Covalent selection of the thiol proteome on activated thiol sepharose: a robust tool for redox proteomics.
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.
Hu
Wentao
W
Proteomics Research Group, Department of Biochemistry, University College Cork, Cork, Ireland.
Tedesco
Sara
S
Faedda
Roberto
R
Petrone
Goffredo
G
Cacciola
Santa Olga
SO
O'Keefe
Anne
A
Sheehan
David
D
D000818:Animals; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D017382:Reactive Oxygen Species; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D020543:Proteome; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; C037851:thiol-sepharose; D012685:Sepharose; D005978:Glutathione; D003545:Cysteine