Method for determining isoelectric point of protein or polypeptide without 280 nm absorption peak

Abstract

The invention relates to a method for determining isoelectric points of proteins and polypeptides, which is characterized in that a capillary isoelectric focusing electrophoresis method or a full-column imaging capillary isoelectric focusing electrophoresis method is used for determining according to a 214nm absorption peak. The method provided by the invention solves the problem that the isoelectric point of the protein or polypeptide which does not contain a benzene ring structure cannot be measured by a capillary isoelectric focusing electrophoresis method because the protein or polypeptide does not have ultraviolet absorption under the wavelength of 280 nm.

Description

Technical Field

[0001] This invention belongs to the field of biomacromolecule separation and analysis technology, and relates to a method for determining the isoelectric point of proteins or peptides. Specifically, it relates to a method for determining the isoelectric point of proteins or peptides without a 280nm absorption peak based on the 214nm absorption peak using capillary isoelectric focusing electrophoresis. Background Technology

[0002] The isoelectric point (pI) is the pH value of a solution at which the net charge of a protein or polypeptide molecule is zero. It is one of the important physicochemical properties of proteins and polypeptides. Accurate determination of the isoelectric point of polypeptides is of great significance for the separation and purification of proteins and polypeptides, structural analysis, bioactivity studies, and quality control in industrial production. For example, in the development of polypeptide drugs, isoelectric point data can guide the selection of appropriate chromatography media and elution conditions, improving separation efficiency and product purity; in proteomics research, the isoelectric point is an important basis for polypeptide identification and classification.

[0003] Currently, methods for determining the isoelectric point of proteins or peptides mainly include isoelectric focusing electrophoresis (CIEF) and slab isoelectric focusing electrophoresis. Among them, capillary isoelectric focusing electrophoresis (CIEF) is widely used for determining the isoelectric point of biomolecules due to its advantages such as small sample volume, fast analysis speed, high resolution, and high degree of automation. The basic principle of this method is that a stable pH gradient is formed in the capillary. When a protein or peptide molecule enters the electric field, it migrates in the pH gradient to a position equal to its isoelectric point (focusing region). At this point, the net charge of the protein or peptide molecule is zero, and it stops migrating. The isoelectric point can be determined by detecting the position of the focusing region.

[0004] Typical CIEF isoelectric point analysis is performed by detecting the characteristic absorption of the target sample at UV 280 nm. At this wavelength, the protein peak is not interfered with by the absorption at the 220 nm end by amphoteric electrolytes and other reagents. For example, proteins such as tryptophan, phenylalanine, and tyrosine contain benzene ring structures and have clear characteristic absorption peaks at UV 280 nm. However, some proteins or peptides do not contain benzene ring structures and have no UV absorption at 280 nm, making it impossible to obtain an absorption peak. Therefore, CIEF is generally considered unsuitable for protein or peptide samples without a UV 280 nm absorption peak. Currently, there is no data available on using capillary isoelectric focusing electrophoresis to determine the isoelectric point of proteins or peptides without a 280 nm absorption peak.

[0005] To address the problems existing in the prior art, this invention provides a method for determining the isoelectric point of proteins or peptides that do not have a 280nm absorption peak. This method utilizes capillary isoelectric focusing electrophoresis, with the determination based on the 214nm absorption peak. 214nm is the characteristic absorption wavelength of peptide bonds in protein or peptide molecules; the vast majority of proteins or peptides exhibit a significant absorption peak at this wavelength, thus enabling the effective detection of most proteins or peptides. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining the isoelectric point of proteins or peptides by using capillary isoelectric focusing electrophoresis through a 214 nm absorption peak. By optimizing the composition of the electrophoresis system, electrophoresis parameters, and data processing methods, the isoelectric point of proteins or peptides with a 214 nm absorption peak can be accurately and rapidly determined. This method also has strong adaptability and solves the problem that capillary isoelectric focusing electrophoresis is conventionally considered unsuitable for samples without UV280 absorption.

[0007] The objective of this invention is achieved through the following technical solution: A method for determining the isoelectric point of a protein or peptide, characterized by: using capillary isoelectric focusing electrophoresis to determine the isoelectric point based on the 214 nm absorption peak; the method includes the following steps: (1) Determine the electrophoretic pattern of sample A at 214 nm based on its isoelectric point. 214 Electrophoretic pattern at 280nm wavelength A 280 The electrophoretic pattern of isoelectric point standard B at a wavelength of 214 nm was determined. 214 ; (2) Compare the electrophoresis spectrum A. 214 Electrophoresis pattern A 280 The migration time of the standard in sample A at its isoelectric point was obtained. (3) Using the migration time of the standard in the isoelectric point sample A as the abscissa and the isoelectric point of the standard as the ordinate, fit a linear curve to obtain the standard curve; (4) Compare the electrophoretic pattern B of the isoelectric point standard B. 214 Electrophoretic pattern A of sample A with isoelectric point 214 The peaks and sample peaks are compared to determine the interference peaks and the migration time of the isoelectric point sample A is determined by the sample peaks. (5) Substitute the migration time of the isoelectric point sample A into the standard curve to calculate its isoelectric point.

[0008] In the method according to the present invention, the capillary isoelectric focusing electrophoresis is performed as follows: 1) Prepare the reagents for capillary electrophoresis as follows: Anode solution: Phosphoric acid, 180-220 mM Catholyte: Sodium hydroxide, 280-320mM Chemical migration solution: Acetic acid, 300-400mM Cathode stabilizer: Arginine, 400-600mM Anode stabilizer: Iminodiacetic acid, 180-220 mM Urea-CIEF Gel: Urea-CIEF Gel, 0-6M The amount of urea-CIEF gel used is 200 μL; the concentration of urea-CIEF gel is determined as follows: take urea solution, prepare urea-CIEF gels with gradient urea concentrations, and prepare a series of isoelectric point samples respectively; test on the instrument and observe the peak shape of the sample at 214 nm. The peak shape that is obvious and sharp is the appropriate urea-CIEF gel concentration.

[0009] 2) Prepare isoelectric point standard B and isoelectric point sample A respectively: (a) Preparation of isoelectric point standard B: Mix the following reagents in a centrifuge tube to prepare the isoelectric point standard. Multiple portions of the isoelectric point standard may be prepared as needed: 200 μL urea-CIEF gel, 10-20 μL 3-10 amphoteric electrolyte, 10-30 μL cathodic stabilizer, 2-8 μL anode stabilizer, and 2.0 μL-4.0 μL of isoelectric point standard; the isoelectric point standard is usually selected from 3 isoelectric points; (b) Preparation of isoelectric point sample A: Dissolve the protein or peptide sample in buffer solution to prepare a sample solution with a concentration of 5-10 mg / mL; the salt concentration in the sample solution shall not exceed 50 mM; the salt ion concentration in the buffer solution shall not exceed 50 mM. Take a certain volume ratio of isoelectric point standard B and mix it with the protein or peptide sample to be tested to prepare isoelectric point sample A for later use; wherein, the total volume of the isoelectric point standard and the isoelectric point sample to be tested is 200μL-260μL, and the volume ratio of the isoelectric point standard to the isoelectric point sample to be tested is 24:1-25:1. The concentration of the urea-CIEF gel is determined as follows: Take a urea solution, prepare urea-CIEF gels with gradient urea concentrations, and make a series of isoelectric point samples; test the sample peaks at 214 nm, and the peaks that are obvious and sharp are the appropriate urea-CIEF gel concentrations.

[0010] 3) Prepare the capillary tube: Select a neutral coated capillary tube, install it, put the cartridge into the system, cover the front cover, and check that the UV detector is installed correctly. The capillary has an inner diameter of 50 μm, a total length of 30.2 cm, an effective distance of 20 cm from the injection port to the detection window, and uses a 200 μm window slit.

[0011] 4) Prepare buffer trays and sample trays: (a) Place deionized water, deionized water, anolyte, urea solution, CIEF gel and chemical migration solution at positions A1, B1, C1, D1, E1 and F1 of the buffer inlet plate, respectively; place deionized water, waste liquid, catholyte, chemical migration solution and waste liquid at positions A1, B1, C1, D1 and E1 of the buffer outlet plate, respectively. Place the specified reagent (1.5 mL) in each buffer bottle and deionized water (0.8 mL) in the waste bottle. Cover with the blue cap, place the buffer bottles on the tray, and then place the tray into the instrument.

[0012] (b) Vortex the CIEF standard and sample thoroughly, transfer 200 μL of the standard and sample into the inner tube respectively, centrifuge at low speed to remove air bubbles, put the inner tube into the sample bottle and cover it with the blue cap, put it into the sample tray, and then put the sample tray into the instrument.

[0013] 5) Balance the capillary: Use the built-in method (CIEF Conditioning - PA 800 plus.met) to rinse with chemical migration solution for 5-10 min, ultrapure water for 2-5 min, and CIEF gel for 5-10 min in sequence.

[0014] 6) Sample separation using amphoteric electrolytes: Utilizing the instrument's built-in method (CIEF Separation-PA800plus.met), the sample solution is injected into the capillary via pressure injection at 25-26 psi for 99-100 seconds. Focusing is then performed at 25-26 kV for 15-16 minutes. After focusing, constant-voltage electrophoresis is performed at 30-32 kV for 30-32 minutes. Simultaneously, online detection is conducted using UV detectors at wavelengths of 214 nm and 280 nm, recording the electrophoretic patterns. 7) Rinse the capillary and turn off the UV lamp: Call the instrument's built-in method (CIEF Shutdown–PA800plus.met), rinse with ultrapure water for 2 minutes, rinse with CIEF gel for 5 minutes, and then turn off the UV lamp.

[0015] The method of this invention can be widely applied in fields such as peptide drug development, proteomics analysis, and industrial quality control.

[0016] The method of this invention is also performed using whole-column imaging capillary isoelectric focusing electrophoresis, for example, using a CEInfinite C01 (Advanced Electrophoresis Solutions, AES) or Maurice capillary electrophoresis apparatus (protein simple). Taking CEInfinite C01 as an example, the operation is as follows: Preparation of electrophoresis solution: Anodic solution: phosphoric acid, 80-100mM; Cathode solution: sodium hydroxide, 80-150mM; Urea: 0-6M; The urea concentration is determined as follows: prepare gradient urea aqueous solutions, prepare isoelectric point samples, test them on a testing machine, and observe the peak shape of the samples at 214 nm. The appropriate urea concentration is the one with obvious and sharp peak shape.

[0017] Prepare isoelectric point standards: 4% by volume of 3-10 amphoteric electrolytes, 0-6M urea, 0.25% by volume of methylcellulose, and 0.5-2.0 μL of each isoelectric point standard, for a total volume of 100 μL; multiple portions of the isoelectric point standards may be prepared as needed. Preparation of isoelectric point sample: Dissolve the protein or peptide sample in buffer solution to prepare a sample solution with a concentration of 5~10 mg / mL, and the salt concentration in the sample should not exceed 50 mM; take 2 μL of sample solution and mix it with 100 μL of isoelectric point standard to obtain the isoelectric point sample.

[0018] The samples were tested on the instrument, and images were acquired at detection wavelengths of 214nm and 280nm. The sample peaks were determined by comparison, and the isoelectric point was calculated using the method described above.

[0019] The present invention has the following beneficial effects: 1. By optimizing the composition of the electrophoresis system and the urea concentration, a distinct peak was formed in the sample at a wavelength of 214nm, reducing the interference of background peaks and improving the accuracy of isoelectric point determination.

[0020] 2. The pressure injection method allows for precise and controllable injection volume. Combined with online detection at the characteristic absorption wavelength of 214nm, it can quickly and efficiently obtain electrophoretic patterns of protein or peptide samples. The entire analysis process is short and requires a small amount of sample, meeting the needs of modern analytical detection for high efficiency and small sample volumes.

[0021] 3. By comparing the electrophoretic spectra at wavelengths of 214 nm and 280 nm, a standard curve was plotted and the isoelectric point of the sample was calculated. This effectively overcame the interference of background peaks at wavelength of 214 nm, further improving the accuracy and reliability of the measurement results. Attached Figure Description

[0022] Figure 1This is the electrophoresis pattern of the KLF peptide sample in Example 1 at a wavelength of 280 nm.

[0023] Figure 2 These are the electrophoretic patterns of the KLF peptide sample in Example 1 at wavelengths of 214 and 280 nm.

[0024] Figure 3 This is the electrophoretic pattern of the KLF peptide sample and isoelectric point standard in Example 1 at a wavelength of 214 nm.

[0025] Figure 4 This is the standard curve of the isoelectric point sample in Example 1 at the 214nm absorption peak. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments. These embodiments are merely illustrative and do not constitute any limitation on the present invention. Within the scope of the technical concept of the present invention, those skilled in the art can make various modifications. Based on the teachings of the present invention, all specific implementation schemes obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention. Example 1

[0027] In this embodiment, KLF peptide (KHNLTLQITKLLAF) was used as the analyte and capillary isoelectric focusing electrophoresis was performed. The capillary isoelectric focusing electrophoresis instrument used was the PA800 plus capillary electrophoresis pharmaceutical analysis system (SCIEX).

[0028] Mucin 1 (MUC1) plays a role in regulating tumor cell proliferation, migration, and stemness maintenance. KLF peptide (KHNLTLQITKLLAF) is a peptide that targets MUC1, exhibiting high specificity and affinity. KLF peptide does not contain a benzene ring structure and shows no UV absorption at 280 nm. The isoelectric point of KLF peptide was determined using the method of this invention. The specific experimental steps are as follows: 1. Preparation of reagents for capillary electrophoresis Prepare the following electrophoresis reagents in sequence: Among them, urea-CIEF gels with urea concentrations of 0, 2, 3.75M and 5M were prepared to form isoelectric point samples; the peak shape of the 3.75M sample at 214nm was observed to be the most obvious and sharp during instrument testing.

[0029] 2. Prepare isoelectric point standard and isoelectric point sample: Isoelectric point standard B: Mix the following reagents in a 0.5 mL centrifuge tube: 200 μL of 3.75 M urea-CIEF gel, 12.0 μL of 3-10 ampholyte (CE Healthcare, 17-0456-01), 20.0 μL of cathode stabilizer, 4.0 μL of anodic stabilizer, and 2.0 μL of standard per pI (SCIEX, A58481, isoelectric points 10.0, 7.0, and 5.5, respectively). Two copies of the isoelectric point standard sample are prepared. Isoelectric point sample A: Dissolve the KLF peptide sample in ultrapure water to prepare a KLF sample solution with a concentration of 5 mg / mL; mix 240 μL of isoelectric point standard with 10 μL of KLF sample solution to prepare an isoelectric point sample for later use.

[0030] 3. Prepare the capillary: Select a neutral-coated capillary (SCIEX, 477441). The neutral-coated capillary has an inner diameter of 50 μm, a total length of 30.2 cm, and an effective distance of 20 cm from the inlet to the detection window. Use a 200 μm window slit. After installing the capillary, insert the cartridge into the system, cover it with the front cover, and check that the UV detector is correctly installed.

[0031] 4. Prepare the buffer solution tray: Place deionized water, deionized water, anolyte, urea solution, CIEF gel, and chemifluid into positions A1, B1, C1, D1, E1, and F1 of the buffer solution inlet plate, respectively. Place deionized water, waste liquid, catholyte, chemifluid, and waste liquid into positions A1, B1, C1, D1, and E1 of the buffer solution outlet plate, respectively. Add 1.5 mL of the designated reagent to each buffer bottle, and 0.8 mL of deionized water to each waste liquid bottle. Seal the containers with the blue caps. After placing the buffer bottles, place the tray into the instrument.

[0032] Vortex the CIEF standard and sample thoroughly, transfer 200 μL of sample to the inner tube, centrifuge at low speed to remove air bubbles, place the inner tube into the sample vial and cover it with the blue cap, place it in the sample tray, and finally place the sample tray into the instrument.

[0033] 5. Balance the capillary: Use the instrument's built-in method, cIEF Conditioning - PA 800 plus.met, and rinse sequentially with chemical migration solution for 5 min, ultrapure water for 2 min, and CIEF gel for 5 min.

[0034] 6. Separate samples using amphoteric electrolytes: Call the instrument's built-in method cIEF Separation - PA 800plus.met. The sample solution was injected into the capillary via pressure injection at a pressure of 25 psi for 99 s. Focusing was then performed at a voltage of 25 kV for 15 min. After focusing, constant-voltage electrophoresis was conducted at a voltage of 30 kV for 30 min. Simultaneously, online detection was performed using ultraviolet detectors at wavelengths of 214 nm and 280 nm, recording the electrophoretic patterns. 7. Rinse the capillary tube and turn off the UV lamp. Use the instrument's built-in method cIEF Shutdown–PA 800 plus.met to rinse with ultrapure water for 2 minutes, then rinse with CIEF gel for 5 minutes, and turn off the UV lamp.

[0035] 8. Isoelectric point calculation: Electrophoretic pattern A of KLF peptide sample at 280 nm wavelength 280 ( Figure 1 As can be seen, the KLF peptide sample has no absorption peak at a wavelength of 280 nm.

[0036] Comparison of electrophoretic patterns of KLF peptide samples at 214 nm and 280 nm wavelengths A 214 and A 280 The migration time of isoelectric point standards can be determined. Figure 2 The migration time for the standard sample with a pI value of 10.0 was 19.80 min; the migration time for the standard sample with a pI value of 7.0 was 30.38 min; and the migration time for the standard sample with a pI value of 5.5 was 34.98 min. Plotting the migration time on the x-axis and the isoelectric point of the isoelectric point standard on the y-axis, a linear curve was fitted: Y = -0.2941*X + 15.85 (…). Figure 4 ). Among them, R 2 The value is 0.9989, indicating a good linear relationship.

[0037] Compare the electrophoretic patterns of isoelectric point standard B and isoelectric point sample A at a wavelength of 214 nm. 214 and A 214 This allows us to identify the interference peaks and sample peaks, thus determining the migration time of the isoelectric point sample to be 26.53 min. Figure 3 Substituting X=26.53 into the fitted linear curve Y=-0.2941*X+15.85, the isoelectric point of the KLF peptide was calculated to be 8.05.

Claims

1. A method for determining the isoelectric point of a protein or polypeptide, characterized in that: The method utilizes capillary isoelectric focusing electrophoresis to determine the absorption peak at 214 nm; the method includes the following steps: (1) Determine the electrophoretic pattern of sample A at 214 nm based on its isoelectric point. 214 Electrophoretic pattern A at 280nm wavelength 280 The electrophoretic pattern of isoelectric point standard B at a wavelength of 214 nm was determined. 214 ; (2) Compare the electrophoresis spectrum A. 214 Electrophoresis pattern A 280 The migration time of the standard in sample A at its isoelectric point was obtained. (3) Using the migration time of the standard in the isoelectric point sample A as the abscissa and the isoelectric point of the standard as the ordinate, fit a linear curve to obtain the standard curve; (4) Compare the electrophoretic pattern B of the isoelectric point standard B. 214 Electrophoretic pattern A of sample A with isoelectric point 214 The peaks and sample peaks are compared to determine the interference peaks and the migration time of the isoelectric point sample A is determined by the sample peaks. (5) Substitute the migration time of the isoelectric point sample A into the standard curve to calculate its isoelectric point.

2. The method according to claim 1, wherein in step (1), the determination is performed as follows: 1) Preparation of capillary electrophoresis reagents, including: The anolyte is phosphoric acid, the catholyte is sodium hydroxide, the chemical migration solution is acetic acid, the catholyte is arginine, the anolyte is iminodiacetic acid, and the urea-CIEF adhesive is used. 2) Prepare isoelectric point standard and isoelectric point sample respectively: (a) Preparation of isoelectric point standard B: Mix 200 μL of urea-CIEF gel, 10-20 μL of 3-10 amphoteric electrolyte, 10-30 μL of cathode stabilizer, 2.0-8.0 μL of anode stabilizer and 2.0-4.0 μL of standard samples for each isoelectric point to prepare isoelectric point standard samples. (b) Preparation of isoelectric point sample A: Dissolve the protein or peptide sample in buffer solution to prepare a sample solution with a concentration of 5-10 mg / mL; Mix isoelectric point standard B with the sample to be tested in a certain volume ratio to prepare isoelectric point sample A for later use; 3) Use neutral-coated capillaries; 4) Prepare the buffer solution tray and sample tray, and then place the trays into the instrument; 5) Rinse the capillary sequentially with chemical migration solution, ultrapure water, and CIEF glue to equilibrate it; 6) The sample solution is injected into the capillary via pressure injection, followed by focusing; After focusing, constant voltage electrophoresis was performed; simultaneously, an ultraviolet detector was used to detect and record the electrophoretic patterns online at wavelengths of 214 nm and 280 nm, respectively. 7) Rinse the capillary tube sequentially with ultrapure water and CIEF glue.

3. The method according to claim 2, wherein in step 1), the concentration of phosphoric acid is 180-220 mM; the concentration of sodium hydroxide is 280-320 mM; the concentration of acetic acid is 300-400 mM; the concentration of arginine is 400-600 mM; the concentration of iminodiacetic acid is 180-220 mM; and the concentration of urea-CIEF gel is 0-6 M.

4. The method according to claim 2, wherein in step 1), the concentration of the urea-CIEF gel is determined as follows: take a urea solution, prepare urea-CIEF gels with gradient urea concentrations, and prepare a series of isoelectric point samples respectively; test on the instrument, observe the peak shape of the sample at 214nm, and determine the appropriate urea-CIEF gel concentration if the peak shape is obvious and sharp.

5. The method according to claim 2, wherein in step 2), the salt concentration in the sample solution is not greater than 50 mM; the salt ion concentration in the buffer solution is not greater than 50 mM; the total volume of the isoelectric point standard and the isoelectric point sample to be tested is 200 μL-260 μL, and the volume ratio of the isoelectric point standard to the isoelectric point sample to be tested is 24:1-25:

1.

6. The method according to claim 5, wherein in step 3), the capillary has an inner diameter of 50 μm, a total length of 30.2 cm, an effective distance from the injection port to the detection window of 20 cm, and a 200 μm window slit is used.

7. The method according to claim 2, wherein in step 6), the injection pressure is 25-26 psi and the injection time is 99-100 s; the focusing voltage is 25-26 kV and the focusing time is 15-16 min; the electrophoresis voltage is 30-32 kV and the electrophoresis time is 30-32 min.

8. The method according to claim 2, wherein in steps (5), (6) and (7), the corresponding built-in instrument methods are invoked for operation.

9. The method of claim 1, wherein the determination is performed using whole-column imaging capillary isoelectric focusing electrophoresis.

10. The application of the method according to any one of claims 1-9 in peptide drug development, proteomics analysis, and industrial quality control.

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