TY - JOUR
T1 - Differential labelling of cysteines for simultaneous identification of thioredoxin h-reducible disulphides in native protein extracts: insight into recognition and regulation of proteins in barley seeds by thioredoxin h
AU - Maeda, Kenji
AU - Finnie, Christine
AU - Svensson, Birte
PY - 2005
Y1 - 2005
N2 - 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 matrixassistend
laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in
vitro reduction of disulphides in recombinant barley a-amylase/subtilisin inhibitor (BASI) by
barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with twodimensional
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 a-amylase/trypsin inhibitors
and a protein of unknown function. Two specific disulphides, located structurally close
to the alpha-amylase binding surfaces of BASI and alpha-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.
AB - 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 matrixassistend
laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in
vitro reduction of disulphides in recombinant barley a-amylase/subtilisin inhibitor (BASI) by
barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with twodimensional
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 a-amylase/trypsin inhibitors
and a protein of unknown function. Two specific disulphides, located structurally close
to the alpha-amylase binding surfaces of BASI and alpha-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.
U2 - 10.1002/pmic.200401050
DO - 10.1002/pmic.200401050
M3 - Journal article
SN - 1615-9853
VL - 5
SP - 1634
EP - 1644
JO - Proteomics
JF - Proteomics
IS - 6
ER -