Two-dimensional electrophoresis analysis of glutathione affinity-selected proteins from the clam Tapes semidecussatus: Evidence for tissue-specific expression of redox proteins V. Dowling a,b , B. McDonagh a,b , E.M. Cotter a,b , N. O'Brien a,c , F. van Pelt a,d , J. O'Halloran a,e , D. Sheehan a,b, ⁎ a Environmental Research Institute, University College Cork, Ireland b Proteomics Research Group, Department of Biochemistry, University College Cork, Ireland c Department of Food Science, University College Cork, Ireland d Department of Pharmacology and Therapeutics, University College Cork, Ireland e Department of Zoology Ecology and Plant Science, University College Cork, Ireland Received 23 August 2005; received in revised form 16 January 2006; accepted 21 January 2006 Available online 28 February 2006 Abstract Proteins from gill, digestive gland and mantle of the clam Tapes semidecussatus were selected on glutathione (GSH) agarose to simplify proteomic comparison. Analysis by two-dimensional gel electrophoresis (2D SDS PAGE) revealed tissue-specific patterns of protein expression with some spots common to all tissues. Immunoblotting of gill and digestive gland separations identified some spots as glutathione transferases (GSTs). In gill and digestive gland several spots were immunoblotted with Pi class GSTs indicating multiple isoenzymes. Selected spots were excised, digested with trypsin and analyzed by reversed phase C-18 high performance liquid chromatography and tandem mass spectrometry. This confirmed that gill and digestive gland share some GST isoenzymes. Our results suggest that this clam expresses a complex tissue-specific pattern of GSH-binding proteins, which may reflect different redox requirements in each tissue. © 2006 Elsevier Inc. All rights reserved. Keywords: Proteomics; Tapes semidecussatus; Glutathione transferase; Affinity chromatography; Ecotoxicology; Redox status 1. Introduction Pollutants such as metals and polyaromatic hydrocarbons (PAHs) can generate reactive oxygen species (ROS) (Halliwell and Gutteridge, 1999; Petropoulos and Friguet, 2005) resulting in toxicity including lipid peroxidation and covalent modification of proteins and DNA (Livingstone et al., 1990). Cellular redox status is determined primarily by the ratio of reduced to oxidised glutathione (GSH/GSSG; Meister and Anderson, 1983). Build-up of ROS due to ageing, chemical pollution or down-regulation of antioxidant enzymes/molecules decreases this ratio, which can trigger apoptosis (Curtin et al., 2002; Robertson and Orrenius, 2000). GSH-agarose has been widely used for isolation of glutathione transferases (GSTs), an important superfamily of Phase II detoxification enzymes some of which also act as Se- independent GSH peroxidases (Sheehan et al., 2001). Several other protein superfamilies (e.g. glutaredoxins and GSH reduc- tases) share the thioredoxin fold with GSTs (Sheehan et al., 2001) and many of these can also bind to GSH-agarose. The clam Tapes semidecussatus has found widespread use in ecotoxicology studies (Bebianno et al., 2004) and further studies conducted in our laboratory have involved the assessment of biochemical endpoints in this animal to study sediment toxicity from polluted and reference sites on the South coast of Ireland (Hartl et al., 2004). While multiple T. semidecussatus GSTs have previously been isolated from whole animals (Hoarau et al., 2002), little is known about their tissue distribution in clams especially in organs likely to be involved in xenobiotic metabolism such as gill and digestive gland. Our laboratory is Comparative Biochemistry and Physiology, Part D 1 (2006) 267 – 272 www.elsevier.com/locate/cbpd ⁎ Corresponding author. Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Mardyke, Cork, Ireland. Tel.: +353 21 4904207; fax: +353 21 4274034. E-mail address: d.Sheehan@ucc.ie (D. Sheehan). 1744-117X/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2006.01.002