The anticancer effects of the trace element selenium have been shown in clinical, preclinical and laboratory studies. Despite mechanisms of action are not completely understood, the tuning of cellular redox state by selenium seems to be of particular relevance. The aim of this work is to study the response to selenium supplementation in a prostate cancer cell line (LNCaP) by comparing protein expression profiles in cells grown without (control) and with (Se supplemented) selenium added to the medium. Selenium supplementation is achieved by growing cells in 100 nM Sodium Selenite for three days. The measurement of selenium peroxidase activities is used as the marker of successful supplementation. Protein expression profiles study was performed by combination of two dimensional liquid chromatography separation with mass spectrometry analysis of chromatographic fractions. Solubilized proteins from pelleted cells are first separated in a two dimensional liquid chromatography system (chromatofocusing and reverse phase chromatography). The profiles of all the chromatographic separations of the two samples are compared by means of a program (DeltaVue) and a differential map expression is generated. Consistent peaks which are reproducibly differently expressed (at least a 100% difference) are analyzed with mass spectrometry (MALDI-TOF and ESI-MS/MS). We obtained high confidence protein identification with different search engine (Mascot and Protein Prospector for MALDI-MS and SEQUEST for ESI-MS) for most of differently expressed chromatographic peaks. This multidimensional protein identification system allows us to identify proteins with about 10-90KDa molecular weight.
Analysis of molecular response to selenium supplementation by multidimensional protein identification technology
ZACCARIN, MATTIA;ROVERI, ANTONELLA
2006
Abstract
The anticancer effects of the trace element selenium have been shown in clinical, preclinical and laboratory studies. Despite mechanisms of action are not completely understood, the tuning of cellular redox state by selenium seems to be of particular relevance. The aim of this work is to study the response to selenium supplementation in a prostate cancer cell line (LNCaP) by comparing protein expression profiles in cells grown without (control) and with (Se supplemented) selenium added to the medium. Selenium supplementation is achieved by growing cells in 100 nM Sodium Selenite for three days. The measurement of selenium peroxidase activities is used as the marker of successful supplementation. Protein expression profiles study was performed by combination of two dimensional liquid chromatography separation with mass spectrometry analysis of chromatographic fractions. Solubilized proteins from pelleted cells are first separated in a two dimensional liquid chromatography system (chromatofocusing and reverse phase chromatography). The profiles of all the chromatographic separations of the two samples are compared by means of a program (DeltaVue) and a differential map expression is generated. Consistent peaks which are reproducibly differently expressed (at least a 100% difference) are analyzed with mass spectrometry (MALDI-TOF and ESI-MS/MS). We obtained high confidence protein identification with different search engine (Mascot and Protein Prospector for MALDI-MS and SEQUEST for ESI-MS) for most of differently expressed chromatographic peaks. This multidimensional protein identification system allows us to identify proteins with about 10-90KDa molecular weight.
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