title: Novel insights into the bottom-up mass spectrometry proteomics approach for the characterization of Pt-binding proteins: The insulin-cisplatin case study.
abstract: 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.
authors.affiliation: Department of Analytical Chemistry, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040, Madrid, Spain.
authors.forename: Estefanía
authors.initials: E
authors.lastname: Moreno-Gordaliza
authors.forename: Benito
authors.initials: B
authors.lastname: Cañas
authors.forename: María A
authors.initials: MA
authors.lastname: Palacios
authors.forename: M Milagros
authors.initials: MM
authors.lastname: Gómez-Gómez
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002945:Cisplatin / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D007328:Insulin / Q000737:chemistry*; D008969:Molecular Sequence Data; D010984:Platinum / Q000737:chemistry*; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D007328:Insulin; D010984:Platinum; D014357:Trypsin; D003545:Cysteine; D002945:Cisplatin