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Updated: May 27, 2026
Ubiquitin Chain Analysis by Parallel Reaction Monitoring
Published on: June 17, 2020
Fragmentation methods on the balance: unambiguous top-down mass spectrometric characterization of
Samuel M Meier1, Yury O Tsybin, Paul J Dyson
1Institute of Inorganic Chemistry, University of Vienna, Waehringer Str. 42, 1090 Vienna, Austria.
Abstract:
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.
Insights
This study used electron transfer dissociation mass spectrometry (ETD-MS/MS) to analyze oxaliplatin binding sites on ubiquitin. ETD-MS/MS precisely identified Met1 and His68 as the metal-binding residues.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Chemical Biology
Background:
- Oxaliplatin is a platinum-based chemotherapy drug.
- Understanding drug-protein interactions is crucial for drug development.
- Ubiquitin (Ub) serves as a model protein for studying drug interactions.
Purpose of the Study:
- To investigate the interaction between oxaliplatin and ubiquitin.
- To identify the specific metal-binding sites of oxaliplatin on ubiquitin.
- To compare the efficacy of different tandem mass spectrometry (MS/MS) techniques for analyzing metallodrug-protein adducts.
Main Methods:
- Top-down mass spectrometry approach using high-resolution electrospray ionization mass spectrometry (ESI-MS).
- Utilized diverse MS/MS techniques: collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD).
- Analyzed oxaliplatin-ubiquitin adducts, specifically [Ub+Pt-(chxn)], where chxn is (1R,2R)-cyclohexanediamine.
Main Results:
- Electron transfer dissociation tandem mass spectrometry (ETD-MS/MS) successfully identified Met1 and His68 as the binding sites for oxaliplatin on ubiquitin.
- ETD-MS/MS outperformed CID and HCD in identifying metallated peptide fragments and localizing binding sites.
- CID-MS/MS experiments on orbitrap and ion cyclotron resonance (ICR)-FT mass spectrometers yielded comparable results to each other but were less effective than ETD-MS/MS.
- Gas-phase protein structure analysis revealed correlations between fragment intensities and solution-phase random coil areas, indicating retention of N-terminal β-hairpin and α-helix structures.
Conclusions:
- ETD-MS/MS is a powerful technique for precise localization of metal-binding sites in metallodrug-protein adducts.
- The study provides the first detailed analysis of metallodrug-protein adducts using ETD-MS/MS for metal-binding site determination.
- Oxaliplatin binds to Met1 and His68 residues of ubiquitin, and protein secondary structures are maintained in the gas phase during MS analysis.
