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Method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivation

A peptide and chemical technology, applied in the field of improving the fragmentation efficiency and response of mass spectrometry based on the chemical derivatization of the C-terminal of the peptide, can solve the problems of unfavorable mass spectrometry identification, poor fragmentation efficiency, etc., and achieve the effect of high reaction efficiency

Active Publication Date: 2020-05-29
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

Due to the lack of basic amino acids at the end of many drug peptides, their fragmentation efficiency in mass spectrometry is poor, which is not conducive to mass spectrometry identification

Method used

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  • Method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivation
  • Method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivation
  • Method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] (1) Weigh 1 mg of the synthesized model peptide CT1200 (sequence LVVSTQTALA), add 1000 μL of a mixed solution of N,N-dimethylformamide and water to make a peptide mother solution, and the volume concentration of water is 5%. Take another centrifuge tube, add 20 μL peptide master solution, 75 μL acetonitrile, 2.5 μL, 1M N-hydroxysuccinimide, 2 μL, 2.5M 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide, 0.5μL, 5M methylamine, react at 25℃ for 2h. After the reaction is completed, after freeze-drying, desalting by liquid chromatography and freeze-drying are carried out.

[0027] (2) The above product was reconstituted by adding 100 μL of tris(hydroxymethyl)aminomethane buffer salt with pH 7.5, adding 0.5 μg of peptide amidase, and hydrolyzing at 25° C. for 12 hours. After the reaction was completed, desalting was carried out by liquid chromatography and freeze-dried.

[0028] (3) The above product was redissolved by adding 93.5 μL of a mixed solution of N,N-dimethylformamide...

Embodiment 2

[0031] (1) Weigh 1 mg of the synthesized model peptide 9094 (sequence KEIFVGI), add 1000 μL of a mixed solution of N,N-dimethylformamide and water to make a peptide mother solution, and the volume concentration of water is 5%. Take another centrifuge tube, add 20 μL peptide stock solution, 74.5 μL acetonitrile, 2.5 μL, 1M N-hydroxysuccinimide, 2 μL, 2.5M 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide, 1μL, 5M methylamine, reacted at 25℃ for 2h. After the reaction is completed, after freeze-drying, desalting by liquid chromatography and freeze-drying are carried out.

[0032] (2) The above product was reconstituted by adding 100 μL of tris(hydroxymethyl)aminomethane buffer salt with pH 7.5, adding 0.5 μg of peptide amidase, and hydrolyzing at 25° C. for 12 hours. After the reaction was completed, desalting was carried out by liquid chromatography and freeze-dried.

[0033](3) The above product was redissolved by adding 90 μL of a mixed solution of N,N-dimethylformamide and wa...

Embodiment 3

[0036] (1) Weigh 1 mg of the purchased drug peptide oxytocin (sequence CYIQNCPLG), add 1000 μL of acetone and water mixed solution to make peptide mother solution, and the volume concentration of water is 5%. Take another centrifuge tube, add 20 μL of peptide stock solution, 65.5 μL of N,N-dimethylformamide, 2.5 μL of 1M N-hydroxysuccinimide, 2 μL of 2.5M 1-(3-dimethylformamide Aminopropyl)-3-ethylcarbodiimide, 10 μL, 5M ethanolamine, reacted at 25°C for 2h. After the reaction is completed, after freeze-drying, desalting by liquid chromatography and freeze-drying are carried out.

[0037] (2) The above product was reconstituted by adding 100 μL of tris(hydroxymethyl)aminomethane buffer salt with pH 7.5, adding 0.1 μg of peptide amidase, and hydrolyzing at 30° C. for 24 hours. After the reaction was completed, desalting was carried out by liquid chromatography and freeze-dried.

[0038] (3) The above product was redissolved by adding 93.5 μL of a mixed solution of N,N-dimethy...

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Abstract

The invention relates to a method for improving mass spectrum fragmentation efficiency and response based on peptide fragment C-terminal chemical derivation. The method comprises three steps of reaction: firstly, sealing carboxyl at the tail end of a peptide fragment C or carboxyl at the tail end of the peptide fragment C and carboxyl of side-chain glutamic acid or aspartic acid; then, using peptide fragment amidase for specific hydrolysis and exposing carboxyl at the tail end of the peptide fragment C; and finally, carrying out chemical derivation of alkaline molecules on the exposed C-terminal carboxyl. Alkaline molecules marked at the tail end of the peptide fragment C can increase the number of y ions in the secondary mass spectrum and improve the fragmentation efficiency and responseof the peptide fragment in the mass spectrum so that the method is applied to improve the detection sensitivity of polypeptide drug peptides.

Description

technical field [0001] The invention relates to a method for improving the fragmentation efficiency and response of mass spectrometry based on the chemical derivatization of the C-terminus of the peptide segment and its application. Background technique [0002] Polypeptide drugs refer to polypeptides with specific therapeutic effects through chemical synthesis, genetic recombination or extraction from animals and plants. Peptide drugs can widely act on the endocrine system, immune system, digestive system, cardiovascular system, musculoskeletal system, etc. The peptide drug market is growing extremely fast, and it is one of the trends in drug market research and development in recent years. At present, more than 80 peptide drugs have been approved for marketing in the world, such as insulin for the treatment of diabetes, human growth hormone for the treatment of senile diseases and dwarfism, and natriuretic hormone for the treatment of cardiovascular diseases. Peptide dru...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/72
CPCG01N30/06G01N30/72G01N2030/067
Inventor 张丽华吴琼单亦初杨开广杨超张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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