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
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[0021] Example 1
[0022] 1. Labeling both ends of the protein enzymatically hydrolyzed peptide based on dimethylation
[0023] Such as figure 1 As shown, mark as follows:
[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, and then add 100 μl of 20 mM iodoacetamide was placed in the dark for 1 hour. Finally, the solution was diluted with 50mM sodium phosphate (pH7.5) to the concentration of urea to 0.8M, and then the intracellular protease lysine-C was added according to the substrate / enzyme ratio of 25 / 1 (w:w), and incubated at 37℃ overnight.
[0025] 2) Dimethylation labeling at both ends of the polypeptide: divide the polypeptide sample obtained in 1) into two equal parts,
[0026] After removing the salt on the first column (C18 pre-column), use formaldehyde solution (5μL 0.6M sodium cyanobo...
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[0031] Example 2
[0032] Labeling both ends of protein enzymatically hydrolyzed peptides based on metabolic and chemical labels
[0033] In this example, a different method of labeling both ends of the polypeptide is adopted, and the method for separation and identification of the polypeptide is the same as the above example.
[0034] 1. Cell culture and metabolic labeling: For Hela cells, use DMEM containing 10% fetal bovine serum for culture. To label lysine, DMEM without L-lysine and dialyzed fetal calf serum were used. L-lysine (146μg / ml) was added to the "light" medium, and the "heavy" medium contained 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. Light-labeled and heavy-labeled proteins were reduced with 10mM dithiothreitol,...
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