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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  • 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

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Example Embodiment

[0024] Example 1:

[0025] Preparation of the capillary monolithic column for rapid enzymolysis of protein:

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

[0027] Chemical modification of the surface of the enzyme molecule: 10mg Trypsin solution is freshly dissolved in 5mL, 100mmol / L, PH=9.3 boric acid buffer solution, and a certain quality of benzidine is added to make the final concentration 0.5mol / L, weighing Dissolve 2.5 mg of NAS in 3...

Example Embodiment

[0029] Example 2:

[0030] Enzymolysis and mass spectrometry of the standard protein equine myoglobin (Myo) by the fast enzymatic hydrolysis monolithic column:

[0031] 50μg MYO is dissolved in 50μL of 25mM ammonium bicarbonate solution, and then driven by a peristaltic pump at a flow rate of 3μL / min through the enzymatic hydrolysis monolithic column. After the enzymatic hydrolysis products flow out, they are directly spotted onto the 192-well MALDI target plate. The volume of each spot is about 1μL, and when it is quickly air-dried at room temperature to reduce to about 0.5μL, add 0.4μL of matrix solution (α-cyano-4-hydroxycinnamic acid CHCA) to each spot, mix the two and air-dry Later used for mass spectrometry identification. 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. by Figure 4A As shown, the main characteristic ...

Example Embodiment

[0032] Example 3:

[0033] The concentration of bovine serum albumin (BSA) is 1μg / μL. The preparation method and other conditions are the same as in Example 2. Repeat the above enzymatic hydrolysis and mass spectrometry experiments. The results are as follows Figure 4B . Similarly, the main characteristic peptide peaks of BSA were detected, and the intensity was strong, indicating that the protein was effectively digested after the capillary enzymatic hydrolysis 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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