Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins

The analysis of the complexes between metal-based chemotherapeutic drugs and proteins in biological samples, such as cisplatin or oxaliplatin, can be a challenge due to metal strong reactivity towards S-donor molecules such as dithiothreitol (DTT) or β-mercaptoethanol (BME), usually employed as reducing agents in electrophoretic separations and proteolytic digestions for LC–MS/MS analysis.
• This protocol describes the use of the thiol-free reducing trialkylphosphines, such as tributylphosphine (TBP) and tris(2-carboxyethyl)phosphine (TCEP) as suitable reagents for the preservation of the metal-protein complexes during OFFGEL-IEF and SDS-PAGE separations, respectively.
• Moreover, the filter-aided sample preparation (FASP) method is presented as an advantageous option to perform tryptic in-solution digestions of metal–protein complexes in combination with OFFGEL-IEF separations.
• The FASP procedure allows including previous reduction and alkylation steps in addition to proteolysis, ensuring the preservation of the metal–protein complexes. The limited time that proteins remain in contact with the reducing agent, either TBP or even DTT, during FASP could be a key factor for its extraordinary performance on the digestion of metal–protein complexes.

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