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

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 [(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.

Exploring metallodrug–protein interactions by mass spectrometry: comparisons between platinum coordination complexes and an organometallic ruthenium compound

“…Typically, ESI MS has been applied to establish the affinity of multiple potential ligands for a single macromolecular target; however, related procedures have been used to study ligand binding to (and affinity for) a protein mixture [38]. Moreover, Will et al [43,44] have recently demonstrated the suitability of an automated method for shotgun proteomics based on ESI MS for establishing some of the protein targets of transition metal complexes in whole-cell systems. Deconvoluted mass spectra of the mixture of the three proteins incubated with each of the selected compounds (5:1 metal-to-protein ratio) in aqueous solution at pH 7.4, for 24 h at 37°C, are shown in Fig.…”

Characterisation of Cisplatin Binding Sites in Human Serum Proteins Using Hyphenated Multidimensional Liquid Chromatography and ESI Tandem Mass Spectrometry

“…[22,23] MudPIT combines 2D SCX (strong cation exchange) and RP (reversedphase) chromatography with ESI-MS-MS, and allows up to 1,500 proteins to be characterised in a 24 h period. We now report the application of this technique to identify cisplatin binding sites in serum proteins.…”

Mass spectrometry as a powerful tool to study therapeutic metallodrugs speciation mechanisms: Current frontiers and perspectives

“…[39] The same approach was described to analyse a Ru(II) complex which is not a drug candidate itself but represents a model for pharmacologically relevant Ru(II) arene class of compounds. [300] The results showed that the complex binds to cold-shock proteins that regulate stress response proteins as well as a DNA damage-inducible helicase. The main binding sites for the ruthenium fragments were identified as aspartic acid, lysine and threonine.…”

“…The main binding sites for the ruthenium fragments were identified as aspartic acid, lysine and threonine. [300] Finally, a combination of MudPIT and metallomic studies was reported by Wolters et al to characterize the effects of the ruthenium-based RAPTA-T drug towards human cancer cells. [301] First, a subcellular fragmentation was realised and the obtained fragments analysed by ICP-MS. Ru(II) was found to accumulate in the particulate containing the organelles rather than in the cytosolic, nucleic or cytoskeleton fractions.…”