PKM2 peptide mapping
Description
Mapping of 15KPF-modified peptides by LC/MS/MS technology.
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Steps to reproduce
Twenty micrograms of recombinant mouse PKM2 was incubated with 15KPF (2.5 μg) in 60 μl PBS at 37℃ for 1 h, and RT for another 1 h. The mixture was separated by 10% SDS-PAGE and stained with Coomassie blue R-250. The expected protein band was carefully recovered and completely destained in 50% acetonitrile containing 50 mM ammonium bicarbonate. Following reduction with 10 mM Tris(2-carboxyethyl) phosphine and alkylation with 55 mM iodoacetamide, trypsin digestion was performed essentially as previously described36. Peptides were recovered, desalted using Pierce C-18 Tips from Thermo Fisher Scientific (Waltham, MA, USA). After the removal of solvent, the residue was solubilized in 0.1% formic acid. Peptides were separated by Dionex 3000 RSLCnano LC system and analysed by Orbitrap Fusion Lumos mass spectrometer. The MS data were processed and searched against Uniprot human protein database with Proteome Discoverer version 2.1.0.81, The parameters were optimized to obtain peptide and protein data at the FDR level of 0.1%. The candidate 15KPF-modified peptides were deduced by applying the addition of the chemical formula C20H32O5 [Monoisotopic mass: 352.22497 Da] to amino acid residues including Cys, His, Lys.