%0 Journal Article
%A Moraleja, I
%A Moreno Gordaliza, Estefanía
%A Mena, María Luz
%A Gómez Gómez, M.Milagros
%T Thiol-free reducing agents in electrophoreticseparations and FASP proteolytic digestions forthe analysis of metal-binding proteins
%D 2014
%@ 2215-0161
%U https://hdl.handle.net/20.500.14352/34099
%X 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 b-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.
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