Electrospray ionization multi-stage mass spectrometric study of the interaction products of the cytotoxic complex [Cu(thp)4][PF6] with methionine-rich model peptides

RATIONALE: The cytotoxic activity of the copper(I) complex [Cu(thp)4][PF6] (CP) (thp = tris(hydroxymethyl) phosphine) is correlated with its high accumulation in cancer cells. Human copper transporter 1 (hCtr1) has been described as the main trans-membrane protein involved in cellular trafficking of physiological copper. Methionine-rich peptide sequences incorporated in the extracellular domain of hCtr1 play a key role in the cellular internalization of copper. We wish to investigate the interaction of CP with model peptides that mimic the extracellular domain of hCtr1. METHODS: The interaction of CP with methionine-rich and methionine-free model peptides has been investigated by electrospray ionization mass spectrometry and the interaction products have been characterized by multiple collisional experiments, using an ion trap mass instrument. RESULTS: The interaction of CP with selected methionine-rich model peptides, Ac-MMMMPMTFK-NH2 (P1) and AcMGMSYMDSK-NH2 (P2), shows that the native copper complex, after sequential loss of phosphines, induces the formation of [Cu(P1)(thp)]+ and [Cu(P1/P2)]+ adducts reasonably by inclusion of the Cu(I) ion in the peptide framework. Collisionally induced fragmentations (MSn) of [Cu(P1/P2)]+ give evidence that the metal is coordinated by the thioetherS of two adjacent methionine residues. Interaction of the same peptides with the isostructural complex [Ag(thp)4] + or AgNO3 yields similar experimental evidence, leading to [Ag(P1/P2)]+. CONCLUSIONS: Methionine sequences incorporated in model peptides are crucial for the recruitment of copper from CP. Such a metal-peptide interaction does not take place when methionine-free Ac-NleGNleSYNleDSK-NH2 (P3) is utilized. A mechanism for tumor cell internalization of CP involving: (i) chemically driven sequential loss of phosphines from the native tetrahedral complex, followed by (ii) transfer of Cu(I) to the methionine-rich sequences typical of the hCtr1 transporter, is proposed.