Covalent selection of the thiol proteome on activated thiol sepharose: A robust tool for redox proteomics

• Published in: Talanta (Oxford) Vol. 80; no. 4; pp. 1569 - 1575
• Main Authors: Hu, Wentao, Tedesco, Sara, Faedda, Roberto, Petrone, Goffredo, Cacciola, Santa Olga, O’Keefe, Anne, Sheehan, David
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.02.2010
• Subjects: Activated thiol sepharose; Animals; Antioxidants; Bivalvia; Cysteine; Cysteine - metabolism; Disulfides - metabolism; Escherichia coli; Glutathione - metabolism; Mytilus edulis; Oxidation-Reduction; Oxidative Stress; Proteome - chemistry; Proteomics - methods; Reactive Oxygen Species; Redox proteomics; Sepharose - analogs & derivatives; Sepharose - chemistry; Sulfhydryl Compounds - chemistry; Sulfhydryl Compounds - metabolism; Thiol-containing proteins; Trichoderma harzianum; Trichoderma harzianum; Oxidative stress; Redox proteomics; Cysteine; Mytilus edulis; Escherichia coli; Thiol-containing proteins; Activated thiol sepharose
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2009.10.047

Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (–SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of –SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching –SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. –SH-containing proteins account for 2.52% ( E. coli), 1.4% ( T. harzianum) and 1.4% ( M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M r thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for –SH-containing proteins in redox proteomics.