title: Characterization of Pt-protein complexes by nHPLC-ESI-LTQ MS/MS using a gel-based bottom-up approach.
abstract: The suitability of in-gel digestion for the characterization of Pt-binding proteins by gel-based bottom-up MS approaches has been evaluated regarding the preservation of Pt-protein bonds during the process. Standard proteins (albumin, transferrin, carbonic anhydrase, myoglobin and cytochome c) incubated with cisplatin were separated by nrSDS-PAGE and in-gel trypsin-digested. The whole in-gel digestion protocol included treatment with reagents such as: ammonium bicarbonate, acetonitrile, formic acid, trypsin as enzyme and alternatively, dithiotreitol and iodoacetamide as reducing and alkylating agents. Digests were analyzed by nHPLC-ESI-LTQ-MS/MS and Pt-peptides were recognized in all the proteins studied on the basis of their isotopic pattern. Only when the reducing and alkylating reagents were used, the amount of detectable Pt-peptides decreased due to the high reactivity of thiol containing reagents towards Pt. Furthermore, the repeated use of acetonitrile could lead to the replacement of ligands originally attached to Pt by CN(-), but does not affect the Pt-protein binding. Platinum-binding sites on the proteins were elucidated from the CID-MS/MS fragmentation spectra and assessed by evaluation of protein structures. Several histidines, cysteines and methionines were identified as platinum binding sites in the different standard proteins. Results were in accordance to those obtained with in-solution digestions.
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; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002945:Cisplatin / Q000737:chemistry*; D056831:Coordination Complexes / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D006639:Histidine / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010984:Platinum / Q000737:chemistry*; D011485:Protein Binding; D059748:Proteolysis; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D001798:Blood Proteins; D056831:Coordination Complexes; D005782:Gels; D010984:Platinum; D006639:Histidine; D008715:Methionine; D014357:Trypsin; D003545:Cysteine; D002945:Cisplatin