Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches |
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Authors: | Charlotte Møller Richard R Sprenger Stefan Stürup Peter Højrup |
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Institution: | 1.Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences,University of Copenhagen,Copenhagen ?,Denmark;2.Department of Biochemistry and Molecular Biology,University of Southern Denmark,Odense M,Denmark |
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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. |
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Keywords: | |
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