Exploring Metallodrug–Protein Interactions by ESI Mass Spectrometry: The Reaction of Anticancer Platinum Drugs with Horse Heart Cytochrome c

Since DNA is commonly believed to be the primary target for platinum metallodrugs, researchers’ interest has mainly focused on the characterisation of platinum–nucleic acid adducts while devoting much less attention to platinum–protein adducts. However, protein-bound platinum fragments probably represent truly active anticancer species—rather than mere drug-inactivation products—provided that metal transfer among distinct binding sites is kinetically allowed. Moreover, platination of specific side chains, which can affect the function of biologically crucial proteins or enzymes through the formation of tight coordinative bonds, might play a relevant role in the overall mechanism and toxicity of platinum drugs. The state of the art of platinum–protein interactions is described in a few articles and reviews; in any case, this issue warrants further experimental work. Thanks to the latest improvements, electrospray ionisation mass spectrometry (ESI-MS) today represents a very powerful method for exploring metallodrug–protein interactions. Owing to the introduction of “soft” ionisation methods, it is possible to transfer the intact metal–protein adduct—whole, in the gas phase—to determine its molecular mass with high accuracy and, thus, obtain its full molecular characterisation. However, much work is still required for the optimisation and the standardisation of experimental ESI-MS procedures directed at these systems. A great variability in ESI-MS responses is generally found in the current literature that depends on many factors, such as the nature of the protein, the nature of the metal, the specific solution conditions, the nature of the metalbound ligands, pH and the kind of buffer. Apparently, the intrinsic “fragility” of the metal–protein coordination bonds represents a major obstacle, often leading to extensive bond cleavage during ionisation and to loss of chemical information. Some pioneering ESI-MS studies of platinum–protein interactions were reported a few years ago by Gibson and co-workers, who used either ubiquitin or myoglobin as model proteins. A number of platinum–protein adducts were identified and characterised in detail. Afterwards, a few additional ESI-MS studies of various metallodrug–protein adducts were reported by other research groups. Cytochrome c is a small electron-carrier heme protein, localised in the mitochondria, that plays a crucial role in apoptotic pathways. Cytochrome c is also known to be an excellent ESI-MS probe and has been the subject of a number of investigations. This led us to choose cytochrome c as the model protein for our study. The following classical platinum drugs were selected: cisplatin, transplatin, carboplatin and oxaliplatin (Scheme 1).