Preparation method of protein quick enzymolysis monolithic column through in situ polymerization and application thereof

An in-situ polymerization and protein technology, applied in the fields of analytical chemistry and proteomics, to achieve the effect of simple operation, simple and effective preparation method and good effect

Inactive Publication Date: 2010-09-08
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main challenge of this technical route is how to achieve rapid enzymatic hydrolysis after protein separation

Method used

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  • Preparation method of protein quick enzymolysis monolithic column through in situ polymerization and application thereof
  • Preparation method of protein quick enzymolysis monolithic column through in situ polymerization and application thereof
  • Preparation method of protein quick enzymolysis monolithic column through in situ polymerization and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Preparation of capillary monolithic columns for rapid enzymatic digestion of proteins:

[0026] Capillary cleaning and activation: Quartz capillary (inner diameter: 250μm) is cut into 6cm sections, washed with methanol for 5 minutes, pure water for 5 minutes, 0.1mol / L sodium hydroxide for 30 minutes, pure water for 5 minutes, and 0.1mol / L hydrochloric acid 30 minutes, wash with pure water for 5 minutes, and methanol for 5 minutes to wash away impurities in the tube and expose the silanol on the inner wall. Use a peristaltic pump to fill the capillary with 50% γ-MAPS methanol solution, and react in the dark for 24 hours at room temperature, so that the inner wall of the capillary is connected with carbon-carbon double bonds.

[0027] Chemical modification on the surface of enzyme molecules: 10 mg of Trypsin solution is freshly dissolved in 5 mL of 100 mmol / L boric acid buffer solution with pH=9.3, and a certain mass of benzamide is added to make the final concentration 0...

Embodiment 2

[0030]Enzymolysis and mass spectrometric determination of the standard protein horse myoglobin (Myo) by the monolithic column for rapid enzymatic digestion of proteins:

[0031] 50 μg of MYO was dissolved in 50 μL of 25 mM ammonium bicarbonate solution, and then passed through the enzymatic monolithic column at a flow rate of 3 μL / min driven by a peristaltic pump. The volume of each spot is about 1 μL, and when it shrinks to about 0.5 μL by rapid air-drying at room temperature, add 0.4 μL of matrix solution (α-cyano-4-hydroxycinnamic acid CHCA) to each spot, and mix and air-dry used for identification by mass spectrometry. In MALDI-TOF / TOF (4700 Proteomics Analyzer, Applied Biosystems); laser is Nd-YAG laser, wavelength 355nm, laser pulse frequency 200Hz; acceleration voltage 20KV; positive ion mode, reflective TOF detection. Depend on Figure 4A As shown, the main characteristic peptide peaks were all detected, and the intensity was strong, indicating that the protein was e...

Embodiment 3

[0033] The concentration of preparing bovine serum albumin (BSA) is 1 μ g / μ L, preparation method and other conditions are the same as example 2, repeat above-mentioned enzymatic hydrolysis and mass spectrometry experiment, the result is as follows Figure 4B . Similarly, the main characteristic peptide peaks of BSA were detected with strong intensity, indicating that the protein was effectively hydrolyzed after the capillary enzymolysis monolithic column.

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Abstract

The invention belongs to the fields of analytical chemistry and proteomics, in particular to a preparation method of a protein capillary quick enzymolysis monolithic column through situ polymerization. The monolithic column is used in enzymolysis of intact protein sample to realize quick enzymolysis of the protein, and analysis and authentication are directly carried out through matrix-assisted laser desorption / ionization mass spectrometry to the peptides after the enzymolysis. The capillary quick enzymolysis monolithic column is suitable to be used in combination with high-efficiency liquid chromatogram and has great application potential in research of proteomics.

Description

technical field [0001] The invention belongs to the fields of analytical chemistry and proteomics, and in particular relates to a capillary rapid enzymolysis monolithic column, including its preparation method and application in protein analysis. Background technique [0002] In the mid-1990s, on the basis of the research and development of the Human Genomic Project (Human Genomic Project) and functional genomics, an emerging discipline that studies the composition and activity of intracellular proteins at the overall level - protein Proteomics, which takes the proteome as the research object. Proteomics has become an important field of functional genomics research, and its importance and strategic significance are becoming more and more significant. [0003] In the past few years, the commonly used method for proteome research is: based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), thousands of proteins are separated and stained to obtain a two-dimension...

Claims

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Application Information

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IPC IPC(8): G01N30/56G01N33/68
Inventor 高明霞张祥民张鹏洪广峰
Owner FUDAN UNIV
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