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@article{0e48430817f34457a3aabac38eadda91,
title = "Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms",
publisher = "Portland Press Ltd.",
author = "K. Maeda and Christine Finnie and Birte Svensson",
year = "2004",
doi = "10.1042/BJ20031634",
volume = "378",
pages = "497--507",
journal = "Biochemical journal",
issn = "0264-6021",

}

RIS

TY - JOUR

T1 - Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms

A1 - Maeda,K.

A1 - Finnie,Christine

A1 - Svensson,Birte

AU - Maeda,K.

AU - Finnie,Christine

AU - Svensson,Birte

PB - Portland Press Ltd.

PY - 2004

Y1 - 2004

N2 - Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization-time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryo-specific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin It in barley seeds

AB - Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization-time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryo-specific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin It in barley seeds

U2 - 10.1042/BJ20031634

DO - 10.1042/BJ20031634

JO - Biochemical journal

JF - Biochemical journal

SN - 0264-6021

VL - 378

SP - 497

EP - 507

ER -