A method for the enrichment and identification of lysine nitrogen-linked phosphorylated post-translational modifications
A post-translational modification, lysine nitrogen technology, applied in the field of enrichment and identification of lysine nitrogen-linked phosphorylation post-translational modifications, can solve problems such as few reports, hydrolysis, dephosphorylation, instability, etc.
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Embodiment 1
[0037] Enrichment and identification of standard lysine phosphorylated peptide A0 (phosphate group located at α-amino group of lysine side chain) in bovine serum albumin (BSA) hydrolyzate.
[0038] First, trypsin is used to enzymatically hydrolyze BSA to obtain an enzymatic hydrolysis product of BSA. Then the standard lysine phosphorylated peptide (A0) was mixed with the BSA hydrolysis product at a mass ratio of 1:100 ( figure 2 a), carry out dimethylation light standard (a chemical label), and then use high pH C18 reversed-phase chromatographic column to carry out the first chromatographic fractionation of the mixed peptides, and collect the fractions every 5 minutes ( figure 2 b) Collect 16 fractions in total, add 2 μL of trifluoroacetic acid (TFA) to each of the collected fractions, react in a water bath at 60°C for 1 h to remove the phosphorylated modification of the peptide, and perform a high-temperature reaction on each fraction after dephosphorylation The second chr...
Embodiment 2
[0051] Enrichment and identification of lysine phosphorylated peptides from E. coli proteolytic digests.
[0052] First, trypsin is used to enzymatically hydrolyze the E. coli protein to obtain an enzymatic hydrolysis product of the E. coli protein. Then the enzymatic hydrolysis product was subjected to dimethylation light labeling, and then the mixed peptide was subjected to the first chromatographic fractionation using a high pH C18 reversed-phase chromatographic column, and the fractions were combined and collected every 5 minutes, and a total of 18 fractions were collected. Add 30,000 units of alkaline phosphatase to each fraction, react in a water bath at 37°C for 1 h to remove peptide phosphorylation, and conduct a second chromatographic fractionation with high pH C18 reverse phase for each fraction after dephosphorylation, and collect relative to the first All peptides with a difference in retention time in the peak time of the secondary chromatogram, where the differen...
Embodiment 3
[0064] Enrichment and identification of lysine phosphorylated peptides from HeLa cell proteolysates.
[0065] Firstly, trypsin is used to enzymatically hydrolyze the HeLa cell protein to obtain the enzymatic hydrolyzate of the HeLa cell protein. Then the enzymatic hydrolysis product was subjected to dimethylation light labeling, and then the mixed peptides were subjected to the first chromatographic fractionation using a strong cation exchange chromatography column, and the fractions were combined and collected every 5 minutes, and 30,000 units of acidic acid was added to each fraction collected For lysine phosphatase, react in a water bath at 37°C for 1 hour to remove peptide phosphorylation modification, and perform a second chromatographic fractionation with high pH C18 reversed phase for each fraction after dephosphorylation, and the collection time is different from the peak time of the first chromatogram. All peptides with a retention time difference, where the retention...
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