Bottom-up mass spectrometry proteomics strategies for the identification of metallodrug binding sites on proteins: the search for a general protocol to assess adduct stability

The bottom-up mass spectrometry approach (reduction, alkylation and enzymatic digestion followed by MS/MS analysis) is one of the main methods used in proteomics to characterize the binding site of metal-based anticancer drugs on proteins. Nevertheless, in our opinion, the study of the stability of the metal fragment-protein coordination bond along the whole process has not received so far adequate attention. Previous studies on myoglobin, that were confirmed in our mass spectrometry facility, showed the instability of Fe-histidine coordination bond under certain preparative (pH, presence of an organic co-solvent) and instrumental (capillary temperature, tube lens voltage) conditions. These findings convinced us to draw up a general protocol to test metal fragment–protein adduct stability under the typical condition of the bottom-up approach (from the solution containing the metal-protein adduct to the MS/MS experiments), especially when the metal complex involved is not the well known and studied cisplatin (cisPt) but a new metal complex containing other metals as Ru and Au. We identified eight critical conditions as potential sources of metal-protein coordination bond impairment during the bottom-up process, when using a LTQ-Orbitrap mass spectrometer: 1) sample permanence in ammonium bicarbonate; 2) dithiothreitol reduction; 3) iodoacetamide alkylation; 4) permanence in loading mobile phases (presence of organic co-solvent and pH); 5) permanence in mobile phases (presence of organic co-solvent and pH); 6) ESI process; 7) transfer through ion transfer tube and tube lens; 8) collision induced dissociation in ion trap. An experimental protocol was thus developed to assess the relevance of the above conditions on the stability of the metal-protein coordinative bond. First of all, the protocol was applied to the well known, model system cisPt-cytochrome C: cisPt-CytC adducts proved their stability toward all conditions listed above and so they showed to be ideal candidates for a binding site investigation using a bottom-up approach. In our opinion it is not necessary to apply again the full protocol for cisPt to test the complex with proteins different from CytC since, during the protocol application, the protein is denatured. Instead, it is strongly suggested to use it studying Pt-complexes different from cisPt or with other metals (Ru, Au).