CN114384194B - Method for high-flux detection of polypeptide - Google Patents
Method for high-flux detection of polypeptide Download PDFInfo
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- CN114384194B CN114384194B CN202111622339.1A CN202111622339A CN114384194B CN 114384194 B CN114384194 B CN 114384194B CN 202111622339 A CN202111622339 A CN 202111622339A CN 114384194 B CN114384194 B CN 114384194B
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a method for detecting polypeptides in high flux, which mainly solves the problems that the detection of batch polypeptide samples is long in time consumption and the method needs to be continuously adjusted. The polypeptide sample selected by the invention is injected into a high performance liquid chromatography analyzer, conventional bonded silica gel is used as chromatographic column filler, an ultraviolet detector is adopted, acetonitrile and water are used as mobile phases, trifluoroacetic acid is used as an ion pair reagent, gradient elution is carried out, the detection wavelength is 210-220nm, the flow rate is 0.3-0.4ml/min, the sample injection volume is 1 mu L, and the column temperature is 35 ℃. The method effectively solves the problem of long time consumption of batch polypeptide detection, has broad spectrum, is suitable for detecting most polypeptide samples, and can greatly improve the detection efficiency.
Description
Technical Field
The invention relates to polypeptide detection, in particular to a method for detecting polypeptide with high flux.
Background
Because the polypeptide medicine has complex structure, poor stability and low concentration, and the target molecule has similar structure with the impurity, and has only one amino acid difference, the separation and purification of the polypeptide medicine are always the most challenging part in the production process of the polypeptide medicine.
The separation and purification technology plays a decisive role in the form, yield and cost of biomolecules. In particular to a polypeptide drug expressed by organisms, because the concentration is low, the impurities are complex, the target product is easy to denature, the separation cost of the target product is more than 60% of the total cost, and the requirement of the polypeptide drug can be met by a plurality of purification processes, so that the separation and purification technology plays an important role in the polypeptide drug industry.
The traditional purification methods of the organic micromolecular medicaments such as recrystallization, rectification and the like are not suitable for the separation and purification of the polypeptides, and the high-performance liquid chromatography or chromatographic technique has extremely high separation and purification efficiency, mild conditions and easy maintenance of the biological activity of target molecules in the separation and purification process, and becomes an important tool for the separation and purification of the polypeptide medicaments. The separation and purification of the polypeptide mainly depend on the preparation of chromatographic packing by high-performance microspheres, and the method has the advantages of good separation effect, high resolution, good repeatability, high recovery rate and the like, and is the main separation and purification method of the polypeptide medicament at present.
Currently, there are three main chromatographic techniques for separating and purifying polypeptide drugs: ion exchange chromatography, reverse phase chromatography and hydrophobic interaction chromatography. The conventional high performance liquid chromatography detection of the polypeptide generally uses a reversed phase chromatographic column, and according to the properties of the polypeptide sequence, the proper composition and proportion of mobile phases, the detection gradient and other conditions are selected for targeted detection. In practical application of biological separation, a mixture of polar polypeptides and polypeptides with different hydrophobicity is often encountered, particularly peptide sequence detection under a complete blind condition, the detection and optimization time is long by the sequence property, or the number of to-be-detected is large, and the efficiency of carefully analyzing and designing detection conditions according to the sequence one by one is very low.
Chromatographic separation is a physical process, and sample molecules are subjected to multiple partitioning, adsorption and desorption between a fixed phase and a mobile phase, and the movement speeds of different molecules in a chromatographic column are different due to the difference of interaction forces between the different molecules and the fixed phase and the mobile phase, so that the purpose of separation is achieved. Usually with-COOH, -NH 2 Compounds with equipolar groups (e.g., polypeptides) tend to be more prone to tailing. The tailing is mainly caused by that for the silicon atom with silicon hydroxyl group connected to the surface, one bond is connected with one hydroxyl group, and the other three bonds are connected with one oxygen atom, so that the electronegativity of oxygen is stronger than that of silicon, and three oxygen atoms generate electron-withdrawing effect on the silicon atom, so that for the hydroxyl group on silicon, the silicon atom is an electron-withdrawing group on the hydroxyl group, so that the silicon hydroxyl group has certain acidity, and the pKa is about 4.5-4.7. pH-pKa of mobile phase according to ionization law>2 is thatpH>At 6.7, more than 99% of the silicon hydroxyl groups should be in the ionic state, i.e., si-O-, while pKa-pH>2, pH<At 2.5, the acidic environment inhibits ionization of the silicon hydroxyl groups, more than 99% of which should be in the molecular state, i.e., si-OH, but still exist in polarity, i.e., si-Odelta-Hdelta+.
The silicon hydroxyl groups in the fixed phase of the chromatographic column have certain polarity Si-O delta-H delta+, and even ionize under certain pH conditions to form ionic states of a-Si-O-form. The forces between Si-O delta-H delta + and-Si-O-are polar electrostatic forces that are much stronger than van der waals forces, and because the surface of the silica gel is bonded with long C18 chains, the opportunity for access to residual silicon hydroxyl groups in the sample molecule is not great due to steric hindrance, and only a small portion of the molecules can interact with the residual silicon hydroxyl groups. Thus, most of the sample molecules move forward uniformly as nonpolar molecules, while a small amount of sample molecules are eluted in a delayed manner due to the existence of the electrostatic force, so that the concentration distribution of the sample molecules changes, and a backward drag is generated. The extent of tailing has a direct relationship with the size of the polarity of the sample molecule and the amount of residual silicon hydroxyl groups. That is, the secondary retention effect of residual silicon hydroxyl groups on the sample is a major cause of peak-shaped drags.
In view of this, there is a need to provide a method that can meet the high throughput detection of a large number of polypeptide samples.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problems of the prior art, provides a method for detecting polypeptides with high flux, mainly solves the problems that the detection of batch polypeptide samples is long in time consumption and the detection conditions are required to be continuously adjusted according to the properties of the polypeptides, can greatly improve the detection flux, and can be used for detecting the polypeptides under the condition of complete blindness.
In order to solve the technical problems, the invention discloses a method for detecting polypeptide with high flux, which is characterized in that a reverse high performance liquid chromatograph is adopted for detection, the detector is an ultraviolet detector, 89.9 to 99.9 percent of water, 0.1 percent of trifluoroacetic acid and 0 to 10 percent of acetonitrile, the mobile phase B is 89.9 to 99.9 percent of acetonitrile, 0.1 percent of trifluoroacetic acid and 0 to 10 percent of water by mass percent, and the gradient elution detection wavelength is 210 to 220nm; the flow rate is 0.3-0.4mL/min; the sample injection volume is 1ul; the column temperature is 35-40 ℃. Preferably, mobile phase a is 99.9% water and 0.1% trifluoroacetic acid by mass, and mobile phase B is 99.9% acetonitrile and 0.1% trifluoroacetic acid by mass.
In some embodiments of the invention an Agilent 1260 ii reversed phase high performance liquid chromatography analyzer is used.
According to Fan Dem special equation, the flow rate of the chromatographic column with the corresponding specification in the method is about 0.35mL/min and is the maximum column efficiency flow rate. And the final flow rate is positioned at 0.3-0.4mL/min by combining the reasons of instrument pump precision, system pressure, retention time and the like. The measured polypeptide samples are compared with HPLC (high Performance liquid chromatography) and the information of the spectrum peaks is basically consistent when the flow rate is 0.3mL/min and 0.4mL/min.
In experiments for separating polypeptides and proteins by reversed phase chromatography, it is often necessary to add ion pair reagents to achieve an optimum peak and retention time, to add acids to the mobile phase to adjust the pH of the mobile phase below 2.5 to help improve peak shape, which is mainly used to improve tailing of compounds with carboxyl groups, and to add acids to suppress ionization of the silicon hydroxyl groups on the one hand and ionization of organic acids on the other hand, thus weakening interaction of-COO-with Si-oδ -hδ+ and reducing tailing, wherein trifluoroacetic acid in the mobile phase is mostly used as ion pair reagent to improve peak shape, overcome peak broadening and tailing problems by interacting with hydrophobic bonding phases and residual polar surfaces in a number of modes.
Trifluoroacetic acid is preferred over other ionic modifiers because it is readily volatile and can be conveniently removed from the prepared sample. On the other hand, the ultraviolet maximum absorption peak of trifluoroacetic acid is lower than 200nm, and the detection interference on the polypeptide at low wavelength is small. The selectivity of the polypeptide on reverse phase chromatography can be finely adjusted by changing the concentration of trifluoroacetic acid. This effect is very beneficial for optimizing the separation conditions, increasing the information content of complex chromatographic analysis (such as the fingerprint of the polypeptide).
The concentration of trifluoroacetic acid added to the mobile phase is typically 0.1%, at which most reverse phase columns produce good peak shapes, and peak broadening and tailing becomes evident when the trifluoroacetic acid concentration is well below this level.
Preferably, the chromatographic column is a chromatographic column with a size of 2.1 x 100mm, a packing particle size of 1.8 μm,specification of the specification. The larger the L/dp value, the higher the theoretical plate number, and therefore the L/dp value of the column to be used should be kept consistent with or higher than that of a conventional column, the column length is usually less than 250mm according to the conventional specification at present, and the filler particle diameter should be as small as possible in order to improve the column efficiency. Through theoretical calculation, the final selection is 2.1 x 100mm,1.8 mu m, and ∈>A chromatographic column of standard.
Preferably, the conditions of the gradient elution are: starting elution with 90-95% of mobile phase A and 5-10% of mobile phase B, gradually reducing the proportion of mobile phase A, increasing the proportion of mobile phase B, reaching 35% of mobile phase A after 6-6.5min, reaching 5% of mobile phase A after 0.3-0.35min of continuous elution, and reaching 95% of mobile phase B; after the continuous elution is carried out for 0.8-0.83min, the mobile phase is adjusted to 90-95% of the mobile phase A, and the mobile phase B is adjusted to 5-10% for continuous elution.
In a preferred embodiment, the conditions for gradient elution are: starting elution with 95% of mobile phase A and 5% of mobile phase B, gradually reducing the proportion of mobile phase A, increasing the proportion of mobile phase B, reaching 35% of mobile phase A after 6.5min, reaching 5% of mobile phase A and 95% of mobile phase B after continuing elution for 0.33 min; after the continuous elution for 0.83min, the mobile phase is adjusted to 95% of the mobile phase A, and the mobile phase B is adjusted to 5% to continue the elution.
As a more preferred embodiment, the column uses a size of 2.1X100 mm, a packing particle size of 1.8 μm,the conditions of the standard chromatographic column and gradient elution are as follows: starting elution with 95% of mobile phase A and 5% of mobile phase B, gradually reducing the proportion of mobile phase A, increasing the proportion of mobile phase B, reaching 35% of mobile phase A after 6.5min, reaching 5% of mobile phase A and 95% of mobile phase B after continuing elution for 0.33 min; after the continuous elution for 0.83min, the mobile phase is adjusted to 95% of the mobile phase A, and the mobile phase B is adjusted to 5% to continue the elution.
Further, the sample was dissolved by the following method: the sample is dissolved by the following method: the freeze-dried polypeptide is dissolved in a mixed solvent of acetonitrile-water-trifluoroacetic acid, preferably, the mixed solvent comprises 5% -20% of acetonitrile, 95% -80% of water and 0.1% of TFA according to the volume ratio, preferably, the volume ratio of the three is 10% of acetonitrile, 90% of water and 0.1% of TFA, the solution is completely dissolved by ultrasonic treatment, and the solution is filtered to be measured by 0.22 mu m.
Preferably, the concentration of the polypeptide sample is 0.1-0.5mg/mL.
The method of the invention is particularly suitable for the detection of polypeptides of 4-85 amino acids in length.
The beneficial effects are that: the invention effectively solves the problem of high flux detection of polypeptide by selecting small particle size and large aperture filler and mobile phase and setting a gradient method, improves the detection efficiency of the product, is convenient to operate, can be used for detecting the polypeptide under a complete blindness condition, can provide basic information for the complete blindness polypeptide sample by HPLC (high performance liquid chromatography) spectrum and other information, is convenient for subsequent further optimization and adjustment, and simultaneously can control the single detection time length to be within 10 minutes without adjusting the method, thereby being capable of meeting the high flux detection of a large number of polypeptide samples. The inventor uses this method, after completing 24 continuous hours, a single instrument is not stopped, the detection is not interrupted, the polypeptide 144 is detected in 24 hours, which is more than 3 times of the daily detection flux of the conventional HPLC.
Drawings
FIG. 1 is a crude analytical map of a # 1 polypeptide using the method of the present invention (other conditions unchanged, flow rate 0.4mL/min, gradient see Table 1);
FIG. 2 is a crude analytical map of the No. 1 polypeptide obtained by the method of the present invention (other conditions are unchanged, the flow rate is 0.4mL/min, and the gradient is shown in Table 2);
FIG. 3 is a crude analytical map of the No. 1 polypeptide obtained by the method of the present invention (other conditions are unchanged, the flow rate is 0.4mL/min, and the gradient is shown in Table 3);
FIG. 4 is a crude analytical map of the 2# polypeptide using the method of the present invention (other conditions unchanged, flow rate 0.3mL/min, gradient see Table 3);
FIG. 5 is a crude analysis of the 2# polypeptide using the method of the present invention (other conditions unchanged, flow rate 0.4mL/min, gradient see Table 3);
FIG. 6 is a crude analysis of 3# polypeptide using the method of the present invention;
FIG. 7 is a crude analysis of 3# polypeptide using a conventional 5 μm column assay;
FIG. 8 is a crude analysis of a No. 4 polypeptide using the method of the invention;
FIG. 9 is a crude analysis of a 4# polypeptide using a conventional 5 μm column assay;
FIG. 10 is a crude analysis of polypeptide # 5 using the method of the present invention;
FIG. 11 is a crude analysis of a No. 6 polypeptide using the method of the invention;
FIG. 12 is a crude analysis of a 7# polypeptide using the method of the present invention;
FIG. 13 is a crude analysis of a polypeptide # 8 using the method of the present invention;
FIG. 14 is a crude analysis of a 9# polypeptide using the method of the present invention;
FIG. 15 is a crude analysis of a 10# polypeptide using the method of the present invention;
FIG. 16 is a crude analysis of 11# polypeptide using the method of the present invention.
Detailed Description
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
Example 1
AKACTPRLHDCSHDRHSCCRGELFKDVCYCFYPEGEDKTEVCSCQQPKSHKYIEKVVDKTKTLVG the polypeptide sequence No. 1 is prepared by dissolving lyophilized polypeptide in acetonitrile 20%, water 80%, and TFA0.1%, controlling sample concentration at 0.25mg/ml, and ultrasonic treating to completely dissolve, and filtering with 0.22um to obtain the final product.
Detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-1.8 μm, the detector being an ultraviolet detector. The mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 1. The detection wavelength was 220nm, the flow rate was 0.4mL/min, the sample volume was 1. Mu.l, and the column temperature was 35 ℃. The analysis results are shown in FIG. 1; the gradients are shown in Table 2, and the analysis results are shown in FIG. 2; the gradients are shown in Table 3 and the results of the analysis are shown in FIG. 3.
TABLE 1
| A | B | |
| 0min | 95 | 5 |
| 8min | 35 | 65 |
| 8.33min | 5 | 95 |
| 9.63min | 5 | 95 |
| 9.96min | 95 | 5 |
| 13min | 95 | 5 |
TABLE 2
| A | B | |
| 0min | 95 | 5 |
| 5.5min | 35 | 65 |
| 5.83min | 5 | 95 |
| 6.13min | 5 | 95 |
| 6.16min | 95 | 5 |
| 10min | 95 | 5 |
TABLE 3 Table 3
| A | B | |
| 0min | 95 | 5 |
| 6.5min | 35 | 65 |
| 6.83min | 5 | 95 |
| 7.63min | 5 | 95 |
| 7.66min | 95 | 5 |
| 10min | 95 | 5 |
Example 2
FGKRSMRDMDTMKYLYDPSLSAADLKTLQKLMENY is a No. 2 polypeptide sequence, the polypeptide after freeze-drying is dissolved in acetonitrile 20%, water 80% and TFA0.1% by volume, the solution is completely dissolved by ultrasound, the concentration of a sample is controlled at 0.25mg/ml, and the sample is filtered and tested by 0.22 um;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-1.8 μm, the detector is uv: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm; the sample injection volume is 1 μl; the column temperature is 35 ℃; the flow rate is 0.3mL/min; the analysis results are shown in FIG. 4; the flow rate is 0.4mL/min; the analysis results are shown in FIG. 5. From the results in the graph, the flow rate of 0.3mL/min and the flow rate of 0.4mL/min are basically consistent in graph peak information, and the peak is faster at the flow rate of 0.4mL/min.
Example 3
LPFLYGSNAGLEMTSGFYGMQAIHTRAHLLQAIYEGVVFSHMTHLNRMRERFTDVHTLRVTGGPAHSDVWMYCF is 3# polypeptide sequence, the freeze-dried polypeptide is dissolved in acetonitrile 20%, water 80%, TFA0.1% by volume, the concentration of the sample is controlled at 0.25mg/ml, the solution is completely dissolved, and the solution is filtered to be measured at 0.22 μm;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-5um, the detector being uv: the mobile phase A is as follows by mass percent: 99.9% water, 0.1% trifluoroacetic acid, streamThe mobile phase B is as follows by mass percent: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 4; the detection wavelength is 220nm; the flow rate is 1.0mL/min; the sample injection volume is 20 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 6.
Detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing1.8um, the detector is ultraviolet; the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm; the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 7.
The method for the total duration of 30min is a conventional detection method, and the comparison purpose is that the method has the efficiency which is 2 times higher than that of the conventional method when the HPLC patterns are basically consistent and the total duration of 10min is one sample.
TABLE 4 Table 4
Example 4
KRRIHCWKFYAESDEQKLMKNRKAMHGVKYEVLEYVLKEWNHQCHSEYMPLNGMLNMKLAKIYHDELKIEGYCF is a No. 4 polypeptide sequence, the polypeptide after freeze-drying is dissolved in acetonitrile 20%, water 80%, TFA0.1% by volume, the concentration of the sample is controlled to be 0.25mg/ml, the solution is completely dissolved, and the solution is filtered to be detected by 0.22 mu m;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-5 μm, the detector being an ultraviolet detector; the mobile phase A is as follows by mass percent: 99.9% water, 0.1% trifluoroacetic acid,the mobile phase B is as follows by mass percent: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 4; the detection wavelength is 220nm; the flow rate is 1.0mL/min; the sample injection volume is 20 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 8.
Detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-1.8 μm, the detector being an ultraviolet detector; the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in Table 3, detection wavelength of 220nm, flow rate of 0.4mL/min, sample volume of 1 μl, column temperature of 35 ℃. The analysis results are shown in FIG. 9.
Example 5
ECIPKHHECTSNKHGCCRGKIFKYKCQCTTVVDQNGEQAERCFCGTATHHKAVELVAGFGKKLFG is 5# polypeptide sequence, the volume ratio of the lyophilized polypeptide to acetonitrile 20%,80% water, 0.1% TFA, ultrasonic wave to complete dissolution, the sample concentration is controlled at 0.25mg/ml, ultrasonic wave to complete dissolution, 0.22um filtration is measured;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-1.8 μm, the detector is uv: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm: the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 10.
Example 6
The 6# polypeptide sequence is: SRRYCVCR, dissolving the lyophilized polypeptide in acetonitrile 5%,95% water, 0.1% tfa, sonicating to complete dissolution, controlling sample concentration to 0.25mg/ml, sonicating to complete dissolution, and filtering to test for 0.22 um;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer, and filling chromatographic columnThe material is Ultimate LP-C18-1.8 μm, the detector is uv: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm: the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 11.
Example 7
The 7# polypeptide sequence is: PGFYKCICWYYVILL dissolving the lyophilized polypeptide in acetonitrile 10%,90% water, 0.1% TFA, controlling sample concentration to 0.25mg/ml, ultrasonic treating to completely dissolve, and filtering to obtain solution of 0.22 μm;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-1.8 μm, the detector is uv: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm: the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 12.
Example 8
The 8# polypeptide sequence is: GCCSTPPCAVLYCGRRR dissolving the lyophilized polypeptide in acetonitrile 10%,90% water, 0.1% TFA, controlling sample concentration to 0.25mg/ml, ultrasonic treating to completely dissolve, and filtering to obtain solution of 0.22 μm;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-1.8 μm, the detector is uv: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm: the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature is 35 DEG C. The analysis results are shown in FIG. 13.
Example 9
The 9# polypeptide sequence is: GCCSHPACAANNQDYC (two pairs of disulfide bonds), dissolving the lyophilized polypeptide in acetonitrile 10%,90% water, 0.1% TFA, controlling sample concentration to 0.25mg/ml, and ultrasonic treating to completely dissolve, and filtering to obtain 0.22 μm;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing-1.8um, the detector is uv: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm: the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 14.
Example 10
The 10# polypeptide sequence is: CLGFGEKCSCCKLCQKHKWCKYD dissolving the lyophilized polypeptide in acetonitrile 10%,90% water, 0.1% TFA, controlling sample concentration to 0.25mg/ml, and ultrasonic treating to completely dissolve, filtering with 0.22um to obtain the final product;
detecting with Agilent 1260 II reversed-phase high performance liquid chromatography analyzer with Ultimate LP-C18 as chromatographic column packing1.8 μm, the detector is ultraviolet: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm: the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 15.
Example 11
The 11# polypeptide sequence is: ACAETGAVCIHNDECCSGACSPVFNYCLPE dissolving the lyophilized polypeptide in 15% acetonitrile, 90% water, 0.1% TFA, controlling sample concentration to 0.25mg/ml, ultrasonic treating to completely dissolve, and filtering to obtain solution of 0.22 μm;
reversed phase high performance liquid chromatography using Agilent 1260 IIDetecting by an analyzer, wherein the chromatographic column packing is Ultimate LP-C18-1.8 μm, the detector is uv: the mobile phase A is as follows by mass percent: 99.9% of water, 0.1% of trifluoroacetic acid, and a mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as shown in table 3; the detection wavelength is 220nm: the flow rate is 0.4mL/min; the sample injection volume is 1 μl; the column temperature was 35 ℃. The analysis results are shown in FIG. 16.
In the gradient elution of the invention, the first 6.5min in the gradient is the effective gradient in the gradient elution, the column flushing and balancing process is carried out after 6.5min, the sample basically shows a peak at the first 6.5min under the method, and the column flushing and balancing only look at whether the sample remains in the column or not and prepare for the next needle detection balancing. The gradient setting is also a method for saving time and improving efficiency of the method for carrying out full-blind condition detection or high-throughput detection, so that the condition that each needle needs to adjust the gradient to measure the sample, then the gradient is adjusted according to the effect, and then the sample is re-detected, and the efficiency is low.
As can be seen from the chromatograms, the method has the advantages of wide application range for polypeptide detection, strong broad spectrum, short detection time consumption, and capability of greatly improving the detection efficiency, and is particularly suitable for full-blind condition detection and high-flux detection. The invention uses small-particle-size and large-aperture filler as a main separation base of chromatography, uses the condition of trifluoroacetic acid ion-containing reagent mobile phase, and forms a set of general detection method for polypeptides meeting the requirements of hundreds to tens of thousands of molecular weights after adjusting and optimizing gradients. The method can be used for polypeptide detection under the condition of complete blindness, can provide basic information for the complete blindness polypeptide sample through information such as HPLC (high Performance liquid chromatography) and the like, is convenient for subsequent further optimization and adjustment, and meanwhile, the single detection duration of the method can be controlled within 10 minutes without adjustment, so that the method can meet the high-throughput detection of a large number of polypeptide samples. The inventor uses this method, after completing 24 continuous hours, a single instrument is not stopped, the detection is not interrupted, the polypeptide 144 is detected in 24 hours, which is more than 3 times of the daily detection flux of the conventional HPLC.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Sequence listing
<110> Hunan Zhongcheng full peptide Biochemical Co., ltd
<120> a method for high throughput detection of polypeptides
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 65
<212> PRT
<213> 1# polypeptide (Artificial Sequence)
<400> 1
Ala Lys Ala Cys Thr Pro Arg Leu His Asp Cys Ser His Asp Arg His
1 5 10 15
Ser Cys Cys Arg Gly Glu Leu Phe Lys Asp Val Cys Tyr Cys Phe Tyr
20 25 30
Pro Glu Gly Glu Asp Lys Thr Glu Val Cys Ser Cys Gln Gln Pro Lys
35 40 45
Ser His Lys Tyr Ile Glu Lys Val Val Asp Lys Thr Lys Thr Leu Val
50 55 60
Gly
65
<210> 2
<211> 35
<212> PRT
<213> 2# polypeptide (Artificial Sequence)
<400> 2
Phe Gly Lys Arg Ser Met Arg Asp Met Asp Thr Met Lys Tyr Leu Tyr
1 5 10 15
Asp Pro Ser Leu Ser Ala Ala Asp Leu Lys Thr Leu Gln Lys Leu Met
20 25 30
Glu Asn Tyr
35
<210> 3
<211> 74
<212> PRT
<213> 3# polypeptide (Artificial Sequence)
<400> 3
Leu Pro Phe Leu Tyr Gly Ser Asn Ala Gly Leu Glu Met Thr Ser Gly
1 5 10 15
Phe Tyr Gly Met Gln Ala Ile His Thr Arg Ala His Leu Leu Gln Ala
20 25 30
Ile Tyr Glu Gly Val Val Phe Ser His Met Thr His Leu Asn Arg Met
35 40 45
Arg Glu Arg Phe Thr Asp Val His Thr Leu Arg Val Thr Gly Gly Pro
50 55 60
Ala His Ser Asp Val Trp Met Tyr Cys Phe
65 70
<210> 4
<211> 74
<212> PRT
<213> 4# polypeptide (Artificial Sequence)
<400> 4
Lys Arg Arg Ile His Cys Trp Lys Phe Tyr Ala Glu Ser Asp Glu Gln
1 5 10 15
Lys Leu Met Lys Asn Arg Lys Ala Met His Gly Val Lys Tyr Glu Val
20 25 30
Leu Glu Tyr Val Leu Lys Glu Trp Asn His Gln Cys His Ser Glu Tyr
35 40 45
Met Pro Leu Asn Gly Met Leu Asn Met Lys Leu Ala Lys Ile Tyr His
50 55 60
Asp Glu Leu Lys Ile Glu Gly Tyr Cys Phe
65 70
<210> 5
<211> 65
<212> PRT
<213> 5# polypeptide (Artificial Sequence)
<400> 5
Glu Cys Ile Pro Lys His His Glu Cys Thr Ser Asn Lys His Gly Cys
1 5 10 15
Cys Arg Gly Lys Ile Phe Lys Tyr Lys Cys Gln Cys Thr Thr Val Val
20 25 30
Asp Gln Asn Gly Glu Gln Ala Glu Arg Cys Phe Cys Gly Thr Ala Thr
35 40 45
His His Lys Ala Val Glu Leu Val Ala Gly Phe Gly Lys Lys Leu Phe
50 55 60
Gly
65
<210> 6
<211> 8
<212> PRT
<213> 6# polypeptide (Artificial Sequence)
<400> 6
Ser Arg Arg Tyr Cys Val Cys Arg
1 5
<210> 7
<211> 15
<212> PRT
<213> 7# polypeptide (Artificial Sequence)
<400> 7
Pro Gly Phe Tyr Lys Cys Ile Cys Trp Tyr Tyr Val Ile Leu Leu
1 5 10 15
<210> 8
<211> 17
<212> PRT
<213> 8# polypeptide (Artificial Sequence)
<400> 8
Gly Cys Cys Ser Thr Pro Pro Cys Ala Val Leu Tyr Cys Gly Arg Arg
1 5 10 15
Arg
<210> 9
<211> 16
<212> PRT
<213> 9# polypeptide (Artificial Sequence)
<400> 9
Gly Cys Cys Ser His Pro Ala Cys Ala Ala Asn Asn Gln Asp Tyr Cys
1 5 10 15
<210> 10
<211> 23
<212> PRT
<213> 10# polypeptide (Artificial Sequence)
<400> 10
Cys Leu Gly Phe Gly Glu Lys Cys Ser Cys Cys Lys Leu Cys Gln Lys
1 5 10 15
His Lys Trp Cys Lys Tyr Asp
20
<210> 11
<211> 30
<212> PRT
<213> 11# polypeptide (Artificial Sequence)
<400> 11
Ala Cys Ala Glu Thr Gly Ala Val Cys Ile His Asn Asp Glu Cys Cys
1 5 10 15
Ser Gly Ala Cys Ser Pro Val Phe Asn Tyr Cys Leu Pro Glu
20 25 30
Claims (5)
1. A method for detecting polypeptide with high flux is characterized in that a reverse high performance liquid chromatography analyzer is adopted for detection, the detector is an ultraviolet detector, a mobile phase A is 99.9% of water and 0.1% of trifluoroacetic acid in percentage by mass, a mobile phase B is 99.9% of acetonitrile and 0.1% of trifluoroacetic acid in percentage by mass, and gradient elution is carried out; the detection wavelength is 210-220nm; the flow rate is 0.3-0.4mL/min; the sample injection volume is 1 μl; the column temperature is 35-40 ℃, wherein the gradient elution conditions are as follows: starting elution with 95% of mobile phase A and 5% of mobile phase B, gradually reducing the proportion of mobile phase A, increasing the proportion of mobile phase B, reaching 35% of mobile phase A after 6-6.5min, and reaching 5% of mobile phase A and 95% of mobile phase B after 0.3-0.35min of continuous elution; continuously eluting for 0.8-0.83min, adjusting the mobile phase to 95% of mobile phase A, adjusting the mobile phase to 5% of mobile phase B, and continuously eluting; the chromatographic column adopts a chromatographic column with the specification of 2.1 x 100mm and the filler particle diameter of 1.8 mu m and 300A; the sample is dissolved by the following method: dissolving the freeze-dried polypeptide in a mixed solvent, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering to be detected; the mixed solvent comprises 5-20% of acetonitrile, 95-80% of water and 0.1% of TFA according to the volume ratio.
2. The method of claim 1, wherein the chromatographic column is a chromatographic column with a size of 2.1 x 100mm, a packing size of 1.8 μm and 300 a specification, and the gradient elution conditions are: starting elution with 95% of mobile phase A and 5% of mobile phase B, gradually reducing the proportion of mobile phase A, increasing the proportion of mobile phase B, reaching 35% of mobile phase A after 6.5min, reaching 5% of mobile phase A and 95% of mobile phase B after continuing elution for 0.33 min; after the continuous elution for 0.8min, the mobile phase is adjusted to 95% of the mobile phase A, and the mobile phase B is adjusted to 5% to continue the elution.
3. The method of claim 1, wherein the polypeptide sample concentration is 0.1-0.5mg/mL.
4. The method of claim 3, wherein the polypeptide sample concentration is 0.25 mg/mL.
5. The method of claim 1, wherein the polypeptide is 4-85 amino acids in length.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103304641A (en) * | 2013-05-28 | 2013-09-18 | 深圳翰宇药业股份有限公司 | Method for purifying WALP peptide |
| CN104897818A (en) * | 2015-06-19 | 2015-09-09 | 重庆医药高等专科学校 | UPLC (ultra-high performance liquid chromatography) method for simultaneously determining six related substances in bicalutamide |
| CN105572234A (en) * | 2014-10-08 | 2016-05-11 | 江苏省中国科学院植物研究所 | Method for rapidly and efficiently determining cysteine content and glutathione content in iris lactea var. chinensis |
| WO2016172855A1 (en) * | 2015-04-28 | 2016-11-03 | 深圳翰宇药业股份有限公司 | High performance liquid chromatography method for polypeptide mixtures |
| CN106526023A (en) * | 2016-10-31 | 2017-03-22 | 安徽瑞思威尔科技有限公司 | Method for quickly detecting alanyl-tyrosine in baijiu |
| CN111624287A (en) * | 2020-05-28 | 2020-09-04 | 江苏吉泰肽业科技有限公司 | Detection method of insoluble polypeptide |
| CN113075310A (en) * | 2021-03-16 | 2021-07-06 | 北京赛升药业股份有限公司 | Method for detecting angiogenesis inhibitory peptide |
-
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Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103304641A (en) * | 2013-05-28 | 2013-09-18 | 深圳翰宇药业股份有限公司 | Method for purifying WALP peptide |
| CN105572234A (en) * | 2014-10-08 | 2016-05-11 | 江苏省中国科学院植物研究所 | Method for rapidly and efficiently determining cysteine content and glutathione content in iris lactea var. chinensis |
| WO2016172855A1 (en) * | 2015-04-28 | 2016-11-03 | 深圳翰宇药业股份有限公司 | High performance liquid chromatography method for polypeptide mixtures |
| CN107531750A (en) * | 2015-04-28 | 2018-01-02 | 深圳翰宇药业股份有限公司 | Mixtures of polypeptides HPLC analytical method |
| CN104897818A (en) * | 2015-06-19 | 2015-09-09 | 重庆医药高等专科学校 | UPLC (ultra-high performance liquid chromatography) method for simultaneously determining six related substances in bicalutamide |
| CN106526023A (en) * | 2016-10-31 | 2017-03-22 | 安徽瑞思威尔科技有限公司 | Method for quickly detecting alanyl-tyrosine in baijiu |
| CN111624287A (en) * | 2020-05-28 | 2020-09-04 | 江苏吉泰肽业科技有限公司 | Detection method of insoluble polypeptide |
| CN113075310A (en) * | 2021-03-16 | 2021-07-06 | 北京赛升药业股份有限公司 | Method for detecting angiogenesis inhibitory peptide |
Non-Patent Citations (1)
| Title |
|---|
| 基于超高效液相色谱的改良直接肽反应试验预测化学物皮肤致敏性;章瑶 等;日用化学品科学;第40卷(第11期);第1-4页 * |
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