CN109239252B - Detection method for continuous multiple proline polypeptides - Google Patents
Detection method for continuous multiple proline polypeptides Download PDFInfo
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- CN109239252B CN109239252B CN201811050158.4A CN201811050158A CN109239252B CN 109239252 B CN109239252 B CN 109239252B CN 201811050158 A CN201811050158 A CN 201811050158A CN 109239252 B CN109239252 B CN 109239252B
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Abstract
The invention relates to a detection method for continuous multiple proline polypeptides, which mainly solves the problem of abnormal peak rows of the polypeptides in a reversed-phase high performance liquid chromatography, and the technical scheme of the invention has the following detection conditions: a chromatographic column: AllsepTM anion chromatography column mobile phase: phase A: NaHCO 23(0.1mol/L) aqueous solution, pH value is 8.3, B phase: regulating the pH value of a mixed solution of methanol and phthalic acid (0.1mol/L) to 6.3 by using triethanolamine; AllsepTM anion chromatography column for 0 to 20 minutes, phase a: the phase B is from 80:20 to 40: 60; flow rate: 0.7ml/min, wavelength: 214nm, sample dissolution method: 70% volume of phase A solution +30% volume of phase B solution. The invention carries out detailed experimental comparison on the aspects of selection of a chromatographic column, selection of a mobile phase reagent and a dissolving method of a sample, and finally solves the problem that the polypeptide can easily and simply obtain a single symmetrical peak in reverse-phase high performance liquid chromatography analysis, and the problem that the polypeptide cannot be subjected to reverse-phase high performance liquid chromatography qualitative analysis due to racemization is solved.
Description
Technical Field
The invention relates to a detection method for continuous multiple proline polypeptides, in particular to a method for detecting the polypeptides by combining a specific chromatographic column with a mobile phase with an unconventional ratio.
Background
Proline (Proline, abbreviated as Pro or P), α -imino acid, neutral, isoelectric point 6.30, water solubility greater than any amino acid, 100g water soluble at 25 ℃ about 162 g, with ninhydrin solution heat, can generate yellow compounds, once entering peptide chain, can generate hydroxylation, thus forming 4-hydroxyproline, is the important component of animal collagen protein.
The proline is widely applied to plants, organisms or the pharmaceutical industry, and the existence of the proline can improve the cold resistance of the plants; in organisms, proline can also play a role in regulating cytoplasmic osmotic balance; and the amino acid medicine containing proline is one of the raw materials of compound amino acid infusion solution, is used for protein supplementation after malnutrition, protein deficiency, severe gastrointestinal diseases, scalds and surgical operations, and has no obvious toxic or side effect.
During routine polypeptide studies, proline-containing polypeptides are involved in the sequence in many ways. Generally, the polypeptide contains a few discontinuous prolines, and the detection in the reversed-phase high-efficiency liquid phase has no influence and forms a good symmetrical single peak. However, as for the polypeptide formed by the condensation reaction of a plurality of continuous prolines, a double peak (namely a cavel peak which we often say) always appears in the reversed-phase high-performance liquid phase, but both the peaks are correct target peaks, and researches find that the polypeptide of the plurality of continuous prolines is easy to racemize under the acidic condition, so that a single symmetrical peak cannot be formed under the conventional acidic system reversed-phase high-performance liquid phase analysis condition. In order to solve the technical problem, the invention provides a specific analytical chromatographic column AllsepTM anion chromatographic column, and a mixture ratio of alkalescence in the selection of two-phase mobile phases aiming at the problem that a plurality of proline polypeptides are easy to racemize continuously, so that the polypeptides can obtain a good symmetrical single peak in reversed-phase high-performance liquid chromatography, and the final detection purity of the products can be determined.
Disclosure of Invention
The invention aims to provide a method for detecting a plurality of continuous proline polypeptides by using a reversed-phase high performance liquid chromatography, which mainly solves the technical problem that the final detection purity of the polypeptides cannot be qualitatively judged due to the fact that the continuous proline polypeptides always have double peaks (goat horn peaks) in the existing detection method.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method of detecting a plurality of proline polypeptides in series, comprising the steps of:
1) proportioning a mobile phase A phase: NaHCO 23(0.1mol/L) water solution, the pH value is 8.3;
2) proportioning mobile phase B: regulating the pH value of a mixed solution of methanol and phthalic acid (0.1mol/L) to 6.3 by using triethanolamine;
3) weighing a plurality of continuous proline polypeptides, ultrasonically dissolving the proline polypeptides in a mixed solution (70% volume of phase A solution and 30% volume of phase B solution) until the proline polypeptides are completely dissolved, filtering the solution by using a filter membrane, and taking filtrate for later use;
4) detecting the filtrate by using a reversed-phase high performance liquid chromatography, wherein the detection conditions are as follows:
a chromatographic column: AllsepTM anion chromatographic column
Mobile phase: phase A: h2O+NaHCO3(0.1mol/L) of a mixed solution, the pH value is 8.3, and the phase B: regulating the pH value of a mixed solution of methanol and phthalic acid (0.1mol/L) to 6.3 by using triethanolamine;
time (min) A B
0 80% 20%
20.00 60% 40%
30.0080% 20%
The percentage is mass percentage concentration, flow rate: 0.7ml/min, wavelength: 214 nm.
More preferably, the size of the chromatographic column is 4.6 × 150mm (I.D), the particle size is 7um, and the pore diameter is 300A.
The invention has the beneficial effects that: the method solves the limitation of the existing method for detecting the polypeptide containing a plurality of continuous proline by using the reversed-phase high performance liquid. The method utilizes a detection method that two mobile phases are both alkalescent ion pair reagents and an AllcepTM anion chromatographic column, not only can solve the defect of abnormal chromatographic peak shapes of the polypeptides containing a plurality of continuous proline, but also can accurately judge the actual purity of the polypeptides. The establishment of the method provides a more convenient and reliable detection way for the purity detection of the polypeptide containing a plurality of continuous proline prepared by HPLC reversed-phase high performance liquid chromatography.
Drawings
FIG. 1 is a diagram of a conventional silica gel packed column in TFA system.
FIG. 2 is a diagram of a conventional silica gel packed chromatographic column under a mixed mobile phase system of two weakly basic ion pair reagents.
FIG. 3 is a photograph of an AllsepTM anion chromatography column in TFA system.
FIG. 4 is a schematic representation of an AllsepTM anion chromatography column of the present invention under a two weakly basic ion pair reagent mixed mobile phase system.
Detailed Description
(for example, PP-12 is abbreviated as polypeptide sequence PPPPPPPPPPPP)
Example 1
1. Sample treatment: weighing 0.2mg PP-12 polypeptide, dissolving with 0.3ml ultrapure water by ultrasonic wave till complete, filtering with 0.45um filter membrane, and collecting filtrate for use.
2. Selecting chromatographic conditions:
1) selecting an instrument: shimadzu liquid chromatograph (model: LC-20A, high pressure double pump, ultraviolet detector), Shimadzu N2000 workstation;
2) selecting chromatographic column of common silica gel filler C18, 5um, 120A, 4.6 × 150mm (I.D);
3) selection of mobile phase: mobile phase: phase A: aqueous solution with 0.1% TFA by mass, phase B: acetonitrile solution with the mass percent concentration of 0.1 percent TFA;
4) selection of detection gradient:
time (min) A B
0 85% 15%
20.00 60% 40%
30.00 85% 15%
5) Selection of other detection conditions: the flow rate was 0.7ml/min and the detection wavelength was 214 nm.
3. And (3) chromatographic analysis: 20ul of the filtered filtrate was subjected to linear gradient liquid phase analysis to obtain a chromatogram of PP-12 (FIG. 1).
Example 2
1. Mixing the phase A mixed solution: 200ml of ultrapure water are weighed out, and 1.68g of NaHCO are weighed out3Dissolving in 200ml ultrapure water solution, stirring thoroughly, mixing, and degassing with helium gas for 10 min.
2. And (3) proportioning the B-phase mixed solution: weighing 200ml of ultra-methanol, weighing 3.32g of phthalic acid in 200ml of methanol solution, fully stirring, dissolving and mixing uniformly, adjusting the pH value to 6.3 by using 98.5% triethanolamine, and degassing for 5min by helium.
3. Sample treatment: 0.2mg of PP-12 polypeptide is weighed, dissolved by ultrasonic wave until complete with 0.3ml of mixed solution (70% volume of phase A solution +30% volume of phase B solution), filtered by a 0.45um filter membrane, and the filtrate is taken for standby.
4. Selecting chromatographic conditions:
1) selecting an instrument: shimadzu liquid chromatograph (model: LC-20A, high pressure double pump, ultraviolet detector), Shimadzu N2000 workstation.
2) The chromatographic column is selected from common silica gel filler chromatographic column C4 (C8), 5um, 300A, 4.6 × 150mm (I.D).
3) Selection of detection gradient:
time (min) A B
0 88% 12%
20.00 55% 45%
30.00 88% 12%
4) Selection of other detection conditions: the flow rate was 0.7ml/min and the detection wavelength was 214 nm.
5. And (3) chromatographic analysis: 20ul of the filtered filtrate was subjected to linear gradient liquid phase analysis to obtain a chromatogram of PP-12 (FIG. 2).
Example 3
1. Sample treatment: weighing 0.2mg PP-12 polypeptide, dissolving with 0.3ml ultrapure water by ultrasonic wave till complete, filtering with 0.45um filter membrane, and collecting filtrate for use.
2. Selecting chromatographic conditions:
1) selecting an instrument: shimadzu liquid chromatograph (model: LC-20A, high pressure double pump, ultraviolet detector), Shimadzu N2000 workstation.
2) The chromatographic column is selected from AllsepTM anion chromatographic column, 7um, 300A, 4.6 × 150mm (I.D).
3) Selection of mobile phase: mobile phase: phase A: aqueous solution with 0.1% TFA by mass, phase B: acetonitrile with a mass percent concentration of 0.1% TFA.
4) Selection of detection gradient:
time (min) A B
0 80% 20%
20.00 55% 45%
30.00 80% 20%
5) Selection of other detection conditions: the flow rate was 0.7ml/min and the detection wavelength was 214 nm.
3. And (3) chromatographic analysis: 20ul of the filtered filtrate was subjected to linear gradient liquid phase analysis to obtain a chromatogram of PP-12 (FIG. 3).
Example 4
1. Mixing the phase A mixed solution: 200ml of ultrapure water are weighed out, and 1.68g of NaHCO are weighed out3Dissolving in 200ml ultrapure water solution, stirring thoroughly, mixing, and degassing with helium gas for 10 min.
2. And (3) proportioning the B-phase mixed solution: weighing 200ml of ultra-methanol, weighing 3.32g of phthalic acid in 200ml of methanol solution, fully stirring, dissolving and mixing uniformly, adjusting the pH value to 6.3 by using 98.5% triethanolamine, and degassing for 5min by helium. 3. Sample treatment: 0.2mg of PP-12 polypeptide is weighed, dissolved by ultrasonic wave until complete with 0.3ml of mixed solution (70% volume of phase A solution +30% volume of phase B solution), filtered by a 0.45um filter membrane, and the filtrate is taken for standby.
4. Selecting chromatographic conditions:
1) selecting an instrument: shimadzu liquid chromatograph (model: LC-20A, high pressure double pump, ultraviolet detector), Shimadzu N2000 workstation.
2) The chromatographic column is selected from AllsepTM anion chromatographic column, 7um, 300A, 4.6 × 150mm (I.D).
3) Selection of detection gradient:
time (min) A B
0 80% 20%
20.00 60% 40%
30.00 80% 20%
4) Selection of other detection conditions: the flow rate was 0.7ml/min and the detection wavelength was 214 nm.
5. And (3) chromatographic analysis: 20ul of the filtered filtrate was subjected to linear gradient liquid phase analysis to obtain a chromatogram of PP-12 (FIG. 4).
Claims (4)
1. A detection method for a plurality of continuous proline polypeptides is characterized by comprising the following steps:
1) proportioning A phase solution: NaHCO 23An aqueous solution; NaHCO 23The concentration of the aqueous solution is as follows: 0.1 mol/L;
2) proportioning the phase B solution: a mixed solution of methanol and phthalic acid; wherein the concentration of the phthalic acid is 0.1 mol/L;
3) weighing a plurality of continuous proline polypeptides, fully dissolving the proline polypeptides by using 70% volume of phase A solution and 30% volume of phase B solution, filtering by using a filter membrane, and taking a filtrate for later use;
4) detecting the filtrate by using a reversed-phase high performance liquid chromatography, wherein the detection conditions are as follows:
a chromatographic column: an AllsepTM anion chromatographic column,
mobile phase: phase A: NaHCO 23Aqueous solution, phase B: methanol + phthalic acid mixed solution, elution conditions:
time min AB
0 80% 20%
20.00 60% 40%
25.00 80% 20%
Flow rate: 0.7ml/min
Wavelength: 214 nm.
2. The method according to claim 1, wherein the method comprises the steps of: said NaHCO3The pH of the aqueous solution was 8.3.
3. The method according to claim 1, wherein the method comprises the steps of: and regulating the pH value of the mixed solution of the methanol and the phthalic acid to 6.3 by using triethanolamine.
4. The method of claim 1, wherein the AllsepTM anion chromatographic column has a size of 4.6 × 150mm, a particle size of 7 μm, and a pore size of 300 Å.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6182167A (en) * | 1984-09-28 | 1986-04-25 | Shimadzu Corp | Analysis of amino acid |
| US6090786A (en) * | 1994-06-10 | 2000-07-18 | Fondatech Benelux N.V. | Serine proteases, their activity and their synthetic inhibitors |
| CN101052656A (en) * | 2004-06-15 | 2007-10-10 | 范德比尔特大学 | New chiral columns with broad chiral selectivity |
| CN104215721A (en) * | 2014-10-09 | 2014-12-17 | 中国科学院上海有机化学研究所 | Application of phthalic acid in amino acid analysis |
-
2018
- 2018-09-10 CN CN201811050158.4A patent/CN109239252B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6182167A (en) * | 1984-09-28 | 1986-04-25 | Shimadzu Corp | Analysis of amino acid |
| US6090786A (en) * | 1994-06-10 | 2000-07-18 | Fondatech Benelux N.V. | Serine proteases, their activity and their synthetic inhibitors |
| CN101052656A (en) * | 2004-06-15 | 2007-10-10 | 范德比尔特大学 | New chiral columns with broad chiral selectivity |
| CN104215721A (en) * | 2014-10-09 | 2014-12-17 | 中国科学院上海有机化学研究所 | Application of phthalic acid in amino acid analysis |
Non-Patent Citations (2)
| Title |
|---|
| Application of cinchona-solfonate-based chiral zwitterionic ion exchangers for the separation of proline-containing dipeptide rotamers and determination of on-column isomerization parameters from dynamic elution profiles;Stefanie Wernisch et al;《Analytica Chimica Acta》;20130808;第795卷;88-98 * |
| Effect of molecular structure and conformational change of proline-containing dipeptides in reversed-phase chromatography;Wayne R. Melander et al;《Journal of Chromatography A》;19820115;第234卷(第2期);269-276 * |
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Inventor after: Xu Hongyan Inventor after: Yan Feng Inventor after: Zhou Min Inventor after: Jiang Ting Inventor before: Xu Hongyan Inventor before: Yan Feng |
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Effective date of registration: 20210609 Address after: Room 603, building 2, 351 GuoShouJing Road, Pudong New Area, Shanghai 201203 Patentee after: GL BIOCHEM (SHANGHAI) Ltd. Patentee after: JIANGSU JITAI PEPTIDE INDUSTRY SCIENCE AND TECHNOLOGY Co.,Ltd. Address before: Room 603d, building 2, 351 GuoShouJing Road, Pudong New Area, Shanghai 201203 Patentee before: GL BIOCHEM (SHANGHAI) Ltd. |
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