TCEP-based rSDS–PAGE AND nLC–ESI-LTQ-MS/MS for oxaliplatin metalloproteomic analysis

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.