A Mass Spectrometry De novo Sequencing Method Based on Peptide C-terminus Selective Enzyme Labeling

An enzyme labeling and selective technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of inability to splice sequence information and obtain fragment ion information, and achieve good selectivity, improve sequencing efficiency, and avoid reactions.

Active Publication Date: 2022-05-17
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, for peptides with long sequences (more than 15 amino acids), complete fragment ion information cannot be obtained in tandem mass spectrometry, resulting in the inability to splice complete sequence information after post-processing of mass spectrometry data

Method used

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  • A Mass Spectrometry De novo Sequencing Method Based on Peptide C-terminus Selective Enzyme Labeling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Weigh 1 mg of peptide powder from bovine pancreas, and add 50 μL of N,N-dimethylformamide to make peptide mother solution. Take another centrifuge tube, add 243 μL of acetonitrile and 2 μL of hydrazine solution, mix well, add 5 μL of peptide mother solution to the centrifuge tube, and mix well. Transfer to 100mg of peptide C-terminal amidase, and react at 50°C for 18h. After the reaction is completed, centrifuge at 16000 g for 10 min, and take the supernatant for freeze-drying. After freeze-drying, 20 μL of 2% formic acid aqueous solution was added to redissolve for mass spectrometric identification. According to the characteristics of the fragmentation law of fragment ions in the secondary mass spectrometry, the sequence of the peptide segment was deduced.

Embodiment 2

[0017] Weigh 5 mg of the peptide mixture powder produced by enzymatic hydrolysis of bovine serum albumin, and prepare the peptide mother solution with 50 μL of dimethyl sulfoxide. Take another centrifuge tube, add 240 μL of acetonitrile and 5 μL of tris(2-aminoethyl)amine solution, mix well, add 5 μL of polypeptide mother solution to the centrifuge tube, and mix well. Transfer to 150mg of peptide C-terminal amidase, and react at 50°C for 24h. After the reaction is completed, centrifuge at 16000 g for 10 min, and take the supernatant for freeze-drying. After freeze-drying, 20 μL of 2% formic acid aqueous solution was added to redissolve for mass spectrometric identification. According to the characteristics of the fragmentation law of fragment ions in the secondary mass spectrometry, the sequence of the peptide segment was deduced.

Embodiment 3

[0019] Weigh 1 mg of peptide powder from mouse brain and add 50 μL of acetonitrile to make peptide mother solution. Take another centrifuge tube, add 243 μL of acetonitrile and 2 μL of ethylenediamine solution, mix well, add 5 μL of peptide mother solution to the centrifuge tube, and mix well. Transfer to 150mg of peptide C-terminal amidase and react at 50°C for 18h. After the reaction is completed, centrifuge at 16000 g for 10 min, and take the supernatant for freeze-drying. After freeze-drying, 20 μL of 2% formic acid aqueous solution was added to redissolve for mass spectrometric identification. According to the characteristics of the fragmentation law of fragment ions in the secondary mass spectrometry, the sequence of the peptide segment was deduced.

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Abstract

The invention relates to a mass spectrometry de novo sequencing method based on peptide C-terminal selective enzyme labeling and its application. The method is that under the catalysis of amidase at the C-terminus of the peptide, the carboxyl group at the C-terminus of the peptide is directly labeled with a small molecule with a basic group through an amidation reaction or is first labeled with an amino group with an amino group through an amidation reaction. Saturated hydrocarbons are further linked to basic functional molecules through the click chemical reaction of azide-alkynyl or mercapto-ene. The basic molecule on the C-terminal label of the peptide increases the number of y ions in the secondary mass spectrometry, and improves the response and fragmentation efficiency of the peptide in the mass spectrometer, thereby realizing peptide sequencing.

Description

technical field [0001] The invention relates to a mass spectrometry de novo sequencing method based on peptide C-terminal selective enzyme labeling and its application. Background technique [0002] Polypeptide is an important constituent unit of protein, widely exists in living systems, and plays an important role in various fields such as cell division and growth. Using peptides as drugs is one of the trends in drug market research and development in recent years. At present, there are more than 80 kinds of peptide drugs approved for marketing in the world. However, the sequences of many active peptides or newly developed peptide drugs are unknown, and the sequencing of peptides and peptide drugs is conducive to understanding the properties of peptides, and is of great significance to their activity and function. The C-terminal sequencing of peptides has high practical value. The early C-terminal sequencing based on the carboxypeptidase method judged the C-terminal seque...

Claims

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Application Information

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