Novel insights into the bottom-up mass spectrometry proteomics approach for the characterization of Pt-binding proteins: The insulin-cisplatin case study.

The characterization of the interaction of platinum drugs with proteins has been previously performed using bottom-up proteomics approaches (enzymatic digestion followed by MS analysis). Nevertheless, the study of the stability of the Pt-protein bonds along the whole process has been obviated for the moment. Herein the suitability of the treatments implied during enzymatic digestion of Pt-protein adducts has been evaluated, focusing on the stability of the Pt bonds. Insulin-cisplatin adducts were generated in vitro and separated from unreacted cisplatin by HPLC, the separation being checked by HPLC-ICP-MS. The chromatographically isolated Pt-insulin adducts have been proved to resist overnight digestion including treatment with Urea, DTT, IAA and trypsin in a Tris buffer. Direct analysis of the peptides generated by nESI-LIT MS allowed the determination of Pt-binding sites in insulin as: B Chain N-terminus, His5, His10, Cys7, Cys19 and A Chain Cys6, Cys7, Cys20. Results have been compared to a previous top-down approach, indicating that more complete information can be obtained with the bottom-up approach. Reactivity of free cysteines has been proved to prevail to N-donor groups, but when cysteines participate in disulfide bonds, their reactivity is comparable to N-donor sites (N-terminus, His). Preliminary results indicate that the use of High Intensity Focused Ultrasound for accelerating the enzymatic digestions is compatible with preserving Pt-protein bonds, allowing a reduction in the total digestion time to 5 min. Pt-containing peptides were fragmented and sequenced by CID, and results were compared with those obtained by the use of ETD, being CID spectra far more informative.