CN114384194A - Method for detecting polypeptide with high flux - Google Patents
Method for detecting polypeptide with high flux Download PDFInfo
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- CN114384194A CN114384194A CN202111622339.1A CN202111622339A CN114384194A CN 114384194 A CN114384194 A CN 114384194A CN 202111622339 A CN202111622339 A CN 202111622339A CN 114384194 A CN114384194 A CN 114384194A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides a method for detecting polypeptide with high flux, which mainly solves the problems that the detection of a batch of polypeptide samples takes long time and the method needs to be continuously adjusted. Injecting the selected polypeptide sample into a high performance liquid chromatography analyzer, using conventional bonded silica gel as chromatographic column packing, adopting an ultraviolet detector, selecting acetonitrile and water as mobile phases, using trifluoroacetic acid as an ion pair reagent, carrying out gradient elution, wherein 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 for batch polypeptide detection, has broad spectrum, is suitable for detection of 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 polypeptide drugs have complex structures, poor stability, low concentration and similar structures of target molecules and impurities, and have only one amino acid difference, the separation and purification of polypeptide drugs are always the most challenging part in the production process of polypeptide drugs.
Separation and purification techniques have a decisive role in the morphology, yield and cost of biomolecules. Especially, biologically expressed polypeptide drugs have low concentration, complex impurities and easy denaturation of target products, so that the separation cost accounts for more than 60% of the total cost, and a plurality of purification processes are required to meet the requirements of the polypeptide drugs, therefore, the separation and purification technology plays an important role in the polypeptide drug industry.
The traditional purification methods of organic small molecule drugs such as recrystallization, rectification and the like are not suitable for the separation and purification of polypeptide, the high performance liquid chromatography or the chromatography technology has extremely high separation and purification efficiency, the conditions are mild, the biological activity of target molecules is easy to maintain in the separation and purification process, and the method becomes an important tool for the separation and purification of polypeptide drugs. The separation and purification of the polypeptide mainly depends on the preparation of chromatographic packing by high-performance microspheres, 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 existing polypeptide medicament.
The current chromatographic or chromatographic techniques for separating and purifying polypeptide drugs are mainly three types: ion exchange chromatography, reverse phase chromatography and hydrophobic interaction chromatography. The conventional high performance liquid chromatography detection of the polypeptide usually uses a reversed phase chromatographic column, and selects suitable conditions such as mobile phase composition, proportion, detection gradient and the like according to the property of the polypeptide sequence, so as to carry out targeted detection. In practical application of biological separation, a mixture of polar polypeptides and polypeptides with different hydrophobicity is frequently encountered, particularly peptide sequence detection under a totally blind condition is long in detection and optimization time through sequence properties, or the number of to-be-detected peptides is large, and the efficiency of carefully analyzing and designing detection conditions according to sequences one to one is low.
Chromatographic separation is a physical process, sample molecules are subjected to multiple distribution, adsorption and desorption between a fixed phase and a mobile phase, and the different molecules have different moving speeds in a chromatographic column due to the difference of interaction forces between the different molecules and the fixed phase and the mobile phase, so that the separation is realized. Usually with-COOH, -NH2Equal polarityCompounds (e.g., polypeptides) of the group are relatively prone to tailing. The tailing reason is mainly that for a silicon atom with silicon hydroxyl connected on the surface, one bond is connected with one hydroxyl, the other three bonds are connected with one oxygen atom, and the electronegativity of oxygen is stronger than that of silicon, and the three oxygen atoms generate an electron-withdrawing effect on the silicon atom, so that for the hydroxyl on the silicon, the silicon atom is an electron-withdrawing group for the hydroxyl, so that the silicon hydroxyl has certain acidity, and the pKa of the silicon hydroxyl is about 4.5-4.7. pH-pKa of the mobile phase according to the ionization law>2 is pH>At 6.7, more than 99% of the Si hydroxyl groups should be in the ionic state, i.e., Si-O-, and pKa-pH>2 is pH<At 2.5, the acidic environment inhibits ionization of the silicon hydroxyl groups, and more than 99% of the silicon hydroxyl groups should be in the molecular state, i.e., Si — OH, but their polarity is still present, i.e., Si — O δ — H δ +.
The silicon hydroxyl in the chromatographic column stationary phase has certain polarity Si-O delta-H delta +, and even can be ionized under certain pH condition to form an ionic state of-Si-O-. Si-O delta-H delta + and-Si-O-are polar electrostatic forces which are much stronger than van der Waals forces for the forces between polar compounds, and because of the long chain C18 bonded to the surface of the silica gel, there are not many opportunities for residual silicon hydroxyl groups to be contacted in the sample molecules due to steric hindrance, and only a small fraction of the molecules can react with the residual silicon hydroxyl groups. Thus, most sample molecules advance uniformly as nonpolar molecules, while a small number of sample molecules are delayed from eluting due to the presence of such electrostatic forces, resulting in a change in the concentration distribution of the sample molecules, resulting in a drag-back. The severity of tailing is directly related to the polarity of the sample molecules and the amount of residual silicon hydroxyl groups. That is, the secondary retention effect of residual silicon hydroxyl groups on the sample is the main cause of the peak-shaped pull-back.
In view of the above, it is desirable to provide a method that can satisfy high throughput detection of a large number of polypeptide samples.
Disclosure of Invention
The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide a method for detecting polypeptide with high flux aiming at the defects of the prior art, mainly solving the problems that the detection of a batch of polypeptide samples takes long time and the detection condition needs to be continuously adjusted according to the property of the polypeptide, greatly improving the detection flux and being used for detecting the polypeptide under the condition of complete blindness.
In order to solve the technical problem, the invention discloses a method for detecting polypeptide with high flux, which is characterized in that a reverse high performance liquid chromatography analyzer is adopted for detection, the detector is an ultraviolet detector, 89.9-99.9 percent of water, 0.1 percent of trifluoroacetic acid and 0-10 percent of acetonitrile, a mobile phase B is 89.9-99.9 percent of acetonitrile, 0.1 percent of trifluoroacetic acid and 0-10 percent of water in percentage by mass, and the detection wavelength of gradient elution is 220nm, wherein the detection wavelength of gradient elution is 210-; the flow rate is 0.3-0.4 mL/min; the sample injection volume is 1 ul; 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.
Agilent 1260 II reverse phase HPLC chromatography was used in some embodiments of the invention.
According to the Van der Nutt equation, the flow rate of the chromatographic column with the corresponding specification in the method is about 0.35mL/min, which is the maximum column efficiency flow rate. The final flow rate is positioned at 0.3-0.4mL/min by combining the reasons of the precision of an instrument pump, the pressure of a system, the retention time and the like. Through the comparative HPLC spectrum analysis of the actually measured polypeptide sample, the peak information of the spectrum is basically consistent when the flow rate is 0.3 and 0.4 mL/min.
In experiments of separating polypeptide and protein by reversed phase chromatography, ion pair reagent is required to be added to achieve the effect of optimizing peak shape and retention time, acid is added into the mobile phase to adjust the pH of the mobile phase to be below 2.5, which is beneficial to improving the peak shape, the mode is mainly used for improving the tailing of carboxyl compounds, the acid is added to inhibit ionization of silicon hydroxyl on one hand and inhibit ionization of organic acid on the other hand, so that the interaction of-COO-and Si-O delta-H delta + is weakened, the tailing is reduced, trifluoroacetic acid is mostly used as the ion pair reagent, and the trifluoroacetic acid in the mobile phase interacts with a hydrophobic bonding phase and a residual polar surface in multiple modes to improve the peak shape and overcome the problems of peak broadening and tailing.
Trifluoroacetic acid is preferred over other ionic modifiers because it is readily volatile and can be easily 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 of the polypeptide at low wavelength is small. The selectivity of the polypeptide on reverse phase chromatography can be finely adjusted by varying the concentration of trifluoroacetic acid. This effect is very beneficial for optimizing separation conditions and increasing the information content of complex chromatographic analyses, such as fingerprinting of polypeptides.
Trifluoroacetic acid is typically added to the mobile phase at a concentration of 0.1% at which most reverse phase chromatography columns produce good peak profiles, and when the trifluoroacetic acid concentration is well below this level, peak broadening and tailing becomes significant.
Preferably, the column is a column with a size of 2.1 x 100mm, a packing particle size of 1.8 μm,
and (5) specification. The larger the value of L/dp, the higher the number of theoretical plates, and therefore the L/dp value of the chromatographic column to be used should be kept consistent with or higher than that of a conventional chromatographic column, and according to the current conventional specification, the column length of the chromatographic column is usually less than 250mm, and the filler particle diameter should be as small as possible in order to improve the column efficiency. By theoretical calculation, 2.1 x 100mm, 1.8 μm,
a standard chromatographic column.
Preferably, the conditions of the gradient elution are: eluting with mobile phase A90-95% and mobile phase B5-10% to gradually reduce the proportion of mobile phase A, increase the proportion of mobile phase B, reach mobile phase A35% after 6-6.5min, reach mobile phase B65%, reach mobile phase A of 5% and mobile phase B of 95% after continuing to elute for 0.3-0.35 min; continuously eluting for 0.8-0.83min, adjusting mobile phase to 90-95% for mobile phase A, and adjusting mobile phase B to 5-10% for continuous elution.
In a preferred embodiment, the conditions for gradient elution are: eluting with mobile phase A95% and mobile phase B5% to gradually reduce the proportion of mobile phase A, increase the proportion of mobile phase B, reach mobile phase A35% and mobile phase B65% after 6.5min, reach mobile phase A of 5% and mobile phase B of 95% after continuously eluting for 0.33 min; after continuously eluting for 0.83min, the mobile phase is adjusted to 95% of mobile phase A, and the mobile phase B is adjusted to 5% for continuous elution.
As a more preferred embodiment, the column is used with a size of 2.1 x 100mm, a packing particle size of 1.8 μm,
standard chromatographic column, gradient elution conditions are: eluting with mobile phase A95% and mobile phase B5% to gradually reduce the proportion of mobile phase A, increase the proportion of mobile phase B, reach mobile phase A35% and mobile phase B65% after 6.5min, reach mobile phase A of 5% and mobile phase B of 95% after continuously eluting for 0.33 min; after continuously eluting for 0.83min, the mobile phase is adjusted to 95% of mobile phase A, and the mobile phase B is adjusted to 5% for continuous elution.
Further, the sample to be injected is dissolved by the following method: the sample injection was dissolved by the following method: dissolving the lyophilized polypeptide in a mixed solvent of acetonitrile-water-trifluoroacetic acid, preferably, the mixed solvent comprises acetonitrile 5% -20%, water 95% -80% and TFA0.1% in volume ratio, preferably, the volume ratio of the acetonitrile 10%, water 90% and TFA0.1%, performing ultrasonic treatment until the three are completely dissolved, and filtering with 0.22 μm to be detected.
Preferably, the concentration of the polypeptide sample is 0.1-0.5 mg/mL.
The method of the invention is particularly suitable for the detection of polypeptides of 4-85 amino acids in length.
Has the advantages that: the invention effectively solves the problem of high-flux detection of polypeptide by selecting the small-particle-size and large-particle-size fillers and the mobile phase and setting the gradient method, improves the detection efficiency of products, is convenient to operate, can be used for detecting the polypeptide under the condition of complete blindness, can provide basic information for the polypeptide sample through information such as HPLC (high performance liquid chromatography) spectrum and the like, is convenient for subsequent further optimization and adjustment, and can meet the requirement of high-flux detection of a large number of polypeptide samples by controlling the detection time length of a single time within 10 minutes without an adjustment method. The inventor uses the method to complete the detection of a single instrument without stopping the machine and without interruption after 24 hours of continuous detection, and detects 144 polypeptides in 24 hours, which is more than 3 times of the daily detection flux of the conventional HPLC.
Drawings
FIG. 1 is a diagram showing the analysis of the crude product of polypeptide # 1 by the method of the present invention (otherwise, the conditions were unchanged, the flow rate was 0.4mL/min, and the gradient is shown in Table 1);
FIG. 2 is a graph showing the analysis of the crude product of polypeptide # 1 by the method of the present invention (otherwise, the conditions were not changed, the flow rate was 0.4mL/min, and the gradient is shown in Table 2);
FIG. 3 is a graph showing the analysis of the crude product of polypeptide # 1 by the method of the present invention (otherwise, the conditions were not changed, the flow rate was 0.4mL/min, and the gradient is shown in Table 3). (ii) a
FIG. 4 is a graph showing the analysis of the crude product of polypeptide # 2 by the method of the present invention (otherwise, the conditions were not changed, the flow rate was 0.3mL/min, and the gradient is shown in Table 3);
FIG. 5 is a graph showing the analysis of the crude product of polypeptide # 2 by the method of the present invention (otherwise, the conditions were not changed, the flow rate was 0.4mL/min, and the gradient is shown in Table 3);
FIG. 6 is a diagram of the analysis of the crude product of polypeptide # 3 using the method of the present invention;
FIG. 7 is a crude analysis of the 3# polypeptide using a conventional 5 μm column detection method;
FIG. 8 is a diagram of crude analysis of polypeptide # 4 using the method of the present invention;
FIG. 9 is a crude analysis of 4# polypeptide using a conventional 5 μm column detection method;
FIG. 10 is a diagram of crude analysis of polypeptide # 5 using the method of the present invention;
FIG. 11 is a diagram of crude analysis of polypeptide # 6 using the method of the present invention;
FIG. 12 is a crude analysis of polypeptide # 7 using the method of the present invention;
FIG. 13 is a diagram of crude analysis of polypeptide # 8 using the method of the present invention;
FIG. 14 is a crude analysis of polypeptide # 9 using the method of the present invention;
FIG. 15 is a crude analysis of polypeptide # 10 using the method of the present invention;
FIG. 16 is a diagram of 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 further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
Example 1
AKACTPRLHDCSHDRHSCCRGELFKDVCYCFYPEGEDKTEVCSCQQPKSHKYIEKVVDKTKTLVG is # 1 polypeptide sequence, the lyophilized polypeptide is dissolved in acetonitrile 20%, 80% water, 0.1% TFA by volume, the sample concentration is controlled at 0.25mg/ml, ultrasonic treatment is carried out until complete dissolution, and 0.22um filtration is carried out for detection.
Detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
-1.8 μm, the detector being an ultraviolet detector. The mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 1. The detection wavelength is 220nm, the flow rate is 0.4mL/min, the sample injection volume is 1 mul, and the column temperature is 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 |
| 8.33 |
5 | 95 |
| 9.63 |
5 | 95 |
| 9.96min | 95 | 5 |
| 13min | 95 | 5 |
TABLE 2
| A | B | |
| 0min | 95 | 5 |
| 5.5min | 35 | 65 |
| 5.83 |
5 | 95 |
| 6.13 |
5 | 95 |
| 6.16min | 95 | 5 |
| 10min | 95 | 5 |
TABLE 3
| A | B | |
| 0min | 95 | 5 |
| 6.5min | 35 | 65 |
| 6.83 |
5 | 95 |
| 7.63 |
5 | 95 |
| 7.66min | 95 | 5 |
| 10min | 95 | 5 |
Example 2
FGKRSMRDMDTMKYLYDPSLSAADLKTLQKLMENY is 2# polypeptide sequence, dissolving lyophilized polypeptide in acetonitrile 20%, 80% water, 0.1% TFA by volume ratio, ultrasonically treating until completely dissolved, controlling sample concentration at 0.25mg/ml, and filtering with 0.22 um;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8 μm, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm; the sample injection volume is 1 mul; the column temperature was 35 ℃; the flow rate is 0.3 mL/min; the analysis results are shown in FIG. 4; the flow rate is 0.4 mL/min; the analytical results are shown in FIG. 5. From the results, the peak information of the flow rate 0.3mL/min and the flow rate 0.4mL/min are basically consistent, and the peak is faster at the flow rate of 0.4 mL/min.
Example 3
LPFLYGSNAGLEMTSGFYGMQAIHTRAHLLQAIYEGVVFSHMTHLNRMRERFTDVHTLRVTGGPAHSDVWMYCF is 3# polypeptide sequence, dissolving lyophilized polypeptide in acetonitrile 20%, 80% water, 0.1% TFA by volume ratio, ultrasonic treating to completely dissolve, controlling sample concentration at 0.25mg/ml, ultrasonic treating to completely dissolve, and filtering with 0.22 μm;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
5um, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 4; the detection wavelength is 220 nm; the flow rate is 1.0 mL/min; the injection volume is 20 mul; the column temperature was 35 ℃. The results of the analysis are shown in FIG. 6.
Detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8um, ultraviolet detector; the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm; the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; the column temperature was 35 ℃. The analytical results are shown in FIG. 7.
The method for the total time of 30min is a conventional detection method, and the comparison aims to compare that the total time of the method is 10min for one sample under the condition that HPLC (high performance liquid chromatography) spectrums are basically consistent, and the efficiency is 2 times higher than that of the conventional method.
TABLE 4
Example 4
KRRIHCWKFYAESDEQKLMKNRKAMHGVKYEVLEYVLKEWNHQCHSEYMPLNGMLNMKLAKIYHDELKIEGYCF is a 4# polypeptide sequence, the lyophilized polypeptide is dissolved in acetonitrile 20%, 80% water, 0.1% TFA by volume ratio, the ultrasonic treatment is carried out until the polypeptide is completely dissolved, the sample concentration is controlled at 0.25mg/ml, the ultrasonic treatment is carried out until the polypeptide is completely dissolved, and the filtration with 0.22 μm is carried out for detection;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
-5 μm, the detector being an ultraviolet detector; the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 4; the detection wavelength is 220 nm; the flow rate is 1.0 mL/min; the injection volume is 20 mul; the column temperature was 35 ℃. The analytical results are shown in FIG. 8.
Detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
-1.8 μm, the detector being an ultraviolet detector; the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile and 0.1% trifluoroacetic acid, and gradient elution, wherein the gradient is shown in Table 3, the detection wavelength is 220nm, the flow rate is 0.4mL/min, the sample injection volume is 1 μ l, and the column temperature is 35 ℃. The analytical results are shown in FIG. 9.
Example 5
ECIPKHHECTSNKHGCCRGKIFKYKCQCTTVVDQNGEQAERCFCGTATHHKAVELVAGFGKKLFG is a 5# polypeptide sequence, the volume ratio of the lyophilized polypeptide solution to acetonitrile is 20%, 80% water and 0.1% TFA, the mixture is subjected to ultrasonic treatment until the lyophilized polypeptide solution is completely dissolved, the sample concentration is controlled at 0.25mg/ml, the mixture is subjected to ultrasonic treatment until the lyophilized polypeptide solution is completely dissolved, and the mixture is filtered by 0.22um to be detected;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8 μm, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm: the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; 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 and 0.1% TFA by volume ratio, performing ultrasonic treatment until the polypeptide is completely dissolved, controlling the sample concentration to be 0.25mg/ml, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering to be detected at 0.22 um;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8 μm, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm: the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; 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 the sample concentration at 0.25mg/ml, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering at 0.22 μm to be detected;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8 μm, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm: the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; the column temperature was 35 ℃. The results of the analysis 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 the sample concentration at 0.25mg/ml, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering at 0.22 μm to be detected;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8 μm, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm: the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; the column temperature was 35 ℃. The analytical 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 by volume, controlling the sample concentration to 0.25mg/ml, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering at 0.22 μm to be detected;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8um, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm: the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; the column temperature was 35 ℃. The analysis results are shown in FIG. 14.
Example 10
The 10# polypeptide sequence is: CLGFGEKCSCCKLCQKHKWCKYD, dissolving the freeze-dried polypeptide in acetonitrile 10%, 90% water and 0.1% TFA by volume ratio, controlling the sample concentration to be 0.25mg/ml, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering with 0.22um to be detected;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8 μm, ultraviolet detector: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm: the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; the column temperature was 35 ℃. The analytical results are shown in FIG. 15.
Example 11
The sequence of the 11# polypeptide is: ACAETGAVCIHNDECCSGACSPVFNYCLPE, dissolving the lyophilized polypeptide in acetonitrile 15%, water 90%, and TFA0.1% by volume, controlling the sample concentration at 0.25mg/ml, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering at 0.22 μm to be detected;
detecting with Agilent 1260 II reversed phase high performance liquid chromatography analyzer with Ultimate LP-C18 as filler
1.8 μm, detector uv: the mobile phase A comprises the following components in percentage by mass: 99.9 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 99.9% acetonitrile, 0.1% trifluoroacetic acid, gradient elution, gradient as in table 3; the detection wavelength is 220 nm: the flow rate is 0.4 mL/min; the sample injection volume is 1 mul; 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, and the column washing and balancing process is performed after 6.5min, so that the sample basically peaks in the first 6.5min, and the column washing and balancing only see whether the sample remains in the column or not and prepare for the next detection and balancing. The gradient setting is also a time-saving and efficiency-improving method for carrying out the full-blind condition detection or the high-flux detection by the method, the condition that the gradient is required to be adjusted at each needle is avoided, the sample is detected, the gradient is adjusted according to the effect, the sample is detected again, and the efficiency is lower.
In conclusion, the obtained chromatogram map shows that the method is suitable for detecting the polypeptide in a wide range, has strong broad spectrum, is short in detection time consumption, can greatly improve the detection efficiency, and is particularly suitable for full-blind condition detection and high-throughput detection. The invention uses the filler with small particle size and large pore size as the main separation basis of the chromatogram, uses the condition of trifluoroacetic acid ion pair reagent-containing mobile phase, and forms a set of polypeptide universal detection method which can meet the requirements from hundreds to tens of thousands of molecular weight after adjusting and optimizing the gradient. The method can be used for polypeptide detection under the condition of complete blindness, can provide basic information for a complete blindness polypeptide sample through information such as an HPLC (high performance liquid chromatography) map and the like, is convenient for subsequent further optimization and adjustment, can control the single detection time within 10 minutes without an adjustment method, and can meet the requirement of high-throughput detection of a large number of polypeptide samples. The inventor uses the method to complete the detection of a single instrument without stopping the machine and without interruption after 24 hours of continuous detection, and detects 144 polypeptides in 24 hours, which is more than 3 times of the daily detection flux of the conventional HPLC.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Sequence listing
<110> Cheng quan peptide Biochemical Co., Ltd in Hunan province
<120> a method for detecting polypeptides with high throughput
<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 (9)
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 comprises 89.9-99.9% of water, 0.1% of trifluoroacetic acid and 0-10% of acetonitrile in percentage by mass, a mobile phase B comprises 89.9-99.9% of acetonitrile, 0.1% of trifluoroacetic acid and 0-10% of water in percentage by mass, and gradient elution is carried out; the detection wavelength is 210-220 nm; the flow rate is 0.3-0.4 mL/min; the sample injection volume is 1 mul; the column temperature is 35-40 ℃.
2. The method of claim 1, wherein the column is a 2.1 x 100mm specification with a filler particle size of 1.8 μm, 300 a.
3. The method of claim 1, wherein the conditions of gradient elution are: eluting with mobile phase A95% and mobile phase B5% to gradually reduce the proportion of mobile phase A, increase the proportion of mobile phase B, reach mobile phase A35% after 6-6.5min, reach mobile phase B65%, and reach mobile phase A of 5% and mobile phase B of 95% after continuously eluting for 0.3-0.35 min; continuously eluting for 0.8-0.83min, adjusting mobile phase to 95% for mobile phase A, and adjusting mobile phase B to 5% for continuous elution.
4. The method of claim 3, wherein the conditions of gradient elution are: eluting with mobile phase A95% and mobile phase B5% to gradually reduce the proportion of mobile phase A, increase the proportion of mobile phase B, reach mobile phase A35% and mobile phase B65% after 6.5min, reach mobile phase A of 5% and mobile phase B of 95% after continuously eluting for 0.33 min; after continuously eluting for 0.83min, the mobile phase is adjusted to 95% of mobile phase A, and the mobile phase B is adjusted to 5% for continuous elution.
5. The method of claim 1, wherein the column is a 2.1 x 100mm size column having a filler particle size of 1.8 μm and a 300 a specification, and the gradient elution conditions are as follows: eluting with mobile phase A95% and mobile phase B5% to gradually reduce the proportion of mobile phase A, increase the proportion of mobile phase B, reach mobile phase A35% and mobile phase B65% after 6.5min, reach mobile phase A of 5% and mobile phase B of 95% after continuously eluting for 0.33 min; after continuously eluting for 0.83min, the mobile phase is adjusted to 95% of mobile phase A, and the mobile phase B is adjusted to 5% for continuous elution.
6. The method of claim 1, wherein the sample is dissolved by: and (3) dissolving the freeze-dried polypeptide in a mixed solvent of acetonitrile-water-trifluoroacetic acid, performing ultrasonic treatment until the polypeptide is completely dissolved, and filtering the solution with the particle size of 0.22 mu m to be detected.
7. The method as claimed in claim 6, wherein the mixed solvent comprises acetonitrile 5% -20%, water 95% -80% and TFA0.1% by volume, and is sonicated to complete dissolution, and 0.22 μm filtration is performed to be measured.
8. The method of claim 6, wherein the concentration of the polypeptide sample is 0.1-0.5 mg/mL.
9. The method of claim 6, wherein the polypeptide is 4-85 amino acids in length.
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| CN114384188A (en) * | 2021-12-28 | 2022-04-22 | 湖南中晟全肽生化有限公司 | Dissolving and detecting method of long peptide |
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 |
-
2021
- 2021-12-28 CN CN202111622339.1A patent/CN114384194B/en active Active
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 |
|---|
| 章瑶 等: "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", 日用化学品科学, vol. 40, no. 11, pages 1 - 4 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114384188A (en) * | 2021-12-28 | 2022-04-22 | 湖南中晟全肽生化有限公司 | Dissolving and detecting method of long peptide |
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