Two-step enzymolysis identification method based on protein reversible fixation

An identification method and protein technology, applied in the field of two-step enzymatic hydrolysis identification based on reversible protein immobilization, can solve problems such as protein loss, inapplicability, and DNA double-strand damage, and achieve good repeatability, high stability and broad application prospects. Effect

Inactive Publication Date: 2016-03-16
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method is not suitable for systems with high concentrations of sodium dodecylsulfonate (SDS)
Because high concentrations of SDS may damage DNA double strands, resulting in protein loss

Method used

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  • Two-step enzymolysis identification method based on protein reversible fixation
  • Two-step enzymolysis identification method based on protein reversible fixation
  • Two-step enzymolysis identification method based on protein reversible fixation

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: The membrane proteomics research route based on the controllable breaking characteristics of disulfide bonds investigated the enzymatic hydrolysis effect of special membrane proteins, and compared with the filter-assisted sample pretreatment method

[0026] Bacteriorhodopsin (bR) solution (concentration: 0.5-1.0 mg / mL) and Fe activated by EDC and NHS 3 o 4 After the PEGADC magnetic nanomaterials were mixed, they were shaken and reacted for 2 hours at 37°C. The resulting material was thoroughly washed and redispersed in 50 μL NH 4 HCO 3 (25mM) solution, two-step enzymatic hydrolysis. Step 1: Add 1.0-2.0 μg Trypsin, and digest overnight at 37°C. Peptide sample No. 1 was obtained after magnetic separation. Step 2: add 10-20mM MDTT solution to the material, react at 56°C for 1 hour, then add IAA to a final concentration of 25-50mM, and react at room temperature for 1 hour in the dark. After magnetic separation, the residual peptide segment of reductive alky...

Embodiment 2

[0028] Example 2: The application of the membrane proteomics research route based on the controlled breakage of disulfide bonds in the large-scale identification of Hela cell membrane proteins

[0029] 10~20mgFe 3 o 4 The PEGADC magnetic nanometer material is dispersed in the MES buffer solution containing 0.1-0.2 mmol EDC and 0.1-0.2 mmol NHS, and reacted at room temperature for 30 minutes. After removing excess EDC and NHS, add 50 μg of Hela cell membrane protein extract (MES solution containing 4% SDS, pH 5.0-6.0), and react at 37°C for 3 hours. After the material was washed with deionized water to remove SDS, it was subjected to a two-step enzymatic hydrolysis. Step 1: Material is dispersed in 50 μL NH 4 HCO 3 (25mM) solution, and add 1μg Trypsin, 37 ℃ overnight enzymatic hydrolysis. Peptide sample No. 1 was obtained after magnetic separation. Step 2: Add 10mM MDTT solution to the material, react at 56°C for 1 hour, then add IAA to a final concentration of 25mM, and ...

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Abstract

The invention belongs to the technical field of biological analysis and detection and particularly provides a two-step enzymolysis identification method based on protein reversible fixation. The method includes the steps that firstly, a formyl group, an epoxy group, trifluoro ethyl sulfonic acid ester base and other active groups are modified on the surface of a Fe<3>O<4>@PEG magnetic nano material, then a group containing a disulfide bond is bonded to the outer layer of the magnetic nano material, and the magnetic nano material Fe<3>O<4>@PEG@ADC is obtained; then a two-step enzymolysis strategy is adopted; firstly trypsin is adopted to conduct first-step digestion, so that hydrophilic carboxyl is exposed out of hydrophobic membrane protein; then reductive alkylation is conducted, and the disulfide bond between the a peptide fragment not subjected to digestion and a material is destroyed; residual peptide fragments eluted out of the surface of the material are continuously subjected to enzymolysis of Glu-C. It is proved by experiments that the two-step enzymolysis strategy has a good enzymolysis effect on special membrane protein, such as rhodopsin bacteria, and a quite good result is further achieved in membrane proteomics research of complex samples. The two-step enzymolysis identification method is practical, efficient, good in repeatability and high in stability and has wide application prospect in scaled identification of membrane protein.

Description

technical field [0001] The invention belongs to the technical field of biological analysis and detection, and in particular relates to a two-step enzymatic hydrolysis identification method based on protein reversible immobilization. technical background [0002] At present, based on the characteristics of good stability and high reproducibility of covalent immobilization, covalent immobilization of proteins on solid phase supports has been widely used. However, the development of reversible covalent immobilization is relatively slow, that is, it is difficult to achieve protein elution and recovery. Most of the existing technologies need to modify the active group on the protein first, and then use the group to interact with the active site on the solid-phase material. For example, in the literature, a DNA single strand is modified on the protein, and the mesoporous silicon material The upper part of the complementary strand is modified, and the protein is reversibly linked ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/06G01N33/68
CPCC12P21/06G01N33/68
Inventor 张祥民刘一颖
Owner FUDAN UNIV
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