Lamas, S.

Martínez-Acedo, Pablo

Martínez-Ruiz, Antonio

Moreno, M.

Núñez, Estefanía

Rodríguez-Pérez, P.

Sánchez-Gómez, F.J.

Vázquez, J.

Repositorio Institucional de la Universidad de Córdoba

S3. Post-Translational Modifications  Poster P47 158  GELSILOX: A NOVEL STRATEGY FOR THE STUDY OF DYNAMIC ALTERATIONS IN THE THIOL REDOX PROTEOME P. Martínez-Acedo (1), F.J. Sánchez-Gómez (1), M. Moreno (1), P. Rodriguez Pérez (1), E. Núñez (1), A. Martínez-Ruiz (2), S. Lamas (1), J. Vázquez (1). (1) Centro de Biología Molecular Severo Ochoa, CSIC-UAM, (2) Instituto de Investigación Sanitaria Princesa, Hospital de la Princesa. Many physiological and pathological conditions are associated with reactive oxygen and nitrogen species accumulation, a condition termed oxidative stress. Redox proteomics, the systematic and exhaustive characterization of oxidized cysteines, should help to establish the physiological scope of Cys oxidation and give clues to its mechanisms. Redox proteomics still remains a technical challenge, mainly because of the labile nature of some thiol-redox modifications, the lack of tools to directly detect the modified residues and the relatively late development of highly sensitive analytical instruments. Here we present a simple and straightforward method for the analysis of the dynamic redox proteome, GELSILOX. This novel method is based on a technology previously developed in our laboratory that allows several treatment steps on proteomes, without sample losses, within concentrating SDS-PAGE gels. Proteomes are subjected to double alkylation to differentially label the oxidation state of Cys, subjected to in-gel digestion, differential 18O/16O-labeling and IEF fractionation followed by LC-LIT-MS/MS analysis. GELSILOX allows the precise quantification of oxidative thiol modifications in several hundreds of peptides using a statistical model developed by our group, determining the exact Cys modified, and the simultaneous differential quantification of the whole proteome in the same experiment. We show the validity of the method with the detection of 341 Cys sites that increase their oxidized state as a response to the treatment with diamide, a thiol-specific oxidant. Using GELSILOX, we have been able to identify the two most sensitive Cys sites that are oxidized, as well as 24 expression changes that take place in endothelial cells treated with low H2O2 concentrations. The results obtained to date, the simplicity and general applicability of the method suggest that GELSILOX may become a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vitro and in vivo, allowing a simultaneous high-throughput quantification of the whole proteome. 

Gelsilox: a novel strategy for the study of dynamics alteration in the thiol redox proteome

Gelsilox: a novel strategy for the study of dynamics alteration in the thiol redox proteome

Abstract

Similar works

Full text

Available Versions

Repositorio Institucional de la Universidad de Córdoba