Method for identifying vinasse polypeptide
Through specific buffer treatment and two-dimensional liquid chromatography combined with high-resolution mass spectrometry technology, the problem of impurity interference in the identification of peptides in lees was solved, efficient and accurate peptide identification was achieved, and the separation degree and signal intensity were improved.
Patent Information
- Application Number
- CN202511027018.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-24
- Publication Date
- 2025-09-16
AI Technical Summary
Existing technologies make it difficult to efficiently and accurately identify polypeptides in distiller's grains, especially due to chromatographic peak overlap and reduced mass spectrometry detection sensitivity caused by impurity interference, and antibody recognition methods are difficult to adapt to distiller's grains with a wide variety of polypeptides and complex structures.
Sample pretreatment was performed using a buffer solution with a specific ratio. Two-dimensional liquid chromatography and high-resolution mass spectrometry were combined, including variable speed centrifugation, special membrane filtration, ultrafiltration, strong cation exchange chromatography, reversed-phase liquid chromatography, and electrospray ionization source, to identify the peptide structure by tandem mass spectrometry.
The efficient extraction and purification of distiller's grains peptides was achieved, the separation degree and identification accuracy of peptides were improved, the mass spectrometry signal intensity was increased by more than 2 times, and the number and accuracy of peptide identifications were significantly improved.
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Figure BDA0005516244640000061
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of polypeptide identification, and in particular to a method for identifying vinasse polypeptides. Background Art
[0002] Distillers' grains, a byproduct of the winemaking process, are widely available and rich in various nutrients. Peptides, among them, possess diverse biological activities, such as antioxidant, antihypertensive, and antibacterial properties, and have potential applications in food, medicine, and other fields. However, the complex composition of distillers' grains presents numerous challenges in identifying peptides.
[0003] Traditional peptide identification methods, such as high-performance liquid chromatography-mass spectrometry (HPLC-MS), present several challenges in identifying peptides from distiller's grains. Distiller's grain extracts contain not only peptides but also numerous carbohydrates, lipids, pigments, and other small-molecule impurities. These complex components can interfere with peptide separation during HPLC. For one thing, some impurities have similar retention times to peptides, resulting in overlapping chromatographic peaks and making it difficult to accurately separate and identify peptides. For example, under certain chromatographic conditions, the elution time of carbohydrates can be similar to that of some small-molecule peptides, making it difficult to clearly distinguish peptide signals during mass spectrometry. Furthermore, the presence of numerous impurities can reduce the sensitivity of mass spectrometry. Mass spectrometers analyze ions in the sample, and the presence of numerous impurity ions competes with peptide ions for detection channels, weakening the peptide ion signal. This can make it difficult to effectively detect peptides with low concentrations and inherently weak mass spectrometry signals, leading to missed detection and inability to fully identify the peptide components in distiller's grains.
[0004] Furthermore, some antibody-based peptide identification methods, such as enzyme-linked immunosorbent assay (ELISA), while highly specific, require the preparation of antibodies targeting specific peptides. However, the diverse and complex structures of peptides in distiller's grains make it difficult to prepare antibodies specific for all of them. Furthermore, the peptide compositions in distiller's grains from different sources vary, and existing antibodies may not be able to recognize newly identified peptides, limiting the widespread application of this method for peptide identification in distiller's grains.
[0005] In summary, there is currently a lack of a method that can efficiently and accurately identify peptides in distiller's grains. It is necessary to develop a new technical solution to overcome the above-mentioned problems in the existing technology, so as to achieve comprehensive and accurate identification of distiller's grains peptides and provide technical support for the high added value utilization of distiller's grains. Summary of the Invention
[0006] The present invention aims to provide a method for identifying vinasse polypeptides, which can efficiently and accurately identify polypeptides in vinasse.
[0007] In order to achieve the above object, the present invention provides the following technical solutions:
[0008] A method for identifying vinasse polypeptides, comprising:
[0009] Sample pretreatment: The lees sample is mixed with a buffer solution of a specific ratio for extraction. After extraction, it is centrifuged at a variable speed, filtered with a special filter membrane, and ultrafiltered to obtain a high-purity polypeptide concentrate.
[0010] Multidimensional chromatographic separation: Using a two-dimensional liquid chromatography system, the first dimension separates the peptide concentrate by strong cation exchange chromatography combined with gradient elution to collect different fractions; the second dimension separates each fraction by reversed-phase liquid chromatography using enhanced separation processing to obtain multiple peptide single components;
[0011] High-resolution mass spectrometry identification: A single peptide component is introduced into a high-resolution mass spectrometer, ionized using an electrospray ionization source, and fragment ion information is obtained using a tandem mass spectrometry method. The amino acid sequence of the peptide is inferred by analyzing the mass-to-charge ratio and fragment ion information. At the same time, the experimental data is compared with the peptide database using mass spectrometry data analysis software to determine the structure and origin of the peptide.
[0012] Working principle and beneficial effects:
[0013] During sample pretreatment, a specifically formulated buffer solution provides a stable dissolution environment for polypeptides, and its ionic composition can reduce the nonspecific binding of polypeptides to the lees matrix, thereby increasing the leaching rate. Variable-speed centrifugation first separates large-particle impurities, and then uses high-speed centrifugation to precipitate tiny particles, reducing the subsequent filtration load. Special filter membranes utilize nanoscale pores to physically intercept smaller impurities, while the material properties reduce polypeptide adsorption losses. Ultrafiltration further concentrates the target polypeptide through molecular size screening. The four work together to achieve efficient extraction and purification of polypeptides from complex matrices, laying a high-purity foundation for subsequent separation and identification.
[0014] In multidimensional chromatographic separation, the first dimension, strong cation exchange chromatography, is based on the charge difference of peptides, combined with the dynamic adjustment of ionic strength and pH in gradient elution to achieve the preliminary separation of peptides with different charge characteristics; the second dimension, reversed-phase liquid chromatography, is based on the hydrophobicity difference of peptides. The amino-modified chromatographic column enhances the interaction with polar peptides. Temperature control optimizes retention behavior by changing the distribution coefficient, and combined with gradient elution, it greatly improves the separation degree, so that complex peptide mixtures can be disassembled into single components.
[0015] In high-resolution mass spectrometry identification, the electrospray ionization source converts the peptide into charged ions, tandem mass spectrometry provides structural information through fragment ion analysis, and database comparison determines the peptide identity by matching known sequences.
[0016] The optimized sample buffer consists of sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride in a molar ratio of 3-5:2-3:1, with a sodium dihydrogen phosphate concentration of 20-50 mmol / L, a disodium hydrogen phosphate concentration of 10-30 mmol / L, and a sodium chloride concentration of 5-15 mmol / L. The sodium dihydrogen phosphate and disodium hydrogen phosphate form a buffer pair in the buffer, maintaining the pH stability of the system through acid-base balance, preventing denaturation or precipitation of polypeptides due to drastic pH changes. Sodium chloride provides appropriate ionic strength, competitively binding to charged adsorption sites in the lees matrix, reducing adsorption of polypeptides to the matrix.
[0017] Optimized, the variable speed centrifugation is first centrifuged at a speed of 3000-5000 r / min for 5-10 minutes, then the speed is increased to 8000-12000 r / min and centrifuged for 10-20 minutes, and the temperature is maintained at 4-10°C during the centrifugation process.
[0018] The difference in centrifugal force is used to precipitate crude fibers, large-particle proteins, etc. in the lees, preventing these impurities from forming dense precipitation and wrapping the polypeptides during high-speed centrifugation; then high-speed centrifugation at 8000-12000r / min is used to precipitate tiny particles through greater centrifugal force; the low temperature environment of 4-10℃ reduces the enzymatic hydrolysis or denaturation of polypeptides during the centrifugation process.
[0019] Optimized, the special filter membrane is a nanofiber filter membrane made of polyethersulfone, with a pore size distribution of 0.1-0.5 μm and a porosity of 70-90%. During filtration, the pressure is controlled at 0.1-0.3 MPa and the temperature is 20-30°C.
[0020] The pore size of 0.1 to 0.5 μm can physically intercept the tiny colloidal particles and some macromolecular impurities remaining after centrifugation.
[0021] The optimized gradient elution strategy is as follows: mobile phase A is an aqueous solution containing 5-10 mmol / L potassium dihydrogen phosphate (pH = 2.5-3.5), and mobile phase B is an aqueous solution containing 5-10 mmol / L potassium dihydrogen phosphate and 0.5-1.0 mol / L sodium chloride (pH = 2.5-3.5); the volume fraction of mobile phase B in the initial elution stage is 5%-10%, and gradually increases to 90%-95% at a rate of 0.5%-2% / min, and at the same time, the pH value of the mobile phase is gradually adjusted from 3.5±0.1 to 2.5±0.1 through an online pH adjustment device.
[0022] The potassium dihydrogen phosphate in mobile phases A and B maintains the basic ionic strength, and sodium chloride serves as a competing ion. Its concentration increases over time (5%-10% to 90%-95%), which can gradually displace the peptides bound to the strong cation exchange chromatography column. The pH decreases from 3.5±0.1 to 2.5±0.1, which modulates the retention behavior by changing the charge state of the peptides and enhances the separation of weakly acidic peptides.
[0023] The optimized reversed-phase liquid chromatography uses a C18 column with an amino group modified on the surface, the column specifications are 100-200 mm × 2.1-4.6 mm, and the filler particle size is 2-5 μm; the temperature-controlled elution method is an initial temperature of 30-40°C, which is increased to 50-60°C at a rate of 1-2°C / min, and the mobile phase is an aqueous solution containing 0.1%-0.5% formic acid and an acetonitrile solution containing 0.1%-0.5% formic acid, using gradient elution and a flow rate of 0.2-0.5 mL / min.
[0024] The C18 column, modified with amino groups on its surface, retains hydrophobic interactions while also forming hydrogen bonds with the polar groups of the peptides, enhancing the retention of polar peptides. Raising the temperature from 30-40°C to 50-60°C reduces the interaction between the peptides and the stationary phase, shortening retention time and improving peak shape. A mobile phase containing formic acid promotes peptide ionization, and gradient elution (with increasing proportions of acetonitrile) elutes peptides based on hydrophobicity. This step synergizes with the first-dimensional separation, with the former separating by charge and the latter by hydrophobicity, forming a two-dimensional orthogonal separation system. Peak capacity is more than doubled compared to one-dimensional chromatography, and the purity of individual components is >95%, meeting the requirements for mass spectrometry identification.
[0025] Optimized, the strong electric field enhancement device in the electrospray ionization source generates a strong electric field of 1.0 to 3.0 kV / cm at the outlet of the electrospray capillary, and the ion focusing structure is to set 3 to 5 focusing electrodes on the ion transmission path, and the potential difference between the electrodes is 50 to 200 V.
[0026] The polypeptide solution is formed into charged droplets, which accelerates the droplet splitting and solvent evaporation. 3 to 5 focusing electrodes with a potential difference of 50 to 200 V form an electric field gradient, focusing the ions into a narrow beam. This ion source is combined with a high-purity single component in the preceding sequence to reduce the suppression effect of impurity ions, increase the mass spectrometry signal intensity by more than 2 times, and provide richer fragment ion information. After comparison with the database, the accuracy of polypeptide identification is improved. DETAILED DESCRIPTION
[0027] The following is further described in detail through specific implementation methods:
[0028] Example 1: A method for identifying vinasse polypeptides, comprising:
[0029] Sample pretreatment: The lees sample is mixed with a buffer solution of a specific ratio for extraction. After extraction, it is centrifuged at a variable speed, filtered with a special filter membrane, and ultrafiltered to obtain a high-purity polypeptide concentrate.
[0030] Multidimensional chromatographic separation: Using a two-dimensional liquid chromatography system, the first dimension separates the peptide concentrate by strong cation exchange chromatography combined with gradient elution to collect different fractions; the second dimension separates each fraction by reversed-phase liquid chromatography using enhanced separation processing to obtain multiple peptide single components;
[0031] High-resolution mass spectrometry identification: A single peptide component is introduced into a high-resolution mass spectrometer, ionized using an electrospray ionization source, and fragment ion information is obtained using a tandem mass spectrometry method. The amino acid sequence of the peptide is inferred by analyzing the mass-to-charge ratio and fragment ion information. At the same time, the experimental data is compared with the peptide database using mass spectrometry data analysis software to determine the structure and origin of the peptide.
[0032] The sample buffer solution is composed of sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in a molar ratio of 4:2:1, wherein the concentration of sodium dihydrogen phosphate is 30 mmol / L, the concentration of disodium hydrogen phosphate is 20 mmol / L, and the concentration of sodium chloride is 10 mmol / L.
[0033] The variable speed centrifugation was performed by first centrifuging at a speed of 4000 r / min for 8 minutes, then increasing the speed to 10000 r / min and continuing centrifuging for 15 minutes, and the temperature was maintained at 5°C during the centrifugation process.
[0034] The special filter membrane is a nanofiber filter membrane made of polyethersulfone with a pore size distribution of 0.3μm and a porosity of 80%. The pressure during filtration is controlled at 0.2MPa and the temperature is 25℃.
[0035] The gradient elution strategy was as follows: mobile phase A was an aqueous solution containing 5 mmol / L potassium dihydrogen phosphate (pH = 2.5), and mobile phase B was an aqueous solution containing 5 mmol / L potassium dihydrogen phosphate and 0.5 mol / L sodium chloride (pH = 2.5); the volume fraction of mobile phase B was 5% at the initial elution stage, and gradually increased to 90% at a rate of 0.5% / min. At the same time, the pH value of the mobile phase was gradually adjusted from 3.5±0.1 to 2.5±0.1 through an online pH adjustment device.
[0036] The reversed-phase liquid chromatography uses a C18 column with an amino group modification on the surface, the column size is 200 mm × 2.1 mm, and the filler particle size is 2 μm; the temperature-controlled elution mode is an initial temperature of 30°C, which is increased to 50°C at a rate of 1°C / min; the mobile phase is an aqueous solution containing 0.1% formic acid and an acetonitrile solution containing 0.1% formic acid, and gradient elution is used with a flow rate of 0.2 mL / min.
[0037] The strong electric field enhancement device in the electrospray ionization source generates a strong electric field of 1.0 to 3.0 kV / cm at the outlet of the electrospray capillary. The ion focusing structure is to set 3 to 5 focusing electrodes on the ion transmission path, and the potential difference between the electrodes is 50 to 200 V.
[0038] Example 2: The difference from Example 1 is that the gradient elution strategy is: mobile phase A is an aqueous solution containing 7 mmol / L potassium dihydrogen phosphate (pH = 3), and mobile phase B is an aqueous solution containing 7 mmol / L potassium dihydrogen phosphate and 0.7 mol / L sodium chloride (pH = 3); the volume fraction of mobile phase B in the initial elution stage is 7%, which is gradually increased to 92% at a rate of 1% / min, and the pH value of the mobile phase is gradually adjusted from 3.5±0.1 to 2.5±0.1 by an online pH regulator.
[0039] Example 3: The difference from Example 1 is that the gradient elution strategy is: mobile phase A is an aqueous solution containing 10 mmol / L potassium dihydrogen phosphate (pH = 3.5), and mobile phase B is an aqueous solution containing 10 mmol / L potassium dihydrogen phosphate and 1.0 mol / L sodium chloride (pH = 3.5); the volume fraction of mobile phase B is 10% at the initial elution stage, and gradually increases to 95% at a rate of 2% / min, and at the same time, the pH value of the mobile phase is gradually adjusted from 3.5±0.1 to 2.5±0.1 by an online pH regulator.
[0040] Example 4: The difference from Example 1 is that the reverse phase liquid chromatography uses a C18 column.
[0041] Example 5: The difference from Example 1 is that a conventional electrospray ionization source is used.
[0042] Blank control group: an equal amount of distilled water was used to replace the lees sample, and the remaining steps were the same as those in Example 1.
[0043] Prior art control group: sample pretreatment used conventional buffer (phosphate buffered saline, pH 7.0), fixed speed centrifugation (8000 r / min, 20 min), ordinary filter paper filtration and conventional ultrafiltration; multidimensional chromatographic separation used one-dimensional reversed-phase liquid chromatography; mass spectrometry identification used a conventional electrospray ionization source, and the remaining parameters were adapted to Example 1.
[0044] The procedure is as follows: 1. Sample pretreatment: Weigh 5 g of vinasse sample and add 50 mL of the corresponding buffer. Ultrasonic extraction is performed at a frequency of 30 kHz, a power of 200 W, a time of 40 minutes, and a temperature of 30°C. Centrifuge according to the corresponding centrifugation parameters, remove the supernatant, filter the supernatant through the corresponding filter membrane, collect the filtrate, and ultrafilter the filtrate to obtain a polypeptide concentrate, and determine the concentration.
[0045] First dimension: 20 μL of peptide concentrate was injected into a strong cation exchange chromatography column and separated using the corresponding set of gradient elution parameters, with fractions collected every 1.5 min. Second dimension: Each fraction was injected into a reversed-phase liquid chromatography column and separated using the corresponding set of parameters. Chromatographic peaks were recorded, and peak capacity and resolution were calculated. The single peptide component was introduced into the mass spectrometer and processed using the corresponding set of ionization parameters. Fragment ion information was obtained using tandem mass spectrometry and compared with the UniProt database. The number of identified peptides, accuracy, and signal intensity were calculated.
[0046] The following data is obtained:
[0047]
[0048] The peptide concentration, peak capacity and other indicators of Examples 1 to 3 are relatively close, indicating that the gradient elution parameters are effective within the range, and Example 1 is the best. The peak capacity and identification number of Example 4 decreased due to the lack of amino modification, insufficient retention of polar peptides, and weakened separation effect. The identification number, accuracy and signal intensity of Example 5 decreased due to the low efficiency of conventional ionization sources and large interference from impurities. Compared with the control group of the prior art, the peptide concentration of Example 1 increased by 113%, the peak capacity increased by 183%, and the identification number increased by 140%, which are significant advantages.
[0049] In sample pretreatment, specific buffers, variable speed centrifugation, special filter membranes and ultrafiltration work together to increase the polypeptide concentration and lay the foundation for subsequent steps. The existing technology has poor buffer adaptability and weak centrifugal filtration effect, resulting in a lot of polypeptide loss. In multidimensional chromatography, strong cation exchange chromatography and reversed-phase liquid chromatography are orthogonally separated, combined with gradient elution and temperature control, to greatly improve peak capacity and separation. Example 4 lacks chromatographic column modification, destroys synergy, and the effect decreases. In mass spectrometry identification, the improved electrospray ionization source works together with high-purity samples to improve signal intensity and accuracy. Example 5 has insufficient ionization source, and although the separation is good, the identification effect is poor.
Claims
1. A method for identifying vinasse polypeptides, characterized in that: include: Sample pretreatment: The lees sample is mixed with a buffer solution of a specific ratio for extraction. After extraction, it is centrifuged at a variable speed, filtered with a special filter membrane, and ultrafiltered to obtain a high-purity polypeptide concentrate. Multidimensional chromatographic separation: Using a two-dimensional liquid chromatography system, the first dimension separates the peptide concentrate by strong cation exchange chromatography combined with gradient elution to collect different fractions; the second dimension separates each fraction by reversed-phase liquid chromatography using enhanced separation processing to obtain multiple peptide single components; High-resolution mass spectrometry identification: A single peptide component is introduced into a high-resolution mass spectrometer, ionized using an electrospray ionization source, and fragment ion information is obtained using a tandem mass spectrometry method. The amino acid sequence of the peptide is inferred by analyzing the mass-to-charge ratio and fragment ion information. At the same time, the experimental data is compared with the peptide database using mass spectrometry data analysis software to determine the structure and origin of the peptide.
2. The method according to claim 1, characterized in that The sample buffer solution is composed of sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium chloride in a molar ratio of 3-5:2-3:1, wherein the concentration of sodium dihydrogen phosphate is 20-50 mmol / L, the concentration of disodium hydrogen phosphate is 10-30 mmol / L, and the concentration of sodium chloride is 5-15 mmol / L.
3. The method according to claim 2, characterized in that The variable speed centrifugation is first performed at a speed of 3000-5000 r / min for 5-10 minutes, and then the speed is increased to 8000-12000 r / min and the centrifugation is continued for 10-20 minutes. The temperature is maintained at 4-10° C. during the centrifugation process.
4. The method according to claim 3, characterized in that The special filter membrane is a nanofiber filter membrane made of polyethersulfone, with a pore size distribution of 0.1-0.5 μm and a porosity of 70-90%. During filtration, the pressure is controlled at 0.1-0.3 MPa and the temperature is 20-30°C.
5. The method according to claim 4, characterized in that The gradient elution strategy is as follows: mobile phase A is an aqueous solution containing 5-10 mmol / L potassium dihydrogen phosphate (pH = 2.5-3.5), and mobile phase B is an aqueous solution containing 5-10 mmol / L potassium dihydrogen phosphate and 0.5-1.0 mol / L sodium chloride (pH = 2.5-3.5); the volume fraction of mobile phase B is 5% to 10% at the initial elution stage, and gradually increases to 90% to 95% at a rate of 0.5% to 2% / min, and at the same time, the pH value of the mobile phase is gradually adjusted from 3.5±0.1 to 2.5±0.1 through an online pH adjustment device.
6. The method according to claim 5, characterized in that The reversed-phase liquid chromatography uses a C18 column with an amino group modification on its surface, the column specifications are 100-200 mm×2.1-4.6 mm, and the filler particle size is 2-5 μm; the temperature-controlled elution method is that the initial temperature is 30-40° C., and the temperature is increased to 50-60° C. at a rate of 1-2° C. / min; the mobile phase is an aqueous solution containing 0.1%-0.5% formic acid and an acetonitrile solution containing 0.1%-0.5% formic acid, and gradient elution is used with a flow rate of 0.2-0.5 mL / min.
7. The method according to claim 6, characterized in that The strong electric field enhancement device in the electrospray ionization source generates a strong electric field of 1.0 to 3.0 kV / cm at the outlet of the electrospray capillary. The ion focusing structure is to set 3 to 5 focusing electrodes on the ion transmission path, and the potential difference between the electrodes is 50 to 200 V.