Fragmentation methods on the balance: unambiguous top–down mass spectrometric characterization of oxaliplatin–ubiquitin binding sites
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The interaction between oxaliplatin and the model protein ubiquitin (Ub) was investigated in a top–down approach by means of high-resolution electrospray ionization mass spectrometry (ESI-MS) using diverse tandem mass spectrometric (MS/MS) techniques, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD). To the best of our knowledge, this is the first time that metallodrug–protein adducts were analyzed for the metal-binding site by ETD-MS/MS, which outperformed both CID and HCD in terms of number of identified metallated peptide fragments in the mass spectra and the localization of the binding sites. Only ETD allowed the simultaneous and exact determination of Met1 and His68 residues as binding partners for oxaliplatin. CID-MS/MS experiments were carried out on orbitrap and ion cyclotron resonance (ICR)-FT mass spectrometers and both instruments yielded similar results with respect to number of metallated fragments and the localization of the binding sites. A comparison of the protein secondary structure with the intensities of peptide fragments generated by collisional activation of the [Ub + Pt-(chxn)] adduct [chxn = (1R,2R)-cyclohexanediamine] revealed a correlation with cleavages in solution phase random coil areas, indicating that the N-terminal β-hairpin and α-helix structures are retained in the gas phase.
KeywordsAnticancer metallodrugs Tandem mass spectrometry Electron transfer dissociation Oxaliplatin Ubiquitin
The authors are indebted to the Austrian Science Fund (FWF; I496-B11), the Hochschuljubiläumsstiftung Vienna and COST D39 and CM0902. We would like to thank Dr. Yue Xuan (Thermo Scientific) and Dr. Jens Fuchser (Bruker Daltonics) for assistance during measurements on the orbitrap FT MS and FT-ICR MS instruments, respectively.
- 7.Casini A, Mastrobuoni G, Temperini C, Gabbiani C, Francese S, Moneti G, Supuran CT, Scozzafava A, Messori L (2007) ESI mass spectrometry and X-ray diffraction studies of adducts between anticancer platinum drugs and hen egg white lysozyme. Chem Commun (2):156–158.Google Scholar
- 15.Allardyce CS, Dyson PJ, Abou-Shakra FR, Birtwistle H, Coffey J (2001) Inductively coupled plasma mass spectrometry to identify protein drug targets from whole cell systems. Chem Commun 2708–2709.Google Scholar
- 17.Timerbaev AR, Aleksenko KS, Polec-Pawlak K, Ruzik R, Semenova O, Hartinger CG, Oszwaldowski S, Galanski M, Jarosz M, Keppler BK (2004) Platinum metallodrug-protein binding studies by capillary electrophoresis-inductively coupled plasma-mass spectrometry: characterization of interactions between Pt(II) complexes and human serum albumin. Electrophoresis 25:1988–1995CrossRefGoogle Scholar
- 36.Mikesh LM, Ueberheide B, Chi A, Coon JJ, Syka JE, Shabanowitz J, Hunt DF (2006) The utility of ETD mass spectrometry in proteomic analysis. Biochim Biophys Acta 1764:1811–1822Google Scholar
- 41.Hartinger CG, Schluga P, Galanski M, Baumgartner C, Timerbaev AR, Keppler BK (2003) Tumor-inhibiting platinum(II) complexes with aminoalcohol ligands: comparison of the mode of action by capillary electrophoresis and electrospray ionization-mass spectrometry. Electrophoresis 24:2038–2044CrossRefGoogle Scholar
- 42.Galanski M, Baumgartner C, Meelich K, Arion VB, Fremuth M, Jakupec MA, Schluga P, Hartinger CG, Von Keyserlingk NG, Keppler BK (2004) Synthesis, crystal structure and pH dependent cytotoxicity of (SP-4-2)-bis(2-aminoethanolato-κ2 N, O)platinum(II)—a representative of novel pH sensitive anticancer platinum complexes. Inorg Chim Acta 357:3237–3244CrossRefGoogle Scholar