Isolation and identification of phosphorylated lysine peptides by retention time difference combining dimethyl labeling strategy

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Protein phosphorylation plays essential roles in various biological procedures. Despite the well-established enrichment strategies for O -phosphoproteomics, the intrinsic acid lability of N–P phosphoramidate bond (phosphorylation of histidine, arginine and lysine) has impaired the progress of N -phosphoproteomics. Herein, we reported a retention time difference combining dimethyl labeling (ReDD) strategy for the isolation and identification of phosphorylated lysine (pLys) peptides. By such a method, pLys peptide could be isolated under 100000-fold interference of non-phosphorylated peptides. Furthermore, ReDD strategy was applied to map pLys sites from E. coli samples, leading to the identification of 11 pLys sites, among which K26p that originating from autonomous glycyl radical cofactor was validated both in mass spectrometry and HPLC co-elution experiments. Furthermore, 112 pLys sites from 100 proteins were identified in HeLa cells. All these results demonstrate that ReDD could provide a first glimpse into Lys phosphorylation, and could be an important step toward the global perspective on protein phosphorylation.