With the unique insight of proteome changes, we next focused on the identification of the corresponding molecular targets responsible for these effects utilizing our FQ-derived probes by AfBPP. We initiated these experiments by optimizing the probe concentration and duration of labeling via a fluorescent gel-based readout using Cipro P1 as proof-of-concept. Intact HEK-293 cells were treated with various concentrations of the Cipro P1 probe ranging from 2 to 75 µM for 1 h at 37 °C in FCS-free medium. After this co-incubation, the cells were irradiated for 15 min at 360 nm, lysed and reacted with TAMRA-azide via copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC) chemistry. After separation via SDS-PAGE, fluorescent scanning revealed concentration-dependent labeling starting already at 2 µM (Figure ). Subsequently, we performed the respective quantitative analysis using biotin azide for avidin bead enrichment of probe-targeted proteins and tryptic digest followed by LC-MS/MS analysis using label-free quantification (LFQ) (Figure ). In MS-based AfBPP, the comparison of proteins in probe-treated and untreated cells provides enrichment ratios and significance values facilitating the ranking of hits (P-value < 0.05, fold-change > log2 2). However, care must be taken with common photo-crosslinker off-targets (photome hits), which have been previously inventoried for human cell lines. In order to narrow down promising targets, we performed concentration-dependent labeling (0.5, 1.0 and 2.5 µM) with an optimal irradiation time of 10 min (Figure ).