Target identification of dioxygenated amide derivatives interacting proteins

To identify the direct binding proteins of dioxygenated amide by chemical proteomics, we firstly evaluated the efficiency of optimized probe 6D-PP probe labelling the E. coli bacterial proteomes by in-gel fluorescence. Briefly, the E. coli bacterial lysates were incubated with 100 M probe 6D-PP under 37 °C for 2 h and exposed to 365 nm UV light for 0.5 h to initiate photo-crosslinking. A blank control with only DMSO, a negative control without UV light and a control sample with pretreatment of 500 M D-PP6-C were also included. After conjugation with the TAMRA-N3 by copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry for another 1 h, the probe 6D-PP labelled proteins were separated with 12% SDS-PAGE and imaged by in-gel fluorescence (Fig.3B). The probe 6D-PP probe showed an efficiently UV-dependent and probe 6D-PP specific labelling, supporting it’s readily applicable in ABPP (activity-based protein profiling) experiment.