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Hybrid protein rapid enzymolysis method

A protein and enzymatic hydrolysis technology, applied in instruments, analytical materials, measuring devices, etc., can solve problems such as the inability to meet the high-throughput analysis requirements of proteomics

Inactive Publication Date: 2011-11-09
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the methods currently used in this field all have technical defects and cannot meet the high-throughput analysis requirements of proteomics. Therefore, there is an urgent need in this field to find a method for efficient and rapid enzymatic hydrolysis and protein identification.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0105] Example 1. Establishment of ultrasound power and ultrasound time safety zone

[0106] Mixed sample of 9 proteins (myosin 200KD, β-galactosidase 116KD, phosphatase b 97.2KD, bovine serum albumin 66.4KD, ovalbumin 44.287KD, carbonic acid 29KD, trypsin inhibitor 20.1KD , lysozyme 14.3KD and aprotinin 6.5KD) take 1μL (18μg / μL) after denaturation by traditional reagents, ultrasonic in the environment without the action of trypsin, the samples are detected by SDS-PAGE, analyzed by Total lab 2.0, according to Equation (1) calculates the relative percentage of protein remaining after sonication.

[0107] As a result, safe regions for ultrasound power and time were established see figure 1 , in the safe area, the protein molecules are basically not broken after being ultrasonicated, and the protein content in the sample is basically unchanged. When the total time of ultrasound is the same as the interval time, the effect of changing the continuous ultrasound time on the protei...

Embodiment 2

[0108] Embodiment 2. Determination of optimal conditions for ultrasonic-assisted enzymatic hydrolysis

[0109] In order to optimize the enzymatic hydrolysis efficiency, the two variables of ultrasonic power and ultrasonic time were selected, and a two-level response surface design (Response Surface Design) was carried out in the safe area. The response surface design is shown in Table 1 (according to the accuracy of the ultrasonic instrument power setting, the The design value of each variable was rounded up before the experiment). 1 μL (18 μg / μL) of the mixed sample of 9 proteins was denatured by traditional reagents, and then ultrasonic-assisted digestion was performed according to the response surface design table. The degree of digestion was detected by SDS-PAGE, analyzed by Total lab 2.0, and calculated according to formula (3) Relative percentage of protein digestion.

[0110] The results showed that the recommended values ​​of each factor were power 22.53W (100μL syste...

Embodiment 3

[0113] Example 3. System pH and organic phase ratio are adjusted to improve protein solubility during thermal denaturation

[0114] Take 0.5 μL (20 μg / μL) of bovine serum albumin (BSA) and cytochrome c and heat denature them in systems with different pH and organic phase ratios, then use SDS-PAGE to detect protein solubility, and use Total lab2.0 to analyze, according to Equation (2) calculates the relative percentage of protein solubilization.

[0115] The result is as figure 2 : When the pH of the system is acidic, the solubility of the protein after thermal denaturation is higher than when it is alkaline, especially the solubility difference of cytochrome c is more significant, and the solubility of BSA in the aqueous phase is higher after alkaline than acidic thermal denaturation.

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Abstract

The invention relates to a method for fast enzymolysis of hybrid proteins comprising: providing protein samples to be enzymolysis; obtaining the denatured protein samples after the degeneration treatment of said protein samples; treating said denatured protein samples by means of protease enzymolysis and assistant with ultrasonic, obtaining the protein polypeptide fragments. The invention also provides a method for fast identifying the protein. The method of the invention can quickly and high efficiently implement the protein enzymolysis and identification with easy operation and high accuracy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a method for rapid enzymatic hydrolysis of mixed proteins. Background technique [0002] Two-dimensional electrophoresis-mass spectrometry and multidimensional chromatography-mass spectrometry are currently used for protein identification. In the former, complex mixed proteins are separated by two-dimensional electrophoresis, and after in-gel enzymatic hydrolysis, the peptides are mainly detected by MALDI-TOF-MS and identified by peptide mass fingerprinting (PMF). sequence information for identification. The latter is that after the mixed protein is digested, the mixed polypeptide is separated by two-dimensional liquid chromatography on-line, and then MS / MS analysis is carried out to realize protein identification. Although two-dimensional gel electrophoresis has improved and its ability to separate proteins has made in-gel digestion suitable for the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/06G01N27/64
Inventor 李友元李雅萍王永红陈长华储炬庄英萍张嗣良
Owner EAST CHINA UNIV OF SCI & TECH
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