Characterization of Pt-protein complexes by nHPLC–ESI-LTQ MS/MS using a gel-based bottom-up approach

► Gel-based bottom-up MS approach for characterizing Pt-protein complexes. ► nrSDS-PAGE separation and conventional in gel digestion preserve Pt-protein bonds. ► Sulfur-containing reagents have a negative impact on Pt-protein bonds preservation. ► Cisplatin binding sites (Met, His, Cys) identified in HSA, TF, CA, MYO and CYT C. 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.