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

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Moraleja, I and Moreno Gordaliza, Estefanía and Mena, María Luz and Gómez Gómez, M.Milagros (2014) Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins. MethodsX, 1 . e175-e180. ISSN 2215-0161

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Official URL: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472949/



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 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.


Item Type:Article
Uncontrolled Keywords:Metal-binding proteins, OFFGEL-IEF, FASP, ICP-MS, Thiol-free reductants, nLC–ESI-LTQ-MS/MS
Subjects:Sciences > Chemistry > Analytic chemistry
ID Code:55183
Deposited On:07 May 2019 10:52
Last Modified:13 May 2019 10:49

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