Competitive LC-MS/MS assay to investigate protein metalation dynamics

glutathione (GSH). Online top-down fragmentation further enabled the localization of binding sites of metallodrugs on proteins in the same run, which could be directly related to complex speciation behaviour. The reactivity of Zeise's salt derivative [Pt(ala)(ASA-But)Cl] (ala = l-alanine; ASA-But = but-3-en-1-yl 2-acetoxybenzoate) with sulfur donor atoms was found to exceed all investigated compounds, including cisplatin. Ruthenium compounds preferred N-donor coordination, which resulted in a strong affinity towards histidine residues. Protein adducts of the somewhat redox-active iridium compounds were quickly transformed into stable adducts with detoxifying nucleophiles, especially with GSH. This model system provides further evidence that metallodrug reactivity is more selective in competitive settings, as would be anticipated from exposure to single biomolecules. This implies that the intracellular selectivity of metallodrugs can be chemically tuned and that this aspect may be accounted for in future metallodrug design. Such design strategies will be supported by expanding the present competitive assay to more complex systems that better mimic physiological intra- and extracellular tumour microenvironments.