A shotgun approach for the identification of platinum–protein complexes

A shotgun approach including peptide-based OFFGEL-isoelectric focusing (IEF) fractionation has been developed with the aim of improving the identification of platinum-binding proteins in biological samples. The method is based on a filter-aided sample preparation (FASP) tryptic digestion under denaturing and reducing conditions of cisplatin–, oxaliplatin–, and carboplatin–protein complexes, followed by OFFGEL-IEF separation of the peptides. Any risk of platinum loss is minimized throughout the procedure due to the removal of the reagents used after each stage of the FASP method and the absence of thiol-based reagents in the focusing buffer employed in the IEF separation. The platinum–peptide complexes stability after the FASP digestion and the IEF separation was confirmed by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The suitability of peptide-based OFFGEL-IEF fractionation for reducing the sample complexity for further nano-liquid chromatographyelectrospray ionization-tandem mass spectrometry (nLC-ESIMS/MS) analysis has been demonstrated, allowing the detection of platinum-containing peptides, with significantly lower abundance and ionization efficiency than unmodified peptides. nLCMS/MS analysis of selected OFFGEL-IEF fractions from tryptic digests with different complexity degrees: standard human serum albumin (HSA), a mixture of five proteins (albumin, transferrin, carbonic anhydrase, myoglobin, and cytochrome-c) and human blood serum allowed the identification of several platinum–peptides from cisplatin–HSA. Cisplatin-binding sites in HSA were elucidated from the MS/MS spectra and assessed considering the protein three-dimensional structure. Most of the potential superficial binding sites available on HSA were identified for all the samples, including a biologically relevant cisplatin-cross-link of two protein domains, demonstrating the capabilities of the methodolog