Direct Determination of the Primary Binding Site of Cisplatin on Cytochrome c by Mass Spectrometry

Protein–cisplatin interactions lie at the heart of both the effectiveness of cisplatin as a therapeutic agent and side effects associated with cisplatin treatment. Because a greater understanding of the protein–cisplatin interactions at the molecular level can inform the design of cisplatin-like agents for future use, mass spectrometric determination of the binding site of cisplatin on a model protein, cytochrome c, was undertaken in this paper. The monoadduct cytochrome c–Pt(NH3)2(H2O) is found to be the primary adduct produced by the cytochrome c–cisplatin interactions under native conditions. To locate the primary binding site of cisplatin, both free cytochrome c and the cytochrome c adducts underwent trypsin digestion, followed by Fourier transform mass spectrometry (FT-MS) to identify unique fragments in the adduct digest. Four such fragments were found in the adduct digest. Tandem mass spectrometry (MS/MS and MS3 indicates that two fragments are Pt(NH3)2(H2O) bound peptides (Gly56-Glu104 and Asn54-Glu104) with one water associated at the peptide bond Lys79∼Met80, and the other two fragments are heme containing peptides (acety1-Gly1-Lys53 and acety1-Gly1-Lys55). The product-ion spectra of the four fragments reveal that Met65 is the primary binding site of cisplatin on cytochrome c.

Graphical AbstractFourier mass spectrometry and tandem mass spectrometry are utilized to directly determine the primary cisplatin binding site on cytochrome c.Figure optionsDownload full-size imageDownload high-quality image (133 K)Download as PowerPoint slide