title: Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches.
abstract: Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached. Identification of several of the binding sites was obtained using matrix-assisted laser desorption/ionization (MALDI)-ToF-ToF-MS and liquid chromatography-nESI-Q-ToF-MS. Upon comparing the top-down and bottom-up approaches, the suitability of the bottom-up approach for determining binding sites was questioned, as the release and possible re-association of Pt(dach) were demonstrated upon enzymatic digestion. The associated advantages and disadvantages of ESI and MALDI were also pointed out.
authors.affiliation: Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Møller
authors.forename: Richard R
authors.initials: RR
authors.lastname: Sprenger
authors.forename: Stefan
authors.initials: S
authors.lastname: Stürup
authors.forename: Peter
authors.initials: P
authors.lastname: Højrup
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001665:Binding Sites; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D000077150:Oxaliplatin; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013552:Swine
chemical_list: D000970:Antineoplastic Agents; D007328:Insulin; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin