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Identification of thioredoxin h‐reducible disulphides in proteomes by differential labelling of cysteines: Insight into recognition and regulation of proteins in barley seeds by thioredoxin h
Author(s) -
Maeda Kenji,
Finnie Christine,
Svensson Birte
Publication year - 2005
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.200401050
Subject(s) - thioredoxin , proteome , labelling , identification (biology) , proteomics , biochemistry , thioredoxin reductase , biology , computational biology , differential (mechanical device) , chemistry , enzyme , botany , gene , engineering , aerospace engineering
Using thiol‐specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h‐reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4‐vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix‐assistend laser desorption/ionisation‐time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley α‐amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two‐dimensional electrophoresis for convenient thioredoxin h‐reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four α‐amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the α‐amylase binding surfaces of BASI and α‐amylase inhibitor BMAI‐1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.