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

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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.