title: TCEP-based rSDS-PAGE AND nLC-ESI-LTQ-MS/MS for oxaliplatin metalloproteomic analysis.
abstract: In this work, the reactivity of the citostatic drugs such as oxaliplatin, cisplatin and carboplatin towards proteins and the stability of Pt-protein complexes along their storage were evaluated. Neither native-PAGE nor nrSDS-PAGE seems to be suitable for the separation of carboplatin-binding proteins. A reducing electrophoretic separation procedure able to maintain the integrity of oxaliplatin-protein complexes has been developed. The method is based on SDS-PAGE under conditions provided by the thiol-free reducing agent tris (2-carboxyethyl) phosphine (TCEP), which allowed the separation of oxaliplatin-binding proteins in narrow bands with almost quantitative recoveries. Different amounts of platinum-bound protein bands covering the range 0.3-2.0 μg were excised and mineralised for platinum determination, showing good linearity. Limits of detection for a mixture of five standard proteins (transferrin, albumin, carbonic anhydrase, myoglobin and cytochrome c) incubated with oxaliplatin were within the range 11.0-44.0 pg of platinum, which were satisfactory for their application to biological samples. The suitability of the TCEP-based SDS-PAGE for the separation of platinum-enriched protein fractions of a kidney cytosol from a rat treated with oxaliplatin was demonstrated. The identification of high Pt to protein ratio cytosolic fractions was carried out by separating the cytosolic platinum-binding proteins by SEC-ICP-MS. Several cytosolic renal proteins were identified in those gel bands containing platinum-enriched protein fractions using nLC-ESI-LTQ-MS/MS after in-gel digested with trypsin. In addition, fractions containing platinum-enriched proteins with lower theorical molecular weight were directly analysed by nLC-ESI-LTQ-MS/MS after in-solution tryptic digestion allowing protein identification.
authors.affiliation: Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain. Electronic address: mariluz@ucm.es.
authors.forename: M L
authors.initials: ML
authors.lastname: Mena
authors.forename: E
authors.initials: E
authors.lastname: Moreno-Gordaliza
authors.forename: M M
authors.initials: MM
authors.lastname: Gómez-Gómez
mesh_terms: D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry; D016190:Carboplatin / Q000737:chemistry*; D002945:Cisplatin / Q000008:administration & dosage / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000737:chemistry / Q000187:drug effects; D057230:Limit of Detection; D009211:Myoglobin / Q000737:chemistry; D009944:Organoplatinum Compounds / Q000737:chemistry*; D000077150:Oxaliplatin; D010446:Peptide Fragments / Q000032:analysis*; D010720:Phosphines / Q000737:chemistry*; D010984:Platinum / Q000032:analysis*; D011485:Protein Binding; D059748:Proteolysis; D051381:Rats; D012709:Serum Albumin / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D014168:Transferrin / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D002256:Carbonic Anhydrases; D016190:Carboplatin; D002945:Cisplatin; D045304:Cytochromes c; D009211:Myoglobin; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin; D010446:Peptide Fragments; D010720:Phosphines; D010984:Platinum; D012709:Serum Albumin; D014168:Transferrin; D014357:Trypsin; C080938:tris(2-carboxyethyl)phosphine
keywords: ICP-MS; Kidney; Platinum-binding proteins; nLC–ESI-LTQ-MS/MS; oxaliplatin; rSDS-PAGE