Identification of (eta6-arene)ruthenium(II) protein binding sites in E. coli cells by combined multidimensional liquid chromatography and ESI tandem mass spectrometry: specific binding of [(eta6-p-cymene)RuCl2 (DMSO)] to stress-regulated proteins and to helicases.
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 [(eta(6)-p-cymene)RuCl(2)(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 (eta(6)-p-cymene)Ru(II) 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 [(eta(6)-p-cymene)RuCl(2)(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.
Will
Joanna
J
Lehrstuhl für Analytische Chemie, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Kyas
Andreas
A
Sheldrick
William S
WS
Wolters
Dirk
D
D000970:Antineoplastic Agents / Q000737:chemistry; D001665:Binding Sites; D002853:Chromatography, Liquid; D004265:DNA Helicases; D004121:Dimethyl Sulfoxide; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins; D040901:Proteomics / Q000379:methods*; D017975:Ruthenium Compounds / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
D000970:Antineoplastic Agents; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D017975:Ruthenium Compounds; D004265:DNA Helicases; D004121:Dimethyl Sulfoxide