Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches
Charlotte Møller1, Richard R Sprenger, Stefan Stürup
1Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark.
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.
Insights
This study compared mass spectrometry techniques to analyze oxaliplatin binding to insulin. The top-down approach identified histidine5 and cysteine6 as primary binding sites, questioning the bottom-up method's reliability.
Area of Science:
- Proteomics
- Analytical Chemistry
- Mass Spectrometry
Background:
- Oxaliplatin is a platinum-based chemotherapy drug.
- Understanding drug-protein interactions is crucial for drug development and efficacy.
- Insulin serves as a model protein to study platinum adduct formation.
Purpose of the Study:
- To compare various mass spectrometric techniques for analyzing oxaliplatin-protein binding.
- To identify the specific binding sites of oxaliplatin on insulin.
- To evaluate the suitability of top-down versus bottom-up mass spectrometry approaches.
Main Methods:
- Top-down analysis of intact insulin-oxaliplatin adducts using nano-electrospray ionization quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS).
- Reduction of disulfide bridges to simplify spectral interpretation.
- Bottom-up analysis involving enzymatic digestion (endoproteinase Glu-C) followed by MALDI-ToF-ToF-MS and LC-nESI-Q-ToF-MS.
- Comparison of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) techniques.
Main Results:
- Observed formation of various platinum adducts, including mono- and di-adducts of Pt(dach).
- Identified histidine5 (B chain) as a major binding site via top-down analysis.
- Identified cysteine6 (A chain) as an additional binding site after disulfide bridge reduction.
- Demonstrated potential release and re-association of Pt(dach) during enzymatic digestion in the bottom-up approach.
- Highlighted advantages and disadvantages of ESI and MALDI ionization techniques.
Conclusions:
- Top-down mass spectrometry is effective for identifying major oxaliplatin binding sites on proteins like insulin.
- The reliability of the bottom-up approach for determining platinum binding sites is questionable due to potential artifact formation during digestion.
- Further investigation into platinum adduct stability during proteomic workflows is warranted.
