A method for dissolving and detecting long peptides

Abstract

The present invention provides a dissolution and detection method for long peptides, which mainly solves the technical problem that the polypeptide samples have poor sample solubility due to a large number of amino acid compositions (more than 50AA) or a large number of hydrophobic amino acids in the sequence, resulting in abnormal test results. The present invention first dissolves the sample with trifluoroacetic acid and acetonitrile at appropriate concentrations, and then injects it into a high performance liquid chromatography analyzer, using C3 bonded silica gel as a chromatographic column filler, using an ultraviolet detector, selecting acetonitrile and water as mobile phases, trifluoroacetic acid as an ion pair reagent, and gradient elution; the detection wavelength is 220 nm; the flow rate is 0.4 ml / min; the injection volume is 1 ul; and the column temperature is 35 ° C. This method effectively solves the detection problem caused by the difficulty in dissolving long peptides and improves the quality control of samples.

Description

Technical Field

[0001] The invention relates to long peptide detection, and in particular to a long peptide dissolution and detection method. Background Art

[0002] A polypeptide is a compound formed by amino acids linked together by peptide bonds. A compound formed by the dehydration condensation of two amino acid molecules is called a dipeptide, and by analogy there are tripeptides, etc. A polypeptide with more than 40 amino acids is usually called a long peptide. Different amino acids have different hydrophilic and hydrophobic properties, which are mainly determined by the hydrophilicity of the amino acid side chains. As the number of amino acids that make up a polypeptide increases, the difficulty of dissolving a long peptide and detecting it by reversed-phase high-performance liquid chromatography increases significantly. Improper dissolution may result in peptide loss or experimental failure.

[0003] The solubility of peptides depends mainly on the sequence of the peptide. For peptides with a hydrophobic amino acid content of 50% to 75%, they may be insoluble in water or partially soluble even if the sequence contains >25% charged amino acids. The commonly used method is to dissolve the peptide in a strong organic solvent such as DMF, ACN, isopropanol, guanidine hydrochloride, urea, or DMSO, and other similar organic solvents.

[0004] The use of dissolving solvents such as DMSO helps to dissolve the polypeptide by destroying the secondary structure of the polypeptide, and can cause side reactions in amino acids such as Cys, Met and Trp that may be contained in the sequence, affecting the original properties or structure of the polypeptide sequence. The dissolution of polypeptides by reagents such as isopropanol mainly relies on the molecular association caused by the polarity of the molecules, but its own large viscosity (2.431) will cause the detection injection pressure to increase to a certain extent after the polypeptide is dissolved, which puts a heavy burden on the instrument and chromatographic column. At the same time, with the increase in the number of polypeptide amino acids, especially when the sequence contains more Ser, Thr, Glu, Asp, Lys, Arg, His, Asn, Gln and Tyr, the polypeptide sequence is prone to form an extra intermolecular hydrogen bond network, which further increases the viscosity of the polypeptide molecules. As a dissolving agent, isopropanol will further increase the viscosity of the polypeptide.

[0005] According to the requirements of the test, the choice of dissolving solvent should not only try not to change the properties of the dissolved substance, but also should not affect the subsequent test purposes and requirements. Therefore, the selected peptide dissolving solvent should be compatible with the experimental system and should not bring about solvent effects, otherwise it may cause large deviations in the test results and other abnormal situations. Patent CN111624287A has a certain effect on dissolving poorly soluble peptides by selecting isopropanol and acetonitrile as solvents, but the high viscosity of isopropanol itself and the viscosity further generated by the intermolecular hydrogen bond network in long peptides put a certain burden on the injection pressure of the subsequent detection system. At the same time, isopropanol and acetonitrile, as strong organic solvents, are prone to solvent effects in subsequent tests, and there are certain differences in compatibility with the experimental system. It is possible that during the test, the dissolved sample is diluted by the mobile phase on the chromatographic column and particles are precipitated again, causing abnormalities in the detection system and inaccurate results.

[0006] In view of this, this application is filed. Summary of the invention

[0007] Purpose of the invention: In order to solve the technical problems existing in the prior art, the present invention provides a dissolution and detection method for long peptides, which mainly solves the technical problems of poor sample solubility, inconvenient handling, and low accuracy of detection results.

[0008] To achieve the above purpose, the present invention proposes a method for detecting long peptides, wherein the long peptide to be detected is dissolved in a solvent, wherein the solvent is a mixed solution of trifluoroacetic acid, acetonitrile and water, ultrasonically dissolved, filtered, and then detected by reversed-phase high performance liquid chromatography, wherein the detector is an ultraviolet detector, acetonitrile and water are used as mobile phases, trifluoroacetic acid is used as an ion pair reagent, and gradient elution is performed; the detection wavelength is 210-220 nm, the flow rate is 0.3-0.4 mL / min; the injection volume is 1-2 μL; and the column temperature is 30-40°C. According to the solvent effect theory: for the same sample, different solvents are used, and due to different effects between the solvent and the solute, the chemical shift also has certain differences. For chromatography, when the solvent strength of the sample solvent is stronger than the mobile phase strength, the chromatographic peak will be extended, the peak will be broadened, and the peak will be bifurcated. The theoretical strength of the solvent in this method is similar to that of the detection mobile phase, which is lower than the strength of the mixed solvent of isopropanol and acetonitrile, thereby avoiding the solvent effect.

[0009] Specifically, in the solvent, the mass percentage of trifluoroacetic acid in the mixed solution is 0.05%-0.2%. The dissolution effect is poor when the TFA ratio is lower than 0.05%, and the upper limit is controlled within 0.2% to prevent the pH of the dissolving solvent from being too low, exceeding the pH tolerance range of the chromatographic column, and damaging the chromatographic column.

[0010] Preferably, the solvent contains 10-25% by mass of acetonitrile, 0.05%-0.2% by mass of trifluoroacetic acid and the balance of water.

[0011] Specifically, mobile phase A in the mobile phase is 94.9%-89.9% water, 5%-10% acetonitrile, and 0.1% trifluoroacetic acid in mass percentage, and mobile phase B is 94.9%-89.9% acetonitrile, 5%-10% water, and 0.1% trifluoroacetic acid in mass percentage, and the elution is performed by gradient.

[0012] Preferably, in the mobile phase, mobile phase A comprises 89.9% by weight water, 10% by weight acetonitrile, and 0.1% by weight trifluoroacetic acid, and mobile phase B comprises 89.9% by weight acetonitrile, 10% by weight water, and 0.1% by weight trifluoroacetic acid.

[0013] In a preferred embodiment, elution is performed according to the gradient in Table 1 below:

[0014] Table 1

[0015]

[0016]

[0017] The first 6.5 minutes in the gradient are the effective gradient in the gradient elution, and the period after 6.5 minutes is the column flushing and equilibrium process. Under this method, the sample basically peaks in the first 6.5 minutes.

[0018] Preferably, the chromatographic column filler of the reversed-phase high performance liquid chromatography analyzer is C3 bonded silica gel with a particle size of 1.8 μm.

[0019] In one embodiment, the chromatographic column packing material used in the present invention is ZORBAX 300SB-1.8 μm.

[0020] Beneficial effects: The present invention improves the dissolution efficiency by optimizing the dissolution solvent. At the same time, by using a detection mobile phase similar to the dissolution solvent, the difficulty of dissolving and detecting long peptides is effectively solved, the effect of product quality control is improved, and the operation is convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the analysis diagram of the crude product detected using isopropanol and acetonitrile as the dissolving solvents in 1#;

[0022] Figure 2 This is the analysis chart of the crude product detected using this method as the dissolving solvent in 1#;

[0023] Figure 3 This is the analysis diagram of the crude product detected using isopropanol and acetonitrile as the dissolving solvent in 2#;

[0024] Figure 4 This is the analysis chart of the crude product detected using this method as the dissolving solvent in 2#;

[0025] Figure 5This is the analysis diagram of the crude product detected using isopropanol and acetonitrile as the dissolving solvents in 3#;

[0026] Figure 6 This is the analysis chart of the crude product detected using this method as the dissolving solvent in 3#;

[0027] Figure 7 This is the analysis diagram of the crude product detected using isopropanol and acetonitrile as the dissolving solvents in 4#;

[0028] Figure 8 This is the analysis chart of the crude product detected using this method as the dissolving solvent in 4#;

[0029] Fig. 9 This is the analysis diagram of the crude product detected using isopropanol and acetonitrile as the dissolving solvents in 5#;

[0030] Fig.10 This is the analysis chart of the crude product detected using this method as the dissolving solvent in 5#;

[0031] Fig.11 This is the analysis diagram of the crude product detected using isopropanol and acetonitrile as the dissolving solvents for 6#;

[0032] Fig.12 This is the analysis chart of the crude product #6 using this method as the dissolving solvent;

[0033] Fig.13 This is a graph showing the analysis of chromatographic column efficiency. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific embodiments. The embodiments will help to understand the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0035] Example 1

[0036] Instrument and reagent HPLC (Agilent 1260)

[0037] 1#-6# peptides were all homemade, acetonitrile, isopropanol and TFA were chromatographically pure, water was purified water, and column efficiency test standards were products sold by Sigma.

[0038] The long peptide sequence is:

[0039] 1# Peptide:

[0040] DGNIQDDPVVRGNRPDFTMQPGVDINGNSDGTVDILKGMNRLSEKMIMALKNEYSRTNLQNKSNITNDEEDYCF;

[0041] 2# peptide:

[0042] AQGDTVKVSSNRGYIKAKAVVTKRIRTLKANGKDIDTIGIPIHWGYEGVAKKGFIANTLTPFVGDANTQTPYCF;

[0043] 3# peptide:

[0044] QCNSSSLTLRTARAGVPINNWRFSPLKPALTKLMAATMLSSPMTAPSIITLFIPTNASRPITQPCRIAPCPYCF;

[0045] 4# polypeptide:

[0046] INMLPELVGDGVLIAGDAAGMCMNLGFTIRGMDLAIAAGEAAAKTVLSAMKSDDFSKQKLAEYRQHLESGPYCF;

[0047] 5# peptide:

[0048] QLETTNSLAKLHALIGVARRIFKGAHRRTVVGEGYQETFMVELFFDAVKAVTFPTEHVFLVQFHVVKGDFTYC;

[0049] 6# peptide:

[0050] ETLILIKSDVNHTLQLTVCFCFIVMRRVQGESVDSPAEYKIFLNIILAPVDDGLRFTYSGDNFFYREISMIYCF.

[0051] Chromatographic column efficiency experiment:

[0052] Take the mixed standard sample for testing, and determine the column efficiency and separation. The results are as follows Fig.13 It shows that both the theoretical plate number and separation degree meet the requirements.

[0053] System stability experiment:

[0054] Six freeze-dried long peptides were taken and tested after being placed for 4 hours and 8 hours respectively. The results showed that there was no obvious change in the peak area and percentage of each chromatographic peak, and the RSD was within 1%.

[0055] Example 1

[0056] ① Dissolve the six freeze-dried long peptides 1#-6# in a solvent containing acetonitrile (25%), TFA (0.1%) and water (74.9%) by mass percentage, sonicate until completely dissolved (5-10 min), and filter through 0.22 μm for testing;

[0057] ② The Agilent 1260Ⅱ reversed-phase high performance liquid chromatograph was used for detection. The column packing was ZORBAX300SB-1.8μm, the detector was UV, the mobile phase A was 89.9% water, 10% acetonitrile, 0.1% trifluoroacetic acid by mass percentage, the mobile phase B was 89.9% acetonitrile, 10% water, 0.1% trifluoroacetic acid by mass percentage, gradient elution, the gradient ratio is shown in Table 1; the detection wavelength was 220nm: the flow rate was 0.4mL / min; the injection volume was 1μl; the column temperature was 35°C. The analysis results are shown in Figure 1 , Figure 3 , Figure 5 , Figure 7 , Fig. 9 and Fig.11 .

[0058] Table 1

[0059] A B 0min 95 5 6.5min 35 65 6.83min 5 95 7.63min 5 95 7.66min 95 5 10min 95 5

[0060] Comparative Example

[0061] ① Dissolve the six freeze-dried long peptides from 1# to 6# in solvents, including isopropanol (25%), acetonitrile (20%) and water (55%) by weight, and filter through 0.22 μm after ultrasonic treatment for 10 minutes;

[0062] ② The Agilent 1260Ⅱ reversed-phase high performance liquid chromatograph was used for detection. The chromatographic column packing was ZORBAX300SB-1.8μm, the detector was ultraviolet, the mobile phase A was 89.9% water, 10% acetonitrile, 0.1% trifluoroacetic acid by mass percentage, and the mobile phase B was 89.9% acetonitrile, 10% water, 0.1% trifluoroacetic acid by mass percentage, gradient elution, the gradient ratio is shown in Table 1; the detection wavelength was 220nm: the flow rate was 0.4ml / min;

[0063] The injection volume was 1 μl and the column temperature was 35°C. The analysis results are shown in Figure 2 , Figure 4 , Figure 6 , Figure 8 , Fig.10 and Fig.12 .

[0064] Table 2

[0065] A B 0min 95 5 6.5min 35 65 6.83min 5 95 7.63min 5 95 7.66min 95 5 10min 95 5

[0066] in conclusion:

[0067] In the same dissolution time, the long peptide dissolved by this method has basically no obvious undissolved matter. The method using isopropanol and acetonitrile as the dissolution conditions observed that some peptides were still undissolved. And after filtration and detection, it can also be clearly observed through the spectrum that the peak area and peak height of each peak in the spectrum of this dissolution method are more significant, which further shows that the dissolution efficiency of this method is higher and the effect is better.

[0068] The present invention provides a thought and method for dissolving and detecting long peptides. There are many methods and approaches to realize the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be realized by existing technologies. Sequence Listing <110> Hunan Zhongsheng Peptide Biochemical Co., Ltd. <120> A method for dissolving and detecting long peptides <140> 2021116235030 <141> 2021-12-30 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 74 <212> PRT <213> 1# Peptide (Artificial Sequence) <400> 1 Asp Gly Asn Ile Gln Asp Asp Pro Val Val Arg Gly Asn Arg Pro Asp 1 5 10 15 Phe Thr Met Gln Pro Gly Val Asp Ile Asn Gly Asn Ser Asp Gly Thr 20 25 30 Val Asp Ile Leu Lys Gly Met Asn Arg Leu Ser Glu Lys Met Ile Met 35 40 45 Ala Leu Lys Asn Glu Tyr Ser Arg Thr Asn Leu Gln Asn Lys Ser Asn 50 55 60 Ile Thr Asn Asp Glu Glu Asp Tyr Cys Phe 65 70 <210> 2 <211> 74 <212> PRT <213> 2# Polypeptide (Artificial Sequence) <400> 2 Ala Gln Gly Asp Thr Val Lys Val Ser Ser Asn Arg Gly Tyr Ile Lys 1 5 10 15 Ala Lys Ala Val Val Thr Lys Arg Ile Arg Thr Leu Lys Ala Asn Gly 20 25 30 Lys Asp Ile Asp Thr Ile Gly Ile Pro Ile His Trp Gly Tyr Glu Gly 35 40 45 Val Ala Lys Lys Gly Phe Ile Ala Asn Thr Leu Thr Pro Phe Val Gly 50 55 60 Asp Ala Asn Thr Gln Thr Pro Tyr Cys Phe 65 70 <210> 3 <211> 74 <212> PRT <213> 3# Polypeptide (Artificial Sequence) <400> 3 Gln Cys Asn Ser Ser Ser Leu Thr Leu Arg Thr Ala Arg Ala Gly Val 1 5 10 15 Pro Ile Asn Asn Trp Arg Phe Ser Pro Leu Lys Pro Ala Leu Thr Lys 20 25 30 Leu Met Ala Ala Thr Met Leu Ser Ser Pro Met Thr Ala Pro Ser Ile 35 40 45 Ile Thr Leu Phe Ile Pro Thr Asn Ala Ser Arg Pro Ile Thr Gln Pro 50 55 60 Cys Arg Ile Ala Pro Cys Pro Tyr Cys Phe 65 70 <210> 4 <211> 74 <212> PRT <213> 4# polypeptide (Artificial Sequence) <400> 4 Ile Asn Met Leu Pro Glu Leu Val Gly Asp Gly Val Leu Ile Ala Gly 1 5 10 15 Asp Ala Ala Gly Met Cys Met Asn Leu Gly Phe Thr Ile Arg Gly Met 20 25 30 Asp Leu Ala Ile Ala Ala Gly Glu Ala Ala Ala Lys Thr Val Leu Ser 35 40 45 Ala Met Lys Ser Asp Asp Phe Ser Lys Gln Lys Leu Ala Glu Tyr Arg 50 55 60 Gln His Leu Glu Ser Gly Pro Tyr Cys Phe 65 70 <210> 5 <211> 73 <212> PRT <213> 5# polypeptide (Artificial Sequence) <400> 5 Gln Leu Glu Thr Thr Asn Ser Leu Ala Lys Leu His Ala Leu Ile Gly 1 5 10 15 Val Ala Arg Arg Ile Phe Lys Gly Ala His Arg Arg Thr Val Val Gly 20 25 30 Glu Gly Tyr Gln Glu Thr Phe Met Val Glu Leu Phe Phe Asp Ala Val 35 40 45 Lys Ala Val Thr Phe Pro Thr Glu His Val Phe Leu Val Gln Phe His 50 55 60 Val Val Lys Gly Asp Phe Thr Tyr Cys 65 70 <210> 6 <211> 74 <212> PRT <213> 6# Polypeptide (Artificial Sequence) <400> 6 Glu Thr Leu Ile Leu Ile Lys Ser Asp Val Asn His Thr Leu Gln Leu 1 5 10 15 Thr Val Cys Phe Cys Phe Ile Val Met Arg Arg Val Gln Gly Glu Ser 20 25 30 Val Asp Ser Pro Ala Glu Tyr Lys Ile Phe Leu Asn Ile Ile Leu Ala 35 40 45 Pro Val Asp Asp Gly Leu Arg Phe Thr Tyr Ser Gly Asp Asn Phe Phe 50 55 60 Tyr Arg Glu Ser Met Tyr Cys Phe 65 70

Claims

1. A method for detecting long peptides, characterized in that: The long peptide to be tested is dissolved in a solvent, wherein the number of amino acids in the long peptide is greater than 40, and the solvent is a mixed solution of trifluoroacetic acid, acetonitrile and water, and ultrasonically dissolved, filtered, and then detected by reversed-phase high performance liquid chromatography, the detector is an ultraviolet detector, acetonitrile and water are used as mobile phases, trifluoroacetic acid is used as an ion pair reagent, and gradient elution: the detection wavelength is 210nm-220 nm, flow rate of 0.3-0.4mL / min; injection volume of 1-2μL; column temperature of 30-40℃, reversed-phase high performance liquid chromatography analyzer, chromatographic column filler of C3 bonded silica gel, particle size of 1.8μm; wherein the mass percentage of trifluoroacetic acid in the mixed solution is 0.05%-0.2%; the solvent contains 10-25% acetonitrile, 0.05%-0.2% trifluoroacetic acid and the remainder water; mobile phase A in the mobile phase includes 94.9%-89.9% water, 5%-10% acetonitrile and 0.1% trifluoroacetic acid by mass percentage, mobile phase B includes 94.9%-89.9% acetonitrile, 5%-10% water and 0.1% trifluoroacetic acid by mass percentage, gradient elution, the conditions of gradient elution are: 。 2. The detection method according to claim 1, characterized in that: The mobile phase A contained 89.9% by weight water, 10% by weight acetonitrile and 0.1% by weight trifluoroacetic acid, and the mobile phase B contained 89.9% by weight acetonitrile, 10% by weight water and 0.1% by weight trifluoroacetic acid.

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