Combining TBP-based rOFFGEL-IEF with FASP and nLC–ESI-LTQ-MS/MS for the analysis of cisplatin-binding proteins in rat kidney

In this work, a methodology based on a reducing IEF separation in combination with a FASP tryptic digestion able to maintain the integrity of cisplatin–protein complexes has been developed. The method is based on OFFGEL-IEF under conditions provided by the thiol-free reducing agent TBP, which allowed the separation of cisplatin-binding proteins in liquid fractions. The FASP procedure is applied as an intermediate stage between the IEF separation and MS analysis where the proteins are retained and concentrated in a commercially available ultrafiltration device. The filter unit acts as a proteomic reactor for detergent removal, buffer exchange, chemical modification (reduction and alkylation) and protein digestion. Finally, purified peptides are recovered by centrifugation. This procedure provides efficiencies comparable to standard in-solution digestion and the risk of platinum-complexes loss is minimized due to the fact that reagents employed along the process are subsequently eliminated before the following step. The stability of platinum–protein complexes under the FASP tryptic digestion, either using TBP or DTT as reducing agents, was maintained, allowing the identification of several platinum-containing peptides from cisplatin–HSA. This methodology was applied to the separation of platinum-enriched protein fractions obtained by SEC–ICP-MS in a kidney tissue extract from a rat treated with cisplatin, followed by further identification by nLC–ESI-LTQ-MS/MS after FASP tryptic digestion of selected platinum-containing liquid fractions.