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Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis : Proteomic identification of target proteins
Author(s) -
McDonagh Brian,
Sheehan David
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700241
Subject(s) - menadione , gelsolin , biochemistry , calreticulin , chemistry , oxidative stress , tropomyosin , mytilus , protein disulfide isomerase , proteomics , peptide mass fingerprinting , biology , actin , enzyme , endoplasmic reticulum , ecology , gene
Protein thiols are targets of oxidative stress. Their modification was analysed in gill extracts of the mussel Mytilus edulis , exposed to menadione. Diagonal gel electrophoresis revealed two clusters of carbonylated proteins involved in interchain disulphide linkages. Immunoblotting identified these as being associated with protein disulphide isomerase (PDI) and actin and this was confirmed by immunoprecipitation. Protein free thiols (–SH) were identified in 2‐DE separations by labelling with 5‐iodoacetamidofluorescein (IAF). Cysteines involved in disulphide bridges were identified by blocking free –SH with N ‐ethylmaleimide, reducing disulphides with DTT and IAF labelling. Several protein spots containing free thiols disappeared on exposure to menadione. Conversely, new protein spots containing disulphides appeared in response to menadione which may be protective against oxidative stress. In‐gel tryptic digestion followed by LC/MS‐MS and database searching identified some of the free thiol targets: PDI; hsp gp96; calreticulin; heavy metal binding protein. Tubulin, PDI, enolase and gelsolin contained new disulphide bridges in response to menadione. Our findings indicate a protein level response to oxidative stress principally involving PDI, chaperone‐like and cytoskeletal proteins. Since many environmental pollutants cause oxidative stress, studies on PDI and structural proteins may be particularly relevant to understanding toxicity in this popular sentinel species.