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Protein identification method based on two-end equal-weight label and database search

A technology of protein identification and database, which is applied in the field of protein identification based on equal weight tags at both ends and database search, can solve the problems of reducing the score of candidate peptides, poor quality of fragment ions, and affecting the identification efficiency of peptides and proteins, etc., to achieve The effect of improving identification efficiency

Inactive Publication Date: 2017-04-26
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

The stable isotope labeling method at both ends of the polypeptide is a commonly used quantitative method in proteomics. By performing stable isotope labeling on the N-terminus and C-terminus of the polypeptide, the peptides in two or more samples have the same or similar quality, and In the process of chromatographic separation, they have the same or similar retention time, so they are fragmented simultaneously in the subsequent mass spectrometry analysis, and the fragment ions formed have a certain quality difference; by comparing the mass spectrum signal intensities of fragment ions in different samples, different samples can be realized Quantitative (Koehler CJ, ArntzenMO, Treumann A, Thiede B, Anal.Bioanal.Chem., 2012, 404 (4), 1103-1114) of polypeptide and protein in the polypeptide, described labeling method utilizes stable isotope labeling to form higher Specific paired fragment ions are quantified, which has good quantitative accuracy; however, the labeling method causes a multiplied increase in the complexity of the peptide mass spectrum, which is not conducive to the identification of the peptide
The current protein identification algorithm only uses the fragment ions formed by one stable isotope labeling form of the peptide to calculate the score value of the candidate peptide, and uses the fragment ions formed by another stable isotope labeling form as interference ions, which will Reduce the score of candidate peptides, seriously affecting the identification efficiency of peptides and proteins

Method used

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  • Protein identification method based on two-end equal-weight label and database search

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Experimental program
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Effect test

Embodiment 1

[0028] (1) Treat the protein as follows: dissolve 100 μg of bovine serum albumin in 1 mL of urea with a concentration of 8 M; then add 100 μL of dithiothreitol with a concentration of 10 mM and place it in a water bath at 56°C for 2 hours; then add 100 μL of dithiothreitol with a concentration of 10 mM 20mM iodoacetamide, placed in the dark for 1h to obtain the treated protein; dilute the urea concentration in the treated protein to 0.8M with 50mM sodium phosphate with a pH value of 7.5, and then add the intracellular protease lysine Acid-C, the mass ratio of bovine serum albumin to intracellular protease lysine-C is 25:1, incubated overnight at 37°C for enzyme digestion to obtain polypeptides.

[0029] (2) Divide the polypeptide obtained in step (1) into two equal parts. After the first part of the polypeptide is desalted with a C18 pre-column, the N-terminus is labeled with a formaldehyde solution containing H. Specifically: 5 μL of 0.6M cyanide Sodium borohydride and 55 μL ...

Embodiment 2

[0044] (1) The protein is processed as follows:

[0045] 1) Protein C-terminal metabolic labeling

[0046] HeLa cells were cultured with cell culture medium containing lysine with different light and heavy isotopes. A cell culture medium containing 13 C-labeled lysine, another cell fluid containing 12 C-labeled lysine; HeLa cells were passaged 8 times to ensure 12 C and 13 C mark completely.

[0047] 2) Extract proteins from the light and heavy isotope-labeled HeLa cells obtained in 1), respectively; take 100 μg of the light and heavy isotope-labeled HeLa cell proteins, respectively, and perform the following operations: dissolve in 1 mL of 8M urea, add 100 μL Dithiothreitol at a concentration of 10 mM was placed in a water bath at 56°C for 1 hour, then 100 μL of iodoacetamide at a concentration of 20 mM was added, and placed in the dark for 0.5 hour to obtain the treated protein, which was treated with 50 mM phosphoric acid at a pH of 7.5 Sodium Dilute the urea concentr...

Embodiment 3

[0059] (1) Treat the protein as follows: Dissolve 100 μg of yeast extract protein in 1 mL of urea with a concentration of 8 M; then add 100 μL of dithiothreitol with a concentration of 10 mM, and place it in a water bath at 56°C for 2 hours; then add 100 μL of dithiothreitol with a concentration of 10 mM 20mM N-ethylmaleimide was placed in the dark for 10min to obtain the treated protein; the urea concentration in the treated protein was diluted to 0.8M with 50mM sodium phosphate with a pH value of 7.5, and then added to the cell Endoprotease lysine-C, the mass ratio of yeast extract protein to intracellular protease lysine-C is 25:1, incubated overnight at 37°C for enzyme digestion to obtain polypeptides.

[0060] (2) Divide the polypeptide obtained in step (1) into two equal parts. After the first part of the polypeptide is desalted with a C18 pre-column, the N-terminus is labeled with succinic anhydride containing H. Specifically: 100 μL of 0.1M The succinic anhydride solut...

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Abstract

The invention relates to a protein identification method based on a two-end equal-weight label and database search and belongs to the technical field of biological information. According to the identification method, a protein enzymolysis product obtained by performing isotoplabelling at the two ends of polypeptide is subjected to separation and detection through liquid chromatogram-mass spectrum combination; when identification is conducted by a database search method, identification of polypeptide is realized by establishing a peptide fragment theory spectrogram containing all the fragment ions of the peptide section formed by two labels and matching with the experimental mass spectrogram, so that the protein is obtained through identification, the identification efficiency of polypeptide and the protein is improved, and the method can be used for efficiently identifying the protein sample of the database.

Description

technical field [0001] The present invention relates to a protein identification method based on isobaric labeling and database search, in particular, relates to a stable isotope labeling of the nitrogen (N) terminal and carbon (C) terminal of the polypeptide respectively, and according to the label formed The light and heavy b (N-terminal) ions and the light and heavy y (C-terminal) ions calculate the score of candidate peptides, and the method for identifying polypeptides and proteins based on a database search method belongs to the field of biological information technology. 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. At present, the most commonly used protein identification method is to hydrolyze the protein to form peptides, perform liquid chromatography separation and mass spectrometry identification, and then compa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/89
CPCG01N30/02G01N30/89
Inventor 郑乃仁
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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