Fig. 2. Activity-based protein profiling (ABPP) using MT16-205 in HEK293 cells (a) activity-based protein profiling (ABPP) workflow in combination with mass-spectrometry proteomics. Briefly, cells are incubated with ABP and lysed. The ABP-labeled proteins are ligated with a capture reagent bearing a fluorophore (TAMRA) and/or affinity tag (biotin) via CuAAC. Labeled proteins are visualized by in-gel fluorescence analysis or affinity enriched and analyzed by immunoblotting. To identify and quantify the labeled proteins, enriched proteins are digested, resulting in peptides which can be analyzed by nanoLC-MS/MS. (b) In-gel fluorescence analysis of ABP MT16-205 labeling. HEK293 cells were incubated with 10, 40, 160, 630 or 2500 nM MT16-205 for 3 h. The lysate was ligated to capture reagent AzTB, separated by SDS-PAGE and visualized by in-gel fluorescence. (c) Affinity enrichment of ABP-labeled proteins was performed using streptavidin magnetic beads. The total lysate before-pulldown (TL) and pulldown (PD) samples were separated by SDS-PAGE and analyzed by in-gel fluorescence and immunoblotting using the indicated antibodies. β-Actin was used as a protein loading control.