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Proteomics analysis method based on SP3 enzymolysis

A technology of proteomics and analysis methods, applied in the field of biological detection, can solve the problems of large sample loss, time-consuming, affecting the effect of enzymatic hydrolysis, etc., and achieve the effect of realizing automation and improving throughput

Pending Publication Date: 2020-06-19
上海中科新生命生物科技有限公司
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Problems solved by technology

[0003] Commonly used proteolysis methods include in-solution enzymatic hydrolysis and Filter-Aided Sample Preparation (abbreviated as FASP). Among them, in-solution enzymatic digestion has restrictions on the composition of the protein solution, and common denaturants (such as SDS, urea, and guanidine hydrochloride) will affect Trypsin activity affects the effect of enzymatic hydrolysis; while FASP enzymatic hydrolysis steps are cumbersome, time-consuming, and sample loss is large, which is not conducive to small samples. Therefore, the invention of new enzymatic hydrolysis methods is of great significance to the development of proteomics

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Embodiment Construction

[0023] The technical solutions of the present invention will be described below in conjunction with the accompanying drawings and embodiments.

[0024] A proteomics analysis method based on SP3 enzymatic hydrolysis. First, add lysate to the cells of the sample to be tested for denaturation, and then add magnetic beads to the denatured sample to replace the lysate. solution, and finally extract the enzymolysis sample for chromatography-tandem mass spectrometry analysis, including the following detailed steps:

[0025] Step S1 For denatured cell samples, take a plate of 293T cells overgrown in a 10cm culture dish, wash the cells with 10mM phosphate buffered saline for 3 times, then add 500μl lysate to the cleaned cell samples, and use a homogenizer After homogenization, a contact ultrasonic instrument was used for assisted denaturation. The ultrasonic power was 200W. The ultrasonic treatment method was 8S, followed by a 5S interval, followed by the second treatment, and the cycl...

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Abstract

The invention aims to provide a proteomics analysis method based on SP3 enzymolysis. The method comprises the following steps: adding lysate into cells of a sample to be detected for denaturation; wherein the component of the lysis solution is 50mM of 4-hydroxyethylpiperazine ethanesulfonic acid; 1% lauryl sodium sulfate, 1% of polyethylene glycol octyl phenyl ether; 1% Tween 20, 1% of ethyl phenyl polyethylene glycol; 5 mM of ethylenediamine tetraacetic acid; 50mM sodium chloride, 1% of glycerol; a 1 * protease inhibitor; and 5 mM dithiothreitol, adding a magnetic bead replacement lysate intothe denatured sample; removing a denaturant influencing trypsin activity; after the replacement is finished, carrying out lysine protease enzymolysis and trypsin enzymolysis on the denatured sample;the method has the advantages that automation can be achieved, the repeatability of experiments of different batches is improved, and meanwhile the flux is improved, and meanwhile the method is not inferior to a traditional protein enzymolysis method in terms of evaluation indexes such as the peptide fragment recovery rate and the sample repeatability.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a proteomics analysis method based on SP3 enzymolysis. Background technique [0002] With the implementation and advancement of the Human Genome Project, life science research has already entered the post-genome era. In this era of greater focus on the function and structure of genes, proteomics has flourished. The concept of proteome (Proteome) was first proposed by Marc Wilkins, which refers to all proteins (Protein) expressed by a genome (genome), or a cell or tissue. Proteomics research mainly includes proteomics qualitative, high-throughput quantitative proteomics, targeted proteomics, and protein post-translational modification. The traditional shotgun method is most widely used in proteomics research. The main process of this method is to hydrolyze protease into peptides by protease, and then analyze it by mass spectrometry, and finally match the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12P21/06G16B35/00G16B25/10
CPCG01N33/6848C12P21/06G16B25/10G16B35/00
Inventor 李嘉玲
Owner 上海中科新生命生物科技有限公司
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