A Metallomics Strategy to Reveal Protein Targets of Auranofin through Direct and Indirect Detection of Gold–Protein Adducts

Auranofin (AF), a gold(I)-thiolate complex, exhibits diverse therapeutic potential, but its precise molecular targets within cells remain incompletely understood. This study aimed to develop and evaluate direct and indirect metallomics-based approaches for identifying AF protein targets. We first explored the stability of gold-protein adducts under standard digestion conditions and mass spectrometry analysis. We evaluated a Precursor Ion Scan-like strategy utilizing High-Collision Energy (HCE) fragmentation to monitor the diagnostic elemental gold ion at m/z 196.96. This direct approach, coupled to the classical data dependent acquisition, successfully identified in vitro AF target in complex protein samples. Direct LC-MS/MS analysis of AF-treated cells presented challenges due to low intracellular gold concentrations. Therefore, we implemented an indirect enrichment strategy, combining biotin labeling of gold-coordinated thiols with SILAC, to enhance detection sensitivity and reduce false positives. This indirect approach detected, in cellulo, known AF target while also revealed endoplasmic reticulum (ER) redox folding proteins as potential targets, consistent with AF-induced ER stress showed in our previous study. The study highlights the complexities of direct gold speciation in cells and contributes to a more comprehensive understanding of AF's multi-target mechanisms of action.