CN111624287A

CN111624287A - Detection method of insoluble polypeptide

Info

Publication number: CN111624287A
Application number: CN202010465230.0A
Authority: CN
Inventors: 徐红岩; 秦敬国; 李阳; 王国军
Original assignee: GL BIOCHEM (SHANGHAI) Ltd; Jiangsu Jitai Peptide Industry Science And Technology Co ltd
Current assignee: GL BIOCHEM (SHANGHAI) Ltd; Jiangsu Jitai Peptide Industry Science And Technology Co ltd
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2020-09-04

Abstract

The invention provides a detection method of insoluble polypeptide, which mainly solves the technical problems of poor solubility and inaccurate detection result of a sample. Dissolving a sample by using isopropanol and acetonitrile, injecting the sample into a high performance liquid chromatography analyzer, using C18 bonded silica gel as a chromatographic column filler, adopting an ultraviolet detector, using the acetonitrile, the isopropanol and water as a mobile phase, using trifluoroacetic acid as an ion pair reagent, and performing gradient elution; the detection wavelength is 220 nm; the flow rate is 1.0 ml/min; the injection volume is 20 mul; the column temperature was 50 ℃. The method effectively solves the problem of detection of the insoluble polypeptide, can ensure the controllability of related substances and improve the product quality.

Description

Detection method of insoluble polypeptide

Technical Field

The invention relates to polypeptide detection, in particular to a detection method of insoluble peptide.

Background

Polypeptides are compounds in which alpha-amino acids are linked together by peptide bonds, and are also intermediates in the hydrolysis of proteins. The compounds obtained by dehydration condensation of two amino acid molecules are called dipeptides, and similarly, tripeptides, tetrapeptides, pentapeptides, and the like are also included. Generally, a compound obtained by dehydrating and condensing three or more amino acid molecules may be called a polypeptide. Hydrophobic polypeptides refer to polypeptides rich in hydrophobic amino acids. The hydrophobic amino acid is a generic term for amino acid side chains having high hydrophobicity, and is represented by subtracting the hydrophobicity of glycine from the hydrophobicity of each amino acid. Such as leucine, isoleucine, valine, are hydrophobic, while lysine, histidine, arginine are hydrophilic. The slightly soluble polypeptide is mainly caused by the slightly soluble amino acid contained in the polypeptide, and generally, the slightly soluble amino acid accounts for more than 75% of the polypeptide, so that the polypeptide is slightly soluble. Solubility is a relatively important problem encountered in the study of proteins and polypeptides, each amino acid having its inherent chemical properties. The reversed-phase high performance liquid chromatography needs to completely dissolve the polypeptide into a solvent, and the most suitable conventional solvents are H2O and ACN, so that the reversed-phase high performance liquid chromatography can be used for analysis and detection, but the insoluble polypeptide cannot be dissolved in a water/acetonitrile solution, and great troubles are brought to the operation of the majority of scientific researchers.

The existing method for solving the problem of difficult-to-dissolve polypeptide is to select an excellent organic solvent (such as DMSO and DMF) to dissolve the polypeptide, and the secondary structure of the polypeptide is destroyed to assist the dissolution, so that the separation effect of the high performance liquid chromatography is greatly reduced, and the dissolving capacity of the peptide chain which is difficult to dissolve is limited.

The patent CN105001298A is to change the synthetic route and add hydrophilic groups to achieve the purpose of dissolution. The method helps to purify the sample, but does not solve the problem of inaccurate detection result of sample analysis.

Disclosure of Invention

The invention aims to provide a method for detecting slightly soluble polypeptide, which mainly solves the technical problems of poor solubility and inaccurate detection result of a sample.

The technical scheme of the invention is as follows: a method for detecting a poorly soluble polypeptide, comprising the steps of:

(1) dissolving the slightly soluble polypeptide crude product in a solvent, performing ultrasonic treatment until the slightly soluble polypeptide crude product is completely dissolved, and filtering to be detected;

(2) detecting with a reversed-phase high performance liquid chromatography analyzer with ultraviolet detector; the mobile phase A comprises the following components in percentage by mass: 94.9 percent of water, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid, and the mobile phase B comprises the following components in percentage by mass: 94.9 percent of acetonitrile, 5 percent of water, 0.1 percent of trifluoroacetic acid, and the mobile phase C comprises the following components in percentage by mass: 94.9% isopropanol, 5% acetonitrile, 0.1% trifluoroacetic acid; gradient elution, with detection wavelength of 220 nm; the flow rate is 1.0 ml/min; the injection volume is 20 mul; the column temperature was 50 ℃.

The slightly soluble polypeptide is selected from polypeptide sequences as follows: one of Pal-VGVAPG, Pal-KTTKS or WILFMVYIIFFW.

The solvent in the step (1) is isopropanol and acetonitrile.

And (3) the reversed-phase high performance liquid chromatography analyzer in the step (2) adopts C18 bonded silica gel as a chromatographic column filler, and the particle size is 5 microns.

The invention has the beneficial effects that: the detection problem of the indissolvable polypeptide is effectively solved through the proportion of three different mobile phases, the controllability of related substances can be ensured, and the product quality is improved. The target peak response value is improved by 10%, the separation degree is greater than 1.0, and the accuracy of the detection result is ensured.

Drawings

FIG. 1 is a crude analysis chart of Pal-VG-6.

FIG. 2 is a crude analysis of Pal-KS-5.

FIG. 3 is a diagram showing a crude analysis of WW-11.

Detailed Description

Example 1

(1) The sequence is as follows: Pal-VGVAPG, dissolving the polypeptide crude product in isopropanol and acetonitrile, performing ultrasonic treatment until the polypeptide crude product is completely dissolved, and filtering to be detected;

(2) detecting with Waters2695 reversed phase high performance liquid chromatography analyzer with Kromaisl C18-5 μm as chromatographic column filler and ultraviolet detector; the mobile phase A is 94.9 percent of water, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent, the mobile phase B is 94.9 percent of acetonitrile, 5 percent of water and 0.1 percent of trifluoroacetic acid by mass percent, and the mobile phase C is 94.9 percent of isopropanol, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent; gradient elution, gradient as shown in Table 1, detection wavelength of 220 nm; the flow rate is 1.0 ml/min; the injection volume is 20 mul; the column temperature was 50 ℃. The analytical results are shown in FIG. 1.

Table 1.

Example 2

(1) The sequence is as follows: dissolving the crude polypeptide product in isopropanol and acetonitrile, performing ultrasonic treatment until the crude polypeptide product is completely dissolved, and filtering to be detected;

(2) detecting with Waters2695 reversed phase high performance liquid chromatography analyzer with Kromaisl C18-5 μm as chromatographic column filler and ultraviolet detector; the mobile phase A is 94.9 percent of water, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent, the mobile phase B is 94.9 percent of acetonitrile, 5 percent of water and 0.1 percent of trifluoroacetic acid by mass percent, and the mobile phase C is 94.9 percent of isopropanol, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent; gradient elution, gradient as shown in Table 2, detection wavelength of 220 nm; the flow rate is 1.0 ml/min; the injection volume is 20 mul; the column temperature was 50 ℃. The analytical results are shown in FIG. 2.

Table 2.

Example 3

(1) The sequence is as follows: WILFMVYIIFFW, dissolving the crude polypeptide product in isopropanol and acetonitrile, performing ultrasonic treatment until the crude polypeptide product is completely dissolved, and filtering to be tested;

(2) detecting with Waters2695 reversed phase high performance liquid chromatography analyzer with Kromaisl C18-5 μm as chromatographic column filler and ultraviolet detector; the mobile phase A is 94.9 percent of water, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent, the mobile phase B is 94.9 percent of acetonitrile, 5 percent of water and 0.1 percent of trifluoroacetic acid by mass percent, and the mobile phase C is 94.9 percent of isopropanol, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent; gradient elution, gradient as shown in Table 3, detection wavelength of 220 nm; the flow rate is 1.0 ml/min; the injection volume is 20 mul; the column temperature was 50 ℃. The analytical results are shown in FIG. 3.

Table 3.

Claims

1. A method for detecting a poorly soluble polypeptide, comprising: the method comprises the following steps:

(2) detecting with a reversed-phase high performance liquid chromatography analyzer with ultraviolet detector; the mobile phase A is 94.9 percent of water, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent, the mobile phase B is 94.9 percent of acetonitrile, 5 percent of water and 0.1 percent of trifluoroacetic acid by mass percent, and the mobile phase C is 94.9 percent of isopropanol, 5 percent of acetonitrile and 0.1 percent of trifluoroacetic acid by mass percent; gradient elution, with detection wavelength of 220 nm; the flow rate is 1.0 ml/min; the injection volume was 20. mu.l.

2. The method for detecting a poorly soluble polypeptide according to claim 1, wherein: in the step (1), the solvents are isopropanol and acetonitrile.

3. The method for detecting a poorly soluble polypeptide according to claim 1, wherein: and (3) in the step (2), the chromatographic column packing of the reversed-phase high-performance liquid chromatography analyzer is C18 bonded silica gel, and the particle size is 5 microns.

4. The method for detecting a poorly soluble polypeptide according to claim 1, wherein: in the step (2), the column temperature is 50 ℃.