title: Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins.
abstract: 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.
authors.affiliation: Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain.
authors.forename: I
authors.initials: I
authors.lastname: Moraleja
authors.affiliation: Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain ; Division of Analytical Biosciences, LACDR, Universiteit Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Moreno-Gordaliza
authors.affiliation: Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain.
authors.forename: M L
authors.initials: ML
authors.lastname: Mena
authors.affiliation: Analytical Chemistry Department, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain.
authors.forename: M M
authors.initials: MM
authors.lastname: Gómez-Gómez
keywords: FASP; FASP proteolytic digestion; ICP-MS; Metal-binding proteins; OFFGEL-IEF; SDS-PAGE; Thiol-free reductants; nLC–ESI-LTQ-MS/MS