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Hydrophobic group modification-based protein N-terminal enrichment method

A hydrophobic group and protein technology, which is applied in the preparation of test samples, material inspection products, biological testing, etc., can solve the problems such as the difficulty of removing materials to eliminate non-specific adsorption, limiting the enrichment selectivity of methods, and the loss of terminal peptides , to achieve high labeling efficiency, promote efficient removal, and high selectivity

Active Publication Date: 2017-10-31
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the limited bonding efficiency and labeling efficiency, it is difficult to completely remove non-N-terminal peptides, which limits the enrichment selectivity of the method; in addition, the removal materials used have non-specific adsorption that is difficult to eliminate, resulting in the loss of terminal peptides. lost

Method used

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  • Hydrophobic group modification-based protein N-terminal enrichment method
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  • Hydrophobic group modification-based protein N-terminal enrichment method

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Such as figure 1 As shown, after the protein is denatured and reductively alkylated, the free amino group is blocked, and then the labeled protein is enzymatically hydrolyzed. The enzymatic hydrolyzed product is labeled with an amino-active reagent with a long carbon chain, and finally the reversed-phase material is used to remove non-N- Telopeptide, thus obtaining the N-terminus of the protein.

[0031] Take bovine serum albumin and lysozyme as samples, dissolve 100 μg protein in 100 μl 100 mM triethylenediamine carbonate buffer at pH 8 containing 6M guanidine hydrochloride, add 2 μl 100 mM dithiothreitol, and denature at 56 ° C for 1 hour , add 2 μl 300mM acrylamide to react for 1 hour, add another 2 μl 100mM dithiothreitol, incubate for 10 minutes, add final concentration of 100mM formaldehyde and sodium cyanoborohydride to dimethylate the free amino group of the blocked protein, and incubate at room temperature for 2 hours, Transfer the solution to a 10 000 Da ultr...

Embodiment 2

[0034] Take yeast cells as samples, dissolve 100 μg of extracted protein in 100 μl of 100 mM phosphate buffer at pH 8 containing 6M guanidine hydrochloride, add 2 μl of 100 mM dithiothreitol, denature at 56°C for 1 hour, add 2 μl of 300 mM acrylamide for 1 hour Afterwards, another 2 μl of 100 mM dithiothreitol was added, and after incubation for 10 min, a final concentration of 100 mM formaldehyde and sodium cyanoborohydride were added to dimethylate the free amino groups of the blocked protein. After incubation at room temperature for 2 h, the solution was transferred to a 10 000 Da ultrafiltration On the membrane, after removing the solvent by centrifugation and washing residual reagents with 50mM pH 8 4-hydroxyethylpiperazineethanesulfonic acid buffer, the protein was dissolved in 200μl 50mM pH 8 4-hydroxyethylpiperazineethanesulfonic acid buffer middle. Trypsin was added to carry out enzymatic hydrolysis on the ultrafiltration membrane, wherein the amount of enzyme was 1 / 5...

Embodiment 3

[0036] Take human serum as a sample, dissolve 100μg protein in 100μl 100mM phosphate buffer with pH 8 containing 6M guanidine hydrochloride, add 2μl 100mM dithiothreitol, denature and reduce at 56°C for 1h, add 2μl 300mM acrylamide for 1h reaction , add 2 μl of 100mM dithiothreitol, incubate for 10min, add final concentration of 100mM acetic anhydride to block the free amino group of protein, incubate at room temperature for 2h, transfer the solution to a 10 000Da ultrafiltration membrane, centrifuge to remove the solvent and use 50mMpH 8 After washing residual reagents with 4-hydroxyethylpiperazineethanesulfonic acid buffer, the protein was dissolved in 200 μl of 50 mM pH 8 4-hydroxyethylpiperazineethanesulfonic acid buffer. Trypsin was added to carry out enzymatic hydrolysis on the ultrafiltration membrane, wherein the amount of enzyme was 1 / 50 (w / w) of the sample mass, the temperature was 37°C, and the enzymatic hydrolysis was performed for 12 hours. The ultrafiltration mem...

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Abstract

The invention relates to a hydrophobic group modification-based protein N-terminal enrichment method, which comprises the steps of sealing free amino groups in protein level, carrying out marking through a strong hydrophobic group, and carrying out affiliative trapping through a reverse phase material. Particularly, the method comprises the steps of firstly sealing free amino groups of an N terminal and a side chain in protein level by adopting chemical modification and carrying out enzymolysis on the protein; introducing the strong hydrophobic group to a non-N-terminal peptide segment through newly generated free amino groups; and finally removing the non-N-terminal peptide segment with the strong hydrophobic group by adopting the reverse phase material in an affiliative manner to obtain an N-terminal peptide segment of the protein. The hydrophobic group modification-based protein N-terminal enrichment method has the advantages of being high in mark efficiency, high in removal efficiency, high in selectivity and simple in processing step, and the sample loss and nonspecific adsorption are reduced.

Description

technical field [0001] The invention relates to a protein N-terminal enrichment method, that is, a protein N-terminal enrichment method based on hydrophobic group modification, so as to realize high-efficiency and high-selectivity enrichment of protein N-terminal. Background technique [0002] The modification of protein N-terminus is one of the common post-translational modifications in organisms. Common N-terminal acetylation and signal peptide cleavage are closely related to protein stability, localization and activity. In addition, the degradation and shearing of proteins in vivo is one of the most common post-translational modifications in vivo, which promotes the generation of new N-terminals of proteins. This process is related to many biological processes and physiological functions such as apoptosis and chemokine processing. closely related. The establishment of terminal proteomics has also greatly promoted the development of proteolytic enzyme substrate identifica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/40G01N33/68
Inventor 张丽华陈玲凡单亦初杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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