Method for extracting and separating polypeptides

A separation method and target extraction technology, applied in the field of high-efficiency desorption polypeptide extraction and separation, can solve the problems of low desorption rate and low selectivity, and achieve the effect of high-efficiency desorption

Inactive Publication Date: 2014-12-10
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It should be noted that although polypeptides can be charged and water-soluble, they all have a certain lipophilicity in nature. This lipophilic effect is one of the importan

Method used

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  • Method for extracting and separating polypeptides
  • Method for extracting and separating polypeptides
  • Method for extracting and separating polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 (Adsorption and Release of Peptides)

[0027] polypeptide amino acid sequence pI molecular weight EE-6 EDAPA E 3.0 630.61 ED-9 ELRDD EAGD 3.4 1018.99 SP-20 SDEDS DGDRP QASPG LGPGP 3.4 1953.95 EL-25 EDDYE DHDGD DKDHD IDQKD DDDEL 3.5 3006.82 PP-6 PPGFS P 6.0 600.67 QF-9 QPLSS PPFF 6.0 1019.16 VE-18 VSEIQ LMHNL GKHLN SME 6.1 2080.42 PH-28 PSKPE DNPGA PAEDA RSALR HINLI TQH 6.1 3035.33 GI-6 GFLRR I 12.4 760.94

[0028] Porous silicon dioxide particles with an average size of 0.5 mm were put into 25 ml of hydrochloric acid aqueous solution, refluxed for 12 hours, filtered, washed with water, and dried. The dried sample was dispersed in 20 ml of toluene, slowly added trimethoxysilyl propyl glyceryl ether (5 ml dissolved in 5 ml of toluene) into the dispersion system under stirring, a few drops of triethylamine were added under nitrogen and refluxed 24 hours. Filter...

Embodiment 2

[0030] Embodiment 2 (polypeptide separation)

[0031] to peptides EE-6 (pI = 3.0) and QF-9 (pI = 6.0) (5.0 × 10 -5 M, 6 mL) in buffered aqueous solution (pH = 5, acetate buffer, 0.01 M) with the adsorbent SiO 2 PEI (30 mg) and allowed to stand for 20 hours. After separation of the solid adsorbent, the residual liquid was lyophilized (concentrated from 6 mL to 300 μL) and tested by high performance liquid chromatography (HPLC). HPLC test conditions: 0.1% trifluoroacetic acid in acetonitrile and 0.1% trifluoroacetic acid in water, the former rises from 18% to 100% within 25 minutes. The solution before adsorption was similarly concentrated and then subjected to control tests. The results are shown in Figure 1, indicating that the peptides with different isoelectric points can be completely separated after extraction.

Embodiment 3

[0032] Embodiment 3 (extraction and elution of trace polypeptide)

[0033] To a concentration of 1.0 × 10 -12 Add the adsorbent to PP-6, QF-9, VE-18 and PH-28 polypeptide aqueous solution (4 ml, pH 7.4 phosphate buffer) and let it stand for 20 hours. The solid adsorbent is adsorbed-separated-eluted (with pH 4 acetate buffer) followed by MALDI-TOF testing. The test uses the regular reflection mode. The results (Fig. 2) showed that all the signals of the four peptides appeared, indicating that all the peptides could be extracted. According to this result, it can be calculated that the adsorption constant reaches 10 12 m -1 , to further reduce the concentration of the peptide concentration to perform a similar test, showing that the extraction is still feasible, that is, the adsorption constant is higher than 10 12 m -1 , indicating that trace amounts of peptides can also be eluted.

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Abstract

The invention relates to a method for extracting and separating polypeptides. According to the method, large-size silicon dioxide (submillimeter scale) covalent loaded polyethyleneimine is adopted as an adsorbent (SiO2@PEI), polypeptides of different isoelectric points can be extracted in different pH environments, and the polypeptides can be efficiently released if the pH values are reduced. The reason is that the adsorbent is easy to protonize to be of positive ion, and the polypeptides can carry positive electricity instead of negative electricity along with reduction of the pH values, so that the polypeptides can be released as static complementary adsorption is changed into static mutual exclusion with the adsorbent. The complexing constant of the adsorbent and the polypeptides is generally greater than 10<12>M<-1>, so that even trace polypeptides with the sub-femtomolar content can be extracted. In addition, the release rate is 79-92% for most polypeptides. By adopting the adsorbent, at least polypeptides of which the relative abundance is only 0.1% can be extracted. The adsorbent is simple to prepare and can be used repeatedly.

Description

technical field [0001] The invention belongs to the fields of separation technology and biological analysis and detection, and specifically relates to a polypeptide extraction and separation method capable of efficiently extracting certain polypeptides in water and capable of efficiently desorbing them. Background technique [0002] The extraction and separation of peptides is an important technology today. For example, some peptides have specific physiological functions, but their content in raw materials is very low, so extraction and concentration are required. Another example is in the analysis of protein and nucleic acid, matrix-assisted laser desorption ionization flight spectroscopy (MALDI-TOF) is an extremely sensitive mass detection technology, suitable for the detection of macromolecular mass, with very high sensitivity, especially suitable for the detection of peptides, proteins, etc. , but the peptides with lower abundance in the system are difficult to be detec...

Claims

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Application Information

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IPC IPC(8): C07K1/36C07K1/30C07K1/14
Inventor 万德成陈峰金明浦鸿汀
Owner TONGJI UNIV
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