LC-MSMS identification of Arabidopsis thaliana heat-stable seed proteins: enriching for LEA-type proteins by acid treatment.

Protein identification in systems containing very highly abundant proteins is not always efficient and usually requires previous enrichment or fractionation steps in order to uncover minor proteins. In plant seeds, identification of late embryogenesis abundant (LEA) proteins is often masked by the presence of the large family of storage proteins. LEA-proteins are predicted to play a role in plant stress tolerance. They are highly hydrophilic proteins, generally heat-stable, and correlate with dehydration in seeds or vegetative tissues. In the present work, we analyze the protein composition of heat-stable Arabidopsis thaliana seed extracts after treatment with trichloroacetic acid (TCA). The composition of the proteins that precipitate and those that remain in solution in 3% TCA was analyzed by two different approaches: 1D SDS-PAGE coupled to LC-ESI-MSMS analysis and a gel-free protocol associated with LC-MALDI-MSMS. Our results indicate that treating total heat-soluble extracts with 3% TCA is an effective procedure to remove storage proteins by selective precipitation and this fractionation step provides a soluble fraction highly enriched in Lea-type proteins. The analysis and determination of protein identities in this acid-soluble fraction by MS technology is a suitable system for large-scale identification of Lea-proteins present in seeds.