein amino acid sequences into polypeptides) reactions in one step, thereby achieving the production of polypeptides from proteins through a one-pot, one-step reaction, thereby reducing preparation time and instrument costs. This will have a wide range of applications in related research and production fields such as protein preparation and analysis.

[0056] Comparative Example 1

[0057] The difference between this comparative example and Example 1 is that the same 2 μL serum was used in this comparative example, and the polypeptide was prepared by sequentially adding reagents and reacting them in sequence according to conventional techniques, including reduction (TCEP, 5 mM, 37° C., 1 h), alkylation (IAA, 10 mM, room temperature, 45 min), and enzymatic digestion (enzyme / protein w / w 1:50, 37° C., 16 h, shaking on a shaker).

[0058] The enzymatically cleaved polypeptide solutions obtained in Example 1 and Comparative Example 1 were centrifuged, the polypeptides were purified and eluted using a C18 solid phase extraction column, and the eluates were vacuum concentrated to prepare pure polypeptide solutions.

[0059] After the peptide solution is separated and analyzed by liquid chromatography-mass spectrometry and data processing, a list of peptides and corresponding proteins with their identification and quantity is given, which is briefly described as follows:

[0060] The digested peptides were analyzed using a Dionex Ultimate 3000 nano-HPLC system coupled to a Q-Exactive mass spectrometer (Thermo Fisher). The mobile phase consisted of 0.1% formic acid in water (Buffer A) and 90% acetonitrile / 0.1% formic acid (Buffer B). Chromatographic separation was performed using a C18 trapping column (5 cm × 200 μm) and an analytical column (60 cm × 75 μm) with a multistep gradient elution program: 12% B (10 min), 20-45% B (95 min), 45-98% B (5 min), and 98% B (3 min), followed by column equilibration. Mass spectrometry parameters included full scan mode (mass range 350-1800 m / z, 70 k resolution) and data-dependent secondary mass spectrometry (Top30 selection of the top 30 most abundant precursor ions, HCD fragmentation, and 17.5 k resolution). Automatic gain control (AGC) targets were set at 1e6 (MS / MS) and 5e5 (MS / MS); the isolation window was 2 m / z, and the dynamic exclusion time was 30 s. The collision energy was 30%, the capillary temperature was 250°C, and the spray voltage was 2.8 kV. Raw data were analyzed using FragPipe software (v22.0) with a precursor ion mass tolerance of 10 ppm and a fragment ion tolerance of 20 ppm. Dynamic modifications included N-terminal acetylation, methionine oxidation, and cysteine carboxylaminomethylation. Confidence in peptide identification was assessed using a 1% false positive rate (FDR) threshold. Experiments were performed in triplicate, and the results are reported as the mean ± relative standard deviation (RSD) of the number of peptide identification IDs. Parameters such as peptide length distribution and missed cleavage site rate were also assessed.

[0061] The polypeptides prepared in Example 1 and Comparative Example 1 were separated by reverse phase chromatography (RPLC), and electrospray ionization (ESI) and mass spectrometry were performed to obtain the original data set. The base peak chromatograms of the primary mass spectra were as follows: Figure 1 and Figure 2 As shown, Example 1 exhibits better separation and resolution than Comparative Example 1. The database group was searched using a peptide search engine in a theoretical protein database to generate a list of peptide identifications. The reaction efficiency of the method of the present invention was compared with that of the traditional method using indicators such as the number of identifications, peptide length, and credibility score distribution. The conclusion is:

[0062] In terms of the number of polypeptides, the values of Example 1 and Comparative Example 1 were 2194±2.6% and 1767±10.1%, respectively, with the former being 24% more than the latter;

[0063] In the distribution of peptide amino acid length, Figure 3 As shown, Example 1 and Comparative Example 1 are relatively close;

[0064] In the distribution of the credibility score hyperscore of shared peptides, such as Figure 4 As shown, Example 1 and Comparative Example 1 are relatively close, with average values of 32 and 34, respectively. It is worth noting that the number of peptide identifications here depends on the liquid chromatography-mass spectrometry instrument used. The latest level of development of the instrument is expected to increase the number of peptide identifications by 4-5 times for the analysis of the two samples.

[0065] In summary, the present invention is based on a mixed reagent and achieves protein reduction, alkylation, and enzymatic cleavage in a one-pot step, efficiently preparing peptides. This effectively increases the throughput of peptide preparation and reduces the corresponding time and instrument costs. It will greatly accelerate the development of basic research and industrial applications related to the preparation of peptides from proteins.

[0066] Parts or structures not specifically described in the present invention may adopt existing technologies or existing products and will not be described in detail here.

[0067] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for rapidly preparing a polypeptide, characterized in that: The following steps are involved: 1) Prepare mixed reagents; 2) The mixed reagent obtained in step 1) is mixed with the protein to react to produce a polypeptide.

2. A method for rapidly preparing a polypeptide according to claim 1, characterized in that: In step 1), the mixed reagent includes at least one of the following components: pH buffer, protein denaturant, stabilizer, protein solubilizing agent I, protein solubilizing agent II, anti-protein adsorption agent by tube wall, enzyme structure stabilizing agent, protein disulfide bond reducing agent, protein sulfhydryl alkylation agent, protein hydrolase.

3. A method for rapidly preparing a polypeptide according to claim 2, characterized in that: The pH buffer is at least one of NH4HCO3 buffer, tris-HCl buffer, and HEPES buffer, with a concentration of 0-5M.

4. A method for rapidly preparing a polypeptide according to claim 2, characterized in that: The protein denaturant is at least one of urea, thiourea, and guanidine hydrochloride, with a concentration of 0-10M.

5. The method for rapidly preparing a polypeptide according to claim 2, wherein: The stabilizer is at least one of glycerol and EDTA-free protease inhibitor.

6. A method for rapidly preparing a polypeptide according to claim 2, characterized in that: The protein dissolving aid I is at least one of sodium lauryl sulfate, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, and 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate.

7. The method for rapidly preparing a polypeptide according to claim 2, characterized in that: The enzyme structure stabilizing reagent is at least one of CaCl2 and MgCl2, with a concentration of 0-100 mM.

8. The method for rapidly preparing a polypeptide according to claim 2, characterized in that: The protein disulfide bond reducing agent is at least one of DTT, TCEP, and β-mercaptoethanol, with a concentration of 0-1000 mM.

9. The method for rapidly preparing a polypeptide according to claim 2, wherein: The protein thiol alkylation reagent is at least one of IAA and iodoacetamide CAA, with a concentration of 0-1000 mM; the protein hydrolase is at least one of trypsin and Lys-C.

10. The method for rapidly preparing a polypeptide according to claim 1, characterized in that: In step 2), the reaction time is 1-60 min, and the reaction temperature is room temperature.

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