title: Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach.
abstract: 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.
authors.affiliation: Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007, Spain; Research Center on Health and Environment (CYSMA), University of Huelva, Spain; International Campus of Excellence on Agrofood (ceiA3), University of Huelva, Spain.
authors.forename: M A
authors.initials: MA
authors.lastname: García-Sevillano
authors.affiliation: Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007, Spain; Research Center on Health and Environment (CYSMA), University of Huelva, Spain; International Campus of Excellence on Agrofood (ceiA3), University of Huelva, Spain.
authors.forename: T
authors.initials: T
authors.lastname: García-Barrera
authors.affiliation: International Campus of Excellence on Agrofood (ceiA3), University of Huelva, Spain; Department of Environmental Biology and Public Health, Cell Biology, Faculty of Experimental Sciences, University of Huelva, Campus El Carmen, Huelva 21007, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Navarro
authors.affiliation: Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, Huelva 21007, Spain; Research Center on Health and Environment (CYSMA), University of Huelva, Spain; International Campus of Excellence on Agrofood (ceiA3), University of Huelva, Spain. Electronic address: ariza@uhu.es.
authors.forename: J L
authors.initials: JL
authors.lastname: Gómez-Ariza
mesh_terms: D000818:Animals; D002850:Chromatography, Gel / Q000295:instrumentation; D002852:Chromatography, Ion Exchange / Q000295:instrumentation; D003600:Cytosol / Q000201:enzymology* / Q000378:metabolism; D004867:Equipment Design; D022781:Hepatocytes / Q000201:enzymology* / Q000378:metabolism; D057230:Limit of Detection; D008628:Mercury / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D052616:Solid Phase Extraction / Q000295:instrumentation; D013482:Superoxide Dismutase / Q000032:analysis* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000295:instrumentation
chemical_list: D013482:Superoxide Dismutase; D008628:Mercury
keywords: Column switching; ICP-MS; Isotopic dilution analysis; Metallomic workflow; Mitochondrial extracts; Superoxide dismutase