title: Characterisation of cisplatin coordination sites in cellular Escherichia coli DNA-binding proteins by combined biphasic liquid chromatography and ESI tandem mass spectrometry.
abstract: Combined multidimensional liquid chromatography and electrospray ionisation tandem mass spectrometry was employed to analyse platinated tryptic peptides from Escherichia coli cells treated with the anticancer drug cis-[PtCl2(NH3)2] at pH 7.0. Prerequisites for the LC/LC/MS/MS analysis of protein targets that are fulfilled by cisplatin are (a) that the original protein binding sites have a high kinetic stability over the range 2.3 < pH < 8.5, and (b) that the metal fragment remains coordinated to a significant number of b+ and y+ peptide ions under MS/MS fragmentation conditions. Matching the MS/MS spectra of the platinated tryptic peptides to sequences of proteins in the E. coli database enabled the identification of 31 protein targets for cisplatin. Whereas six of these are high-abundance enzymes and ribosomal proteins in E. coli cells, five low-abundance DNA-binding proteins were also identified as specific targets. These include the DNA mismatch repair protein mutS, the DNA helicase II (uvrD) and topoisomerase I (top1). Two efflux proteins (acrD, mdtA), the redox regulator thioredoxin 1 (thiO) and the external filament-like type-1 fimbrial protein A chain (fimA1) were also characterised as specific cisplatin-binding proteins. Kinetically favoured carboxylate (D, E) and hydroxy (S, T, Y) O atoms were identified as the Pt coordination sites in 18 proteins and methionyl S atoms in 9 proteins.
authors.affiliation: Lehrstuhl für Analytische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
authors.forename: Joanna
authors.initials: J
authors.lastname: Will
authors.forename: William S
authors.initials: WS
authors.lastname: Sheldrick
authors.forename: Dirk
authors.initials: D
authors.lastname: Wolters
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D002945:Cisplatin / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D002945:Cisplatin