Detection and analysis method of protein polymer
Through the combination of capillary isoelectric focus electrophoresis technology and premix solution, the detection and analysis of protein polymers is optimized, the problems of poor detection accuracy and repetition are solved, and efficient isoelectric point detection of protein polymers isoelectric point, especially The separation effect was significantly improved at high concentration samples.
Patent Information
- Application Number
- CN202411324098.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2024-12-27
AI Technical Summary
The existing protein polymer detection and analysis methods have problems with poor detection accuracy and repetition, especially when the separation time is too long and the peak time is offset, which affects the reliability of the detection results.
Capillary isoelectric focusing electrophoresis technology is adopted to conduct electrophoresis analysis by mixing premixed liquid composed of cathode stabilization liquid, anode stabilization liquid, urea solution and amphoteric electrolyte with protein polymer, forming a PH gradient and focusing separation, combining with UV-visible spectrophotometer Perform detection to optimize isoelectric point detection of protein polymers.
Improve the detection accuracy and repeatability of protein polymers, shorten the detection time, and ensure the separation effect of high-concentration samples. Especially when the protein polymer concentration is 10mg/ml, the peak time and repeatability achieve good consistency. .
Smart Images
Figure CN119198879A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a detection and analysis method for protein polymers. Background Art
[0002] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely present in tissues such as bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, and urine. They have the characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and easy collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN), etc., and participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, immune maturation and other processes. They can be used for immune regulation, tissue repair and treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome.
[0003] The inventors have discovered that by stimulating MSCs and then lysing them, a protein polymer with nerve repair activity can be separated and purified from intracellular proteins. This protein polymer has achieved significant results in completed animal and clinical trials, especially for ALS, and has a clear potential for drug development, and has now entered the clinical trial stage.
[0004] In order to further explore the drug-making possibility of this protein polymer, the study of its physical and chemical properties is crucial, among which the isoelectric detection and analysis of protein polymers is a very important aspect. Capillary isoelectric focusing electrophoresis (CIEF) can separate proteins or peptides according to their isoelectric points, and has the advantages of high resolution and high accuracy. Using capillary isoelectric focusing electrophoresis technology to develop an isoelectric point detection and analysis method with high accuracy and reliability for the above-mentioned protein polymers is a very valuable research direction. Summary of the invention
[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for detecting and analyzing protein polymers.
[0006] The technical solution adopted by the present invention is:
[0007] A method for detecting and analyzing protein polymers, the method comprising the following steps:
[0008] S1: Measure the cathode stabilizing solution, the anode stabilizing solution, the urea solution, the ampholyte and the isoelectric point marker, and mix them into a premixed solution for later use;
[0009] S2: mixing the protein polymer with the premix prepared in step S1 and injecting the mixture into a capillary electrophoresis instrument, applying voltage to form a pH gradient in the capillary of the capillary electrophoresis instrument and performing focusing separation;
[0010] S3: Obtaining the isoelectric focusing map of the protein polymer through a detector and performing detection and analysis.
[0011] The concentration of the protein polymer in step S2 is 5-60 mg / ml, the concentration of the cathode stabilizing solution in step S1 is 100-800 mM, and preferably, the concentration of the protein polymer in step S2 is 10 mg / ml.
[0012] According to the experimental results in the examples of this application, a protein polymer concentration of 5 to 60 mg / ml and a cathode stabilizing solution concentration of 100 to 800 mM can obtain a better peak shape and reduce the peak time, further shortening the detection time, thereby improving the detection accuracy of the protein polymer of this application and obtaining better consistency in repeated detection.
[0013] In some possible implementations, the cathode stabilizing liquid in step S1 is arginine, and the anode stabilizing liquid is iminodiacetic acid.
[0014] In some possible implementations, the cathode stabilizing solution is prepared by measuring arginine, transferring it to a centrifuge tube, adding ionized water to mix thoroughly and vibrating to completely dissolve the solid, adding deionized water, and storing at 2-8° C. for 14 days.
[0015] In some possible implementations, the urea solution is 4M urea gel.
[0016] In some possible embodiments, the protein polymer needs to be subjected to ultrafiltration and liquid exchange treatment before step S2, wherein the ultrafiltration and liquid exchange treatment uses a 3KD ultrafiltration tube to add water for ultrafiltration and liquid exchange, and the salt concentration of the protein polymer is lower than 80 mM.
[0017] In some possible embodiments, the amphoteric electrolytes in step S1 can be divided into amphoteric electrolytes with a pH of 3 to 10 and amphoteric electrolytes with a pH of 5 to 8 according to the pH value, and the mass ratio of the amphoteric electrolytes with a pH of 3 to 10 to the amphoteric electrolytes with a pH of 5 to 8 is 1:(1-3).
[0018] In some possible implementations, the capillary electrophoresis instrument is a capillary electrophoresis instrument of model PA800Plus produced by SCIEX, and the inner wall of the capillary is coated with a neutral coating.
[0019] In some possible implementations, the focusing separation condition is that the separation voltage is 25-50 KV and the duration is 5-10 min.
[0020] In some possible embodiments, the detector is one of an ultraviolet-visible spectroscopic detector, a laser induced fluorescence detector, an electrochemical detector, a mass spectrometric detector, a nuclear magnetic resonance detector, an LED detector, and a chemiluminescence detector.
[0021] In some possible embodiments, the detector is an ultraviolet-visible spectroscopic detector, and its detection wavelength is 280 nm. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 It is the isoelectric focusing spectrum of the sample to be tested with a protein polymer concentration of 5 to 15 mg / ml in the present application.
[0023] Figure 2 It is the isoelectric focusing spectrum of the sample to be tested with a protein polymer concentration of 15 to 30 mg / ml in the present application.
[0024] Figure 3 It is the isoelectric focusing spectrum of the sample to be tested with a protein polymer concentration of 30 to 100 mg / ml in the present application.
[0025] Figure 4 It is the isoelectric focusing spectrum of the samples of the present application with arginine concentrations of 100mM, 200mM, 400mM and 500mM respectively.
[0026] Figure 5 It is the isoelectric focusing spectrum of the sample prepared by mixing ampholytes with pH values of 3 to 10 and ampholytes with pH values of 5 to 8 in a mass ratio of 1:0, 1:1, 1:2 and 1:3.
[0027] Figure 6 It is the isoelectric focusing spectrum of 6 samples prepared in parallel when the protein polymer concentration of the present application is 30 mg / ml, the arginine concentration is 500 mM, and the amount of 3 to 10 ampholytes.
[0028] Figure 7 It is the isoelectric focusing spectrum of three samples prepared in parallel when the concentration of the protein polymer of the present application is 10 mg / ml, the concentration of arginine is 500 mM, and the amount of ampholytes is 3 to 10. DETAILED DESCRIPTION
[0029] The technical solution of the present invention is further described below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.
[0030] The production process of the protein polymer samples used in the following experiments includes:
[0031] Cultivating mesenchymal stem cells and using ultraviolet irradiation to create a stress environment;
[0032] The mesenchymal stem cells are lysed and separated and purified using size exclusion chromatography to obtain protein polymers; wherein, under the size exclusion chromatography condition of a flow rate of 0.2 ml / min, the elution volume of the first component peak is 12-13.2 ml, the elution volume of the second component peak is 15.2-17 ml, the elution volume of the third component peak is 17-20 ml, the elution volume of the fourth component peak is 29-31 ml, and the elution volume of the fifth component peak is 31-34 ml.
[0033] Capillary electrophoresis is used to detect the isoelectric point of protein polymer samples
[0034] S01: Pre-solution preparation
[0035] Prepare cathode stabilizing solution: weigh 6.5 g of arginine, transfer to a 15 ml centrifuge tube, add 8 ml of deionized water, mix thoroughly and shake to completely dissolve the solid, add deionized water to the 10 ml mark, and store at 2-8°C for 14 days.
[0036] Prepare anode stabilizing solution: weigh 3 g of iminodiacetic acid on an analytical balance, transfer to a 15 ml centrifuge tube, add 8 ml of deionized water, mix thoroughly and shake to completely dissolve the solid, add deionized water to the 10 ml mark and mix well, and store at room temperature for 30 days.
[0037] Prepare 4M urea gel: weigh 5g urea and transfer to a 15ml centrifuge tube, add 7ml CIEF gel, vortex for 5 minutes to completely dissolve the solid, add CIEF gel to the 10ml mark, vortex for 1 minute, invert the centrifuge tube three times, mix the solution, filter with a filter membrane, and collect the filtrate into a new 15ml centrifuge tube. Store at 2-8℃ for 30 days.
[0038] The anode stabilizing solution, cathode stabilizing solution, urea gel, amphoteric electrolytes and isoelectric point markers prepared above are mixed to obtain a pre-solution.
[0039] S02: Test sample preparation
[0040] The protein polymer sample to be tested is ultrafiltered with water using a 3KD ultrafiltration tube to replace the solution, ensuring that the protein polymer salt concentration is less than 80mM, and then prepared according to the following table to obtain the test solution:
[0041] Reagent name Reagent dosage 4M urea glue 300μl 100 mM arginine 30.0μl 300 mM iminodiacetic acid 5.0μl Amphoteric electrolytes 18.0μl Isoelectric point markers (3) 3.0 μl each 5mg / ml sample 10.0μl
[0042] Among them, the main instruments and equipment
[0043]
[0044] Main reagents and consumables
[0045]
[0046]
[0047] S03: Sample loading test
[0048] The prepared test solution was sent to the machine for detection, and the detection conditions were set as follows: acquisition frequency: 6 Hz, focusing voltage 25-50 KV, injection volume 25-100 μl, capillary temperature 10-40 ° C, elution pressure 10-40 psi; separation conditions were set as follows: separation voltage: 25-30 KV, duration 5-10 minutes.
[0049] Regarding the storage conditions of the capillaries, in some embodiments, for short-term storage of the capillaries (1 to 3 days), the coated capillaries can be left in the instrument with both ends immersed in deionized water; for long-term storage (more than 3 days), the capillaries should be placed in the original capillary storage box with both ends immersed in deionized water, and then the capillary storage box should be placed vertically in a 4°C refrigerator for storage.
[0050] Acid and alkali are added to the two electrode grooves respectively. After voltage is applied, the electrolyte solution in the capillary gradually forms a pH gradient. When each solute migrates to its own isoelectric point in the capillary, it becomes neutral and forms a focused zone. The solute is passed into the UV-visible spectrophotometer for detection. The isoelectric focusing spectrum of the sample is obtained by the above detection method.
[0051] Protein polymer sample concentration analysis
[0052] Samples with a protein polymer concentration of 5 to 15 mg / ml were taken and tested separately. The results are shown in Table 1 and Figure 1 It can be seen that the isoelectric point ranges from 5.3 to 6.0, and the peak shape gradually improves with the increase of sample concentration.
[0053] Sample concentration Peak1 Peak2 Peak3 Peak4 5mg / ml 5.34 5.41 5.90 5.99 7.5mg / ml 5.35 5.41 5.93 5.99 10mg / ml 5.36 5.42 5.94 6.00 15mg / ml 5.34 5.42 5.95 6.03
[0054] Continue to take samples with protein polymer concentrations in the range of 15 to 30 mg / ml and test them separately. The results are shown in the table below and Figure 2 It can be seen that the isoelectric point ranges from 5.1 to 6.1, and the peak shape gradually improves with the increase of sample concentration.
[0055] Sample concentration Peak1 Peak2 Peak3 Peak4 Peak5 Peak6 Peak7 15mg / ml 5.33 5.40 5.47 5.93 6.00 N / A N / A 20mg / ml 5.12 5.31 5.39 5.45 5.93 5.99 N / A 25mg / ml 5.11 5.30 5.38 5.44 5.94 6.00 6.09 30mg / ml 5.10 5.29 5.37 5.44 5.95 6.02 6.10
[0056] Continue to take samples with protein polymer concentrations in the range of 30 to 100 mg / ml and test them separately. The results are shown in the table below and Figure 3It can be seen that after the sample concentration reaches 60 mg / ml, the peak time gradually shifts as the sample concentration increases. Even when the sample concentration reaches 80 mg / ml, no peak appears within the separation time.
[0057]
[0058] Analysis of arginine concentration in protein polymer samples
[0059] Samples with arginine concentrations of 100mM, 200mM, 400mM and 500mM were taken and tested respectively. The results are shown in the table below and Figure 4 .
[0060]
[0061] Continue to take samples with arginine concentrations of 600mM, 700mM, and 800mM and test them respectively. The results are shown in the table below. Figure 4 It can be seen that under different arginine concentrations, the isoelectric point ranges from 5.1 to 6.0.
[0062]
[0063] Analysis of ampholytes in protein polymer samples
[0064] Samples prepared by mixing pH 3-10 ampholytes with pH 5-8 ampholytes in a mass ratio of 1:0, 1:1, 1:2 and 1:3 were tested respectively. The results are shown in the table below and Figure 5 ,It can be seen that with the increase of the ratio of amphoteric electrolytes from 5 to 8, the isoelectric point distribution of the samples shifts toward the base, and the separation effects of 1:1, 1:2 and 1:3 are generally similar, and there is no significant improvement compared to the 1:0 ratio.
[0065]
[0066] Repeatability analysis of detection method
[0067] Take the protein polymer sample concentration of 30mg / ml, arginine concentration of 500mM, and select 3-10 ampholytes. Prepare 6 samples in parallel and test them separately. The results are shown in the table below and Figure 6 It can be seen that the isoelectric point ranges from 5.2 to 6.4, but the peak time deviates seriously and the repeatability is poor. It may be because the sample concentration is too high, resulting in sample residue after multiple injections, causing the peak time to shift later.
[0068] name StartpI StoppI StartTime StopTime Repeatability 1 5.26 6.31 27.46 32.79 Repeatability 2 5.24 6.37 34.58 39.78 Repeatability 3 5.25 6.20 34.64 39.40 Repeatability 4 5.24 6.18 35.23 39.69 Repeatability 5 5.25 6.20 34.24 38.73 Repeatability 6 N / A N / A N / A N / A
[0069] Take the protein polymer sample concentration of 10mg / ml, arginine concentration of 500mM, and select 3-10 ampholytes. Prepare 3 samples in parallel and test them separately. The results are shown in the table below and Figure 7 It can be seen that the isoelectric point ranges from 5.4 to 6.1, with RSD < 1%, and the peak time is between 23.7 and 25.9, with RSD < 1%, with good repeatability. Therefore, when the concentration of the sample to be tested is 10 mg / ml, the repeatability of the isoelectric point and peak time is better.
[0070] name StartpI StoppI StartTime StopTime Repeatability 1 5.41 6.09 23.74 25.94 Repeatability 2 5.40 6.09 23.73 25.92 Repeatability 3 5.40 6.09 23.77 25.94 RSD 0.1％ 0.0％ 0.1％ 0.0％
[0071] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.
Claims
1. A method for detecting and analyzing protein polymers, characterized in that: The method comprises the following steps: S1: Measure the cathode stabilizing solution, the anode stabilizing solution, the urea solution, the ampholyte and the isoelectric point marker, and mix them into a premixed solution for later use; S2: mixing the protein polymer with the premix prepared in step S1 and injecting the mixture into a capillary electrophoresis instrument, applying voltage to form a pH gradient in the capillary of the capillary electrophoresis instrument and performing focusing separation; S3: Obtaining the isoelectric focusing map of the protein polymer through a detector and performing detection and analysis. The concentration of the protein polymer in step S2 is 5-60 mg / ml, the concentration of the cathode stabilizing solution in step S1 is 100-800 mM, and preferably, the concentration of the protein polymer in step S2 is 10 mg / ml.
2. The detection and analysis method according to claim 1, characterized in that: The cathode stabilizing liquid in step S1 is arginine, and the anode stabilizing liquid is iminodiacetic acid.
3. The detection and analysis method according to claim 2, characterized in that: The preparation method of the cathode stabilizing solution is as follows: measure arginine, transfer it to a centrifuge tube, add ionized water, mix it thoroughly and shake it to completely dissolve the solid, add deionized water, and store it at 2-8° C. for 14 days.
4. The detection and analysis method according to claim 1, characterized in that: The urea solution is 4M urea gel.
5. The detection and analysis method according to claim 1, characterized in that: The protein polymer needs to be subjected to ultrafiltration and liquid replacement treatment before step S2. The ultrafiltration and liquid replacement treatment adopts 3KD ultrafiltration tube with water and ultrafiltration and liquid replacement, and the salt concentration of the protein polymer is lower than 80mM.
6. The detection and analysis method according to claim 1, characterized in that: The amphoteric electrolytes in step S1 are divided into amphoteric electrolytes with a pH of 3 to 10 and amphoteric electrolytes with a pH of 5 to 8 according to the pH value, and the mass ratio of the amphoteric electrolytes with a pH of 3 to 10 to the amphoteric electrolytes with a pH of 5 to 8 is 1:(1-3).
7. The detection and analysis method according to claim 1, characterized in that: The capillary electrophoresis instrument is a PA 800Plus capillary electrophoresis instrument produced by SCIEX, and the inner wall of the capillary is coated with a neutral coating.
8. The detection and analysis method according to claim 1, characterized in that: The conditions for the focusing separation are that the separation voltage is 25-50 KV and the duration is 5-10 min.
9. The detection and analysis method according to claim 1, characterized in that: The detector is one of an ultraviolet-visible spectroscopic detector, a laser-induced fluorescence detector, an electrochemical detector, a mass spectrometric detector, a nuclear magnetic resonance detector, an LED detector, and a chemiluminescence detector.
10. The detection and analysis method according to claim 1, characterized in that: The detector is an ultraviolet-visible spectroscopic detector, and its detection wavelength is 280nm.