Christine Finnie

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

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, embryospecific 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 h in barley seeds.

Feasibility study of a tissue-specific approach to barley proteome analysis: aleurone layer, endosperm, embryo and single seeds

Abstract Proteome analysis in the pI 4–7 range of aqueous extracts from whole barley seeds results in complex 2D-gel spot patterns that represent proteins from all seed tissues. Proteins were extracted from dissected aleurone layer, embryo and endosperm for separate analysis of these components and comparison with single seed extracts. The analysis showed that while the starchy endosperm comprises approximately 85% of the mature seed dry weight, it contributes less than 50% of the soluble protein to seed extracts. Although the aleurone layer and embryo are minor components of the seed in terms of dry weight, their proteins contribute significantly to the 2D-gel pattern. Distinct but overlapping patterns were observed for the dissected tissues. While whole seed gels contained about 850 spots, endosperm, aleurone layer and embryo gels had about 575, 850 and 1000 spots, respectively, representing a total increase of at least 15%. Protein patterns were also reduced in complexity and this enrichment of subsets of proteins facilitated identification by mass spectrometry of proteins of interest in mature seed tissues and developing endosperm. These techniques can therefore be used with advantage to describe the proteomes of dissected seed tissues.

Aspects of the barley seed proteome during development and germination

Analysis of the water-soluble barley seed proteome has led to the identification of proteins by MS in the major spots on two-dimensional gels covering the pI ranges 4-7 and 6-11. This provides the basis for in-depth studies of proteome changes during seed development and germination, tissue-specific proteomes, cultivar differences related to quality parameters, analysis of the genetic basis for spot variations and targeted investigations of specific proteins.