RT Journal Article
T1 Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach
A1 García Sevillano, Miguel Ángel
A1 García Barrera, Tamara
A1 Navarro, Francisco
A1 Gómez Ariza, José Luis
AB In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3-5% and detection limits is 0.21 ngCug(-1). The application of the methodology to hepatic cells from mice exposed to inorganic mercury reveals decreased levels of Cu,Zn-SOD in cytosolic and mitochondrial extracts, as a consequence of the oxidative stress caused by this toxic metal. Additionally, the quantification of mitochondrial Cu,Zn-SOD in hepatic cells from Mus musculus has been carried out for the first time.
PB Elsevier Masson
SN 0003-2670
YR 2014
FD 2014
LK http://hdl.handle.net/10272/14381
UL http://hdl.handle.net/10272/14381
LA eng
NO García Sevillano, M.A., García Barrera, T., Navarro, F., Gómez Ariza, J.L.: "Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach".  Analytica Chimica Acta. Vol. 842, págs. 42-50, (2014). DOI: 10.1016/j.aca.2014.07.014
NO This work was supported by the project CTM2012-38720-C03-01 from the Spanish Ministry of Economy and Competitiveness, and P12-FQM-0442 from the Regional Ministry of Economy, Innovation, Science and Employment (Andalusian Government, Spain). Miguel Ángel García Sevillano thanks Ministerio de Educación, Cultura y Deporte for a predoctoral scholarship. We also thank to the group of Prof. Sanz-Medel at the University of Oviedo, especially to Dr. M.L. Fernández-Sánchez and Dr. Nuevo-Ordóñez, for introducing and training in copper isotope dilution analysis.
DS Repositorio Institucional de la Universidad de Huelva
RD 1 jun 2026