Mass spectrometric quantification of the binding ratio of metal-based anticancer complexes with protein thiols

The binding ratio of metal complexes with cysteinyl thiols in proteins plays an important role in deciphering the mechanisms of action of therapeutic metal complexes, but its analysis is still a significant challenge. In this work, a quantitative mass spectrometry method is developed to determine the binding ratio of metal-based anticancer complexes with cysteines in human copper chaperone protein Atox1.

A novel strategy based on a thiol-specific stable isotopic labelling reagent was developed to determine the binding ratio of metal-based anticancer complexes, namely cisplatin and organometallic ruthenium complex [(η⁶ -biphenyl)RuCl(en)]PF₆ (en = ethylenediamine), with the cysteinyl residues of Atox1.

Both cisplatin and the ruthenium complex were reactive not only to Cys15 and/or Cys18, the copper(I) binding site of Atox1, but also to Cys44. The binding ratios of the ruthenium complex with the cysteinyl residues were much higher than those of cisplatin. However, the addition of copper(I) could markedly increase the binding ratios of cysteinyl residues of Atox1 with cisplatin, but not with the ruthenium complex.

This strategy can not only precisely determine the binding ratios of metal complexes to protein thiols, but also be helpful in distinguishing thiol-binding sites from other binding sites of metal complexes in proteins. We expect wide application of this method to the research of covalent/coordinative interactions between metal complexes and protein thiols.