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-QToF-
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 reassociation
of Pt(dach) were demonstrated upon enzymatic
digestion. The associated advantages and disadvantages of
ESI and MALDI were also pointed out.