De Novo Sequencing Method Based on Chemical Modification and Isotope Labeling of Peptide Amino Acid Sequence

A technology of isotope labeling and chemical modification, applied in the field of de novo sequencing of protein amino acid sequences to identify fragment ions, can solve the problems of affecting the accuracy of de novo sequencing, complex peptide fragmentation mechanism, and reduced probability and intensity, so as to improve specificity, improve Accuracy and efficiency

Active Publication Date: 2019-06-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

However, because the fragmentation mechanism of the polypeptide is relatively complicated, even if the polypeptide is subjected to the aforementioned treatment, other fragment ions will still be produced when the polypeptide is fragmented, so the specificity is not high, which affects the accuracy of de novo sequencing; in addition, with the length of the amino acid sequence The probability and intensity of larger fragment ions will decrease, which is not conducive to de novo sequencing

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  • De Novo Sequencing Method Based on Chemical Modification and Isotope Labeling of Peptide Amino Acid Sequence
  • De Novo Sequencing Method Based on Chemical Modification and Isotope Labeling of Peptide Amino Acid Sequence
  • De Novo Sequencing Method Based on Chemical Modification and Isotope Labeling of Peptide Amino Acid Sequence

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Embodiment 1

[0024] 1. Peptide N-terminal labeling and C-terminal labeling based on arginine labeling

[0025] Such as figure 1 As shown, mark according to the following process:

[0026] 1) Guanylation of polypeptide lysine amino groups: Add 40 μL of 100 mM sodium bicarbonate solution containing 2M O-methylisourea to 1 ml of polypeptide solution, adjust the pH value to 11 with 2M sodium hydroxide solution, and incubate at 37 °C React for 2 hours. The reaction was then terminated by adding 10% trifluoroacetic acid and the pH of the solution was adjusted to 8.0.

[0027] 2) Polypeptide N-terminal arginine labeling: 1) the amino group of arginine is blocked with 50 mM sodium phosphate buffer solution (pH 7.5) containing 4% formaldehyde and 60 mM sodium cyanoborohydride; use 1-(3- Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activate the carboxyl group of arginine; 2) The obtained peptide samples are divided into two equal In the first batch,...

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Abstract

The invention relates to a method for de novo sequencing of polypeptide amino acid sequences based on chemical modification and isotope labeling. Using the correlation between the N-terminal and C-terminal labeled polypeptide molecular mass and retention time, the MS / MS spectra of the same polypeptide with different labels are correlated. Using the N-terminal and C-terminal fragment ion pairs formed during the fragmentation process of peptides in different labeled samples, de novo sequencing of the amino acid sequences of peptides is carried out. The present invention modifies and modifies the polypeptide with positively charged reagents, which facilitates the formation of abundant fragment ions when the polypeptide is fragmented in the mass spectrum; Fragment ions formed in pairs during cleavage are easy to distinguish, thereby reducing the influence of interference signals, improving the specificity of fragment ion selection and the speed of de novo sequencing; using the complementarity of N-terminal and C-terminal fragment ions to improve the accuracy and accuracy of peptide sequencing efficiency.

Description

technical field [0001] The present invention relates to a protein amino acid sequence de novo sequencing method assisted by chemical modification and isotope labeling, in particular to a protein amino acid sequence de novo sequencing method for identifying fragment ions by performing stable isotope labeling on the N-terminus and C-terminus of a polypeptide respectively . Background technique [0002] Protein is an important biomacromolecule and plays an important role in life activities. Sequencing proteins is essential for the analysis of protein primary structure. Although there are protein databases for some species, the amino acid sequence of proteins can be analyzed by searching the database. However, most species still do not have protein databases available for searching, and for species that already have databases, the amino acid sequence of their proteins will also change due to genetic variation. Therefore, it is very necessary to develop de novo protein amino a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02
Inventor 张丽华单亦初张珅陈玲凡张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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