Abstract
Recent genomic analyses revealed that plants contain a large number of genes that are predicted to be involved in the ubiquitin/26S proteasome pathway. For example, nearly 700 F-box proteins have been annotated in Arabidopsis. These observations strongly indicate that ubiquitination contributes to various biological processes in plants. Indeed, genetic analyses have shown that the ubiquitin/26S proteasome pathway components regulate embryogenesis, hormonal responses, circadian rhythms, floral homeosis, photomorphogenesis, trichome differentiation, senescence, and pathogen defense. However, only a few target proteins for ubiquitination were so far identified.
Here, we present a novel method for identifying ubiquitinated proteins from plants. Ubiquitinated proteins were enriched by an affinity column that utilizes ubiquitin-binding domains isolated from Arabidopsis. After trypsin digestion, peptides were separated and analyzed by 2DLC-MS/MS. 2D separation was performed online with a strong cation-exchange chromatography followed by a reverse-phase chromatography.
