Fig 2: Chemoproteomic profiling reveals VDAC3 as a target of dankastatin B.(A) isoTOP-ABPP analysis of dankastatin B in MDA-MB-231 breast cancer cells. MDA-MB-231 breast cancer cells were treated with DMSO vehicle or dankastatin B (10 µM) for 1 h. Resulting cell lysates were treated with the cysteine-reactive alkyne-functionalized iodoacetamide probe (IA-alkyne) and subsequently taken through the isoTOP-ABPP procedure and LC-MS/MS analysis. Control (isotopically light) versus treated (isotopically heavy) probe-modified peptide ratios and adjusted p-values were calculated and shown. Shown is red are the targets that showed control/treated ratio >4 with adjusted p-values <0.05 from n=6 biological replicates/group. Only C65 of VDAC3 fit these criteria. (B) Gel-based ABPP analysis of dankastatin B against pure VDAC3 protein. Pure VDAC3 protein was pre-incubated with DMSO or dankastatin B for 30 min prior to labeling of protein with a rhodamine-functionalized iodoacetamide (IA-rhodamine) probe (1 µM) for 1 h. Proteins were resolved on SDS/PAGE and subsequently visualized by in-gel fluorescence and protein loading was assessed by silver staining. (C) Shown is a structure of the alkyne-functionalized dankastatin B probe, dankastatin B-alkyne. Shown in red are the potential electrophilic sites. (D) Dankastatin B-alkyne probe labeling of VDAC3 and competition of probe labeling by dankastatin B. Pure VDAC3 protein was pre-incubated with DMSO or dankastatin B for 30 min prior to incubation with dankastatin B-alkyne (50 µM) for 30 min. An azide-functionalized rhodamine was subsequently appended onto probe-labeled proteins by copper-catalyzed azide-alkyne cycloaddition, proteins were resolved by SDS/PAGE, and visualized by in-gel fluorescence. Protein loading was assessed by silver staining. Shown in (B, D) are representative gels from n=3 biological replicates/group.