A method for the enrichment and identification of lysine nitrogen-linked phosphorylated post-translational modifications
A technology of post-translational modification and identification method, which is applied in the field of enrichment and identification of lysine nitrogen-linked phosphorylation post-translational modifications, can solve problems such as few reports on abundance and biological function, and achieve a simple enrichment strategy. Effect
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Embodiment 1
[0036] 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.
[0037] 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) Perform dimethylation labeling, then add 2 μL of trifluoroacetic acid (TFA) to the peptide mixture, and react in a water bath at 60° C. for 1 h to remove phosphorylated peptide modifications. Cleavage-hydrophobic derivatization of dephosphorylated peptide mixtures, derivatization reagents such as image 3 as shown in a. Next, the solution was lyophilized and redissolved in solution A (2% acetonitrile, 0.1% TFA), and an appropriate amount of XBP C18 filler was added thereto, incubated at room temperature, washed and eluted ( figure 2 b), adding ...
Embodiment 2
[0048] Enrichment and identification of lysine phosphorylated peptides from E. coli proteolytic digests.
[0049] 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 dimethylated, and then the mixed peptides were chromatographically fractionated using a high pH C18 reversed-phase chromatographic column, and the fractions were combined and collected every 5 minutes, and a total of 7 fractions were collected. Add 2 μL of TFA, react at 60°C for 1 h to remove the phosphorylation modification of the peptide, freeze-dry each fraction after dephosphorylation, redissolve in 80% acetonitrile (50 mM HEPES) solution, and carry out cleavable hydrophobization derivatization ( image 3 a). Next, the solution was lyophilized and redissolved in solution A (2% acetonitrile, 0.1% TFA), and an appropriate amount of XBP C18 filler was added thereto. After incubation at ro...
Embodiment 3
[0063] Enrichment and identification of lysine phosphorylated peptides from HeLa protein digests.
[0064] Firstly, trypsin is used to enzymatically hydrolyze the HeLa protein to obtain an enzymolyzed product of the HeLa protein. Then the enzymatic hydrolysis product was marked with dimethylation, and then the mixed peptides were chromatographically fractionated using a strong cation exchange column. A total of 10 fractions were collected, and 2 μL TFA was added to each fraction collected, and reacted at 60°C for 1 hour to remove Phosphorylation modification of the peptide, desalination of each fraction after dephosphorylation, redissolving in 70% acetonitrile (50mM HEPES) solution, and derivatization by cleavable hydrophobization ( image 3 b). Next, the solution was lyophilized and redissolved in solution A (2% acetonitrile, 0.1% TFA) with a 5 μm, 4.6mm i.d.×150mm XBP C18 chromatographic column for high performance liquid chromatography for enrichment, after cleaning and ...
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