Characterisation of cisplatin binding sites in human serum proteins using hyphenated multidimensional liquid...

Cisplatin binding sites in human serum proteins have been characterised by using combined multidimensional liquid chromatography and ESI tandem mass spectrometry (MudPIT). Following incubation periods of 3 h for cisplatin-blood serum mixtures and subsequent trypsin digestion, MS-MS spectra were recorded for individual peptides that had been separated by SCX and RP liquid chromatography. Matching of the MS-MS spectra to theoretical sequences that were generated for human proteins in the SWISS-PROT database led to the identification of specific binding sites in human serum albumin (HSA), serotransferrin (Trfe) and other abundant serum proteins (A2mg, A1at, Apoa1, Apoa2). The cisplatin coordination sites in HSA and Trfe were confirmed by independent MudPIT studies on cisplatin reaction mixtures with the individual proteins. A total of five specific binding sites were identified for HSA, including the cysteine residue C34, two methionine sites (M329, M548) and the tyrosine and aspartate O-donor sites Y150 (or Y148) and D375 (or E376). Methionine-256 was established as a cisplatin coordination site for Trfe in addition to the O-donor sites E265, Y314, E385 and T457. Inspection of the protein structures indicates that the preferred residues belong either to peripheral alpha helices or to flexible loops within the protein-binding pockets. O-donor residues dominate as cisplatin binding sites for other abundant serum proteins.