Identification of (η6-arene)ruthenium(II) protein binding sites in E. coli cells by combined multidimensional liquid chromatography and ESI tandem mass spectrometry: specific binding of [(η6-p-cymene)RuCl2(DMSO)] to stress-regulated proteins and to helicases

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Abstract
An automated multidimensional protein identification technology, which combines biphasic liquid chromatography with electrospray ionisation tandem mass spectrometry (MS/MS), was employed to analyse tryptic peptides from Escherichia coli cells treated with the antiproliferation agent [(η6-p-cymene)RuCl2(DMSO)], where DMSO is dimethyl sulfoxide. MS/MS spectra were recorded for molecular ions generated by neutral loss of p-cymene from intensive peptide ions coordinated by the (η6-p-cymene)RuII fragment. Matching of the MS/MS spectra of the ruthenated peptides to spectra of proteins in the E. coli database enabled the identification of five protein targets for [(η6-p-cymene)RuCl2(DMSO)]. One of these is the constitutive cold-shock protein cspC, which regulates the expression of genes encoding stress-response proteins, and three of the other targets, ppiD, osmY and sucC, are proteins of the latter type. The DNA damage-inducible helicase dinG was likewise established as a protein target. Aspartate carboxylate functions were identified as the probable Ru binding sites in cspC, ppiD and dinG, and threonine and lysine side chains in osmY and sucC, respectively.

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