Interaction of transferrin with non-cognate metals studied by native electrospray ionization mass spectrometry

Transferrin (Tf) is a key player in iron homeostasis which transports otherwise insoluble ferric ion and delivers it to cells via receptor-mediated endocytosis. Since Tf is also capable of binding a range of other metals with very high affinity, it remains in the focal point of multiple studies aiming at understanding the mechanism of biodistribution of clinically relevant non-ferric metals. The latter included toxic metals, as well as metals that can be exploited for medicinal or therapeutic benefit (e.g., radionuclides). More recently, there was a growing interest in using non-cognate metals as tracers of Tf-based therapeutics in animal-based disease models. In this work interaction of seven metal ions in their physiologically relevant oxidation states (Cr3+, In3+, Ti4+ and four lanthanides) with Tf were studied using native electrospray ionization mass spectrometry (ESI MS). Cr3+, In3+ and Ti4+ display Tf-binding properties that are similar to those of the cognate metal (Fe3+), while Tf association with lanthanides leads to formation of complexes that are very unstable in the gas phase, indicating the protein/metal binding mechanism and structure in this case are very different from those leading to formation of the Fe2Tf complex. Native ESI MS also allowed the receptor binding competence of Tf loaded with non-ferric metals to be evaluated. Lastly, behavior of these complexes under conditions mimicking the endosomal environment (where the release of the cognate metal actually occurs) was also evaluated. This analysis revealed striking similarities between Cr2Tf, In2Tf and Ti2Tf on one hand, and the diferric form of Tf on the other hand, strongly suggesting that Tf is indeed a critical player in homeostasis of these metals and must be considered both in the analysis of metal toxicity and in the design of metal-based therapeutics.