Protein N-terminal peptide fragment reverse enrichment method based on guanidination labeling

A guanidine and protein technology, which is applied in the field of reverse enrichment of protein N-terminal peptides based on guanidine labeling, can solve the problems of complex secondary fragment information, unfavorable peptide ionization, and interference with peptide identification, etc. Achieve the effect of improving secondary ion fragment information, improving ionization efficiency, and efficient enzyme cleavage rate

Inactive Publication Date: 2020-05-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the lysine ε-amino group of the protein is marked by dimethylation, acetylation, etc., trypsin cannot digest lysine, which will cause a serious enzyme missed cleavage rate, making the peptides obtained by enzymatic digestion too high. Long, when using mass spectrometry for analysis, the secondary fragment information is complex, which is not conducive to identification; at the same time, after the N-terminus of the protein is marked by dimethylation, acetylation, etc., it is not conducive to the ionization of the peptide segment, which further interferes with the N-terminal peptide segment of the protein Identification

Method used

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  • Protein N-terminal peptide fragment reverse enrichment method based on guanidination labeling

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Embodiment 1

[0026] like figure 1 As shown, after reductive alkylation of the protein, the N-terminal amino group of the protein and the ε-amino group of lysine were guanylated using 1H-pyrazole-1-carboxamidine, and the guanidinylated Enzymatic digestion of protein to obtain peptides, use HPG-ALD or sixteen circles to label the enzyme-digested peptides, and use filter membrane or C18 reverse chromatography to remove non-protein N-terminal peptides, so as to realize the identification of protein N-terminal peptides Inverse enrichment.

[0027] Taking yeast as a sample, the yeast was first ground by liquid nitrogen grinding method, and the protein was extracted by using 6M guanidine hydrochloride. Take 200 μl of yeast protein with a concentration of 1 μg / μl for enrichment of N-terminal peptides. Add 2 μl of 1M dithiothreitol, denature at 56°C for 30 minutes, add 6 μl of 1M iodoacetamide and react in the dark for 30 minutes at room temperature, then add 6 μl of 1M dithiothreitol to the reac...

Embodiment 2

[0029] Taking yeast as a sample, the yeast was first ground by liquid nitrogen grinding method, and the protein was extracted by using 6M guanidine hydrochloride. Take 200 μl of yeast protein with a concentration of 1 μg / μl for enrichment of N-terminal peptides. Add 2 μl of dithiothreitol with a concentration of 1M, denature and reduce at 56°C for 30 minutes, add 6 μl of iodoacetamide with a concentration of 1M and react in the dark for 30 minutes at room temperature, and then add 6 μl of 100 with a concentration of 1M dithiothreitol to the reaction system, Incubate at room temperature for 30 min to terminate excess iodoacetamide, add 1H-pyrazole-1-carboxamidine and 500 mM triethylamine at a final concentration of 500 mM to the solution, and label at 95° C. for 15 min. Use formic acid to adjust the pH of the reaction system to 7-8, use a 10kDa filter to remove unreacted guanidinylation reagents, dissolve the protein in 200 μl of 50 mM HEPES, adjust the pH to 8.0, and add 4 μg ...

Embodiment 3

[0031]Taking yeast as a sample, the yeast was first ground by liquid nitrogen grinding method, and the protein was extracted by using 6M guanidine hydrochloride. After the protein was reductively alkylated, 1H-pyrazole-1-carboxamidine and 500 mM triethylamine were added to the solution at a final concentration of 500 mM, and labeled at 95° C. for 30 min. Add acetone with a final concentration of 80 to the reaction system, and place it at -20°C for more than 2 hours. Centrifuge to get the protein pellet, and wash three times with acetone at -20°C. Finally, the protein was dissolved in 50 mM TEAB solution, the amount of trypsin used was 1 / 50 of the protein mass, and digested at 37 degrees overnight. Add N-hydroxysuccinimide ester active agarose to the peptide generated after enzymatic hydrolysis, react at 25°C for 1 hour, then use 100mM Tris-Cl to terminate the active group on the amino active material, react at 37°C for 1 hour, Centrifuge at 2000rcf for 10min to separate the ...

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Abstract

The invention relates to an analysis method for protein N-terminal peptide fragment reverse enrichment based on guanidination labeling and application. The analysis method comprises the steps of guanidination labeling of lysine epsilon-amino and polypeptide N-terminal amino, removal of a guanidination reagent, trypsin digestion of guanidination lysine C-terminal peptide bonds and selective removalof non-protein N-terminal peptide fragments. Firstly, labeling conditions capable of efficiently guanidinating and labeling lysine epsilon-amino and polypeptide N-terminal amino are established, andexcessive labeling reagents are selectively removed; and then the optimized labeling conditions are applied to N-terminal amino and lysine epsilon-amino guanidination labeling in the protein, and theamino active material is utilized to react with N-terminal amino of a peptide fragment generated by enzyme digestion so as to realize reverse enrichment of the N-terminal peptide fragment of the protein. The method has the advantages that lysine epsilon-amino and peptide end N-segment amino guanidination labeling is efficient, the ionization rate is efficient, the protein N-end peptide segment reverse enrichment rate is increased and the identification depth is increased.

Description

technical field [0001] The invention relates to a reverse-phase analysis method for protein N-terminal peptide segments based on guanidinylation labeling, which improves the identification sensitivity and identification depth of protein N-terminal information. Background technique [0002] The N-terminus of protein is generated during protein synthesis and is extensively modified after synthesis, such as N-terminal promoter methionine removal and N-terminal acetylation modification, as well as conversion into mature and active proteins through specific hydrolysis. Specific proteolytic treatment produces new protein forms, which may significantly change the stability, activity and function of the protein. For example, after caspase-1 or caspase-11 is activated, it can specifically cut the two domains of GSDMD protein The released N-terminal domain can recognize and bind the phospholipid molecules on the cell membrane, making holes in the cell membrane, resulting in changes in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8831
Inventor 张丽华孙明伟单亦初梁振杨开广梁玉张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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