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Protein amino acid sequence de novo sequencing method based on unequal stable isotope labeling at two ends of polypeptide

A protease lysine and protein technology, applied in the field of de novo sequencing of protein amino acid sequences to identify fragment ions, can solve the problems of reduced probability and intensity, low specificity, complex peptide fragmentation mechanism, etc. Efficient and selective effect

Active Publication Date: 2016-02-03
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

Method used

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  • Protein amino acid sequence de novo sequencing method based on unequal stable isotope labeling at two ends of polypeptide
  • Protein amino acid sequence de novo sequencing method based on unequal stable isotope labeling at two ends of polypeptide
  • Protein amino acid sequence de novo sequencing method based on unequal stable isotope labeling at two ends of polypeptide

Examples

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Embodiment 1

[0022] 1. Two-terminal labeling of protein enzymatically digested polypeptides based on dimethylation markers

[0023] Such as figure 1 As shown, mark according to the following process:

[0024] 1) Denaturation, reduction, alkylation and enzymatic hydrolysis of protein samples: Dissolve 100 micrograms of protein in 1 milliliter of 8M urea, add 100 microliters of 10mM dithiothreitol, place in a water bath at 56°C for 2 hours, then add 100 μl of 20 mM iodoacetamide was left for 1 hour in the dark. Finally, the solution was diluted with 50mM sodium phosphate (pH7.5) to urea concentration to 0.8M, then the intracellular protease lysine-C was added according to the substrate / enzyme ratio 25 / 1 (w:w), and incubated at 37°C overnight.

[0025] 2) Dimethylation labeling at both ends of the polypeptide: Divide the polypeptide sample obtained in 1) into two equal parts,

[0026] After desalting the first column (C18 pre-column), use formaldehyde solution (5 μL 0.6M sodium cyanoboroh...

Embodiment 2

[0032] Labeling of both ends of protein enzymatic peptides based on metabolic labeling and chemical labeling

[0033] In this example, different methods for labeling both ends of the polypeptide are adopted, and the separation and identification methods for the polypeptide are the same as those in the previous example.

[0034] 1. Cell culture and metabolic labeling: For Hela cells, use DMEM containing 10% fetal bovine serum for culture. For labeling of lysine, L-lysine-free DMEM and dialyzed fetal bovine serum were used. L-lysine (146 μg / ml) was added to the "light" culture solution, and the same concentration of [ 13 C] L-Lysine.

[0035] 2. Protein extraction and pretreatment: After the cells are collected, they are dissolved in a buffer solution containing 8M urea and protease inhibitors. After the lysate was sonicated three times, the supernatant was collected by centrifugation. Lightly and heavily labeled proteins were reduced using 10 mM dithiothreitol in 50 mM sodi...

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Abstract

The invention relates to a protein amino acid sequence de novo sequencing method based on unequal stable isotope labeling at two ends of polypeptide. The method comprises the following steps: conducting enzyme digestion on protein subjected to denatured reduced alkylation by intracellular protease lysine-C to form peptide fragments containing N-terminal alpha-amino and C-terminal lysine side chain epsilon-amino; after the sample is divided into two parts, conducting selective derivatization on the N-terminal alpha-amino of the polypeptide by using a labeling reagent containing different isotopes; then conducting derivatization on C-terminal lysine side chain epsilon-amino of the polypeptide by using the labeling reagent containing different isotopes, or in cell culture, labeling lysine in the protein by using a culture solution containing isotope labeling lysine; finally mixing the sample, and conducting separation and identification by using high performance liquid chromatography- mass spectrometry. According to the method, paired polypeptide fragment ions are formed through labeling for resolution, the influence of interference signals is reduced, the selective specificity of the fragment ions and the accuracy of polypeptide sequencing are improved, and the speed of de novo sequencing is improved.

Description

technical field [0001] The invention relates to a marker-assisted protein amino acid sequence de novo sequencing method, in particular to a protein amino acid sequence de novo sequencing method for identifying fragment ions by performing stable isotope labeling on the N-terminal and C-terminal of the 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 acid sequence sequencing methods that do not rel...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 张丽华单亦初张珅吴琪张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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