After exclusion of 4 known photome hits, 12 putative proteins of interest remained, featuring concentration-dependent enrichment (Figure ). Interestingly, almost the same scope of protein hits was also observed in A549 cells suggesting a conserved set of targets within these different cell lines (Figure ). With those promising initial results for Cipro P1, we again shifted our attention to PDL-hTERT cells as our model system for tendons/ligaments. For these studies, we applied our full set of diversified Cipro P1, Cipro P2 as well as Levo P1 and Levo P2 probes. Gel-based studies revealed an optimal labeling concentration of 5 µM with the P1-probes and 25 µM for the P2-probes attributed to the branched photo-crosslinker, which was reported to require higher concentrations for sufficient labeling (Figure ). AfBPP of Cipro P1 in PDL-hTERT cells revealed a strong overlap of 6 proteins which are in common with the respective experiment in HEK-293 and A549 cells and three proteins that were solely enriched in PDL-hTERT cells (Figure ). Despite the structural differences between their cyclic scaffolds, Cipro P1 and Levo P1 enriched a mutual set of 8 proteins including ClpP, AIFM1, NENF, TRIM21, SLC19A1, TEX264, GRAMD1A and IDH2 (Figure ). Importantly, largely different protein hits were obtained with Cipro P2 and Levo P2, with 9 shared hits between both and no common overlap of the P2-with the P1-probes, suggesting a significant effect on the labeling specificity by de-functionalizing the free acid (Figure ). Interestingly, a majority of targets are lysosomal proteins suggesting that the modification of the free acid enhances the direction or trapping of the scaffold into the acidic lysosomal compartment (Figure ). This notion was confirmed by co-localization of Cipro-P2 with LAMP1 in the lysosome via immuno-cytochemistry corroborating previous reports about lysosomotropic effects of Ciprofloxacin (Figure ). Thermal protein profiling (TPP) is based on protein stabilization (or destabilization) upon small molecule binding. Treated and untreated proteomes are heated to several temperatures and the resulting fraction of soluble proteins is quantified by LC-MS/MS resulting in characteristic protein melting curves (Figure ). This method bears the advantage that the unmodified FQ can be directly applied without any derivatization, however, the detection of intrinsically stabilized proteins (e.g. in the membrane or protein dimers) is limited. When performing TPP in situ using intact cells rather than lysate, the method is also suitable to detect changes in protein stabilization due to down-stream and secondary effects on whole pathways. Notably, TPP with Ciprofloxacin has been performed before in bacteria by Mateus et al., highlighting the feasibility of this method for FQs. We thus used this method as a complementary approach to AfBPP and our whole proteome studies providing additional insights into general proteome stability upon Ciprofloxacin treatment. HEK-293 cells were treated with Ciprofloxacin (75 µM) for 1 h at 37 °C, processed and analyzed via LC-MS/MS. Interestingly even after only 1 hour of treatment, numerous mitochondrial ribosomal subunits were destabilized indicating impaired mitochondrial translation and, again, mitochondrial dysfunction (Figure ). Vice versa, we obtained few proteins with increased stability upon Ciprofloxacin treatment, including NUDT1 and ADAL (Figure ). Interestingly, NUDT1 is the single overlapping protein found in both AfBPP and TPP experiments. The limited overlap between AfBPP and TPP could be attributed to the discussed complementary design of both approaches and highlights the value of performing both methods. With both protein enrichment (AfBPP) and protein stabilization (TPP) data in hand we prioritized putative targets for validation studies. Based on the results we decided to focus on ClpP (mitochondrial proteostasis) , AIFM1 (respiratory chain complexes I and IV biogenesis) , IDH2 (mitochondrial ROS homeostasis, TCA cycle and mitochondrial metabolism and connection to MMP activity) , SCARB1 (cholesterol homeostasis) , PTGR2 (inflammation and lipid metabolism) , NUDT1 (oxidized purine nucleoside salvage) and ADAL1 (methylated purine nucleoside salvage) . In addition to the discussed photome hits, we decided to exclude general transporter-like proteins (e.g. for xeniobiotics) and proteins frequently found enriched with the minimalist photo-crosslinker appended to diverse scaffolds from target validation, as they are likely transiently labeled by the photo-crosslinker (SCCPDH, SLC25A20, SLC19A1, ABCC4). We excluded NENF, as the protein is secreted and reported to be frequently overexpressed in cancer and immortalized cell lines. Notably, those proteins could, however, give an idea of how the probes and potentially also the parent FQs are translocated into the cells and organelles.