Plants have gene families encoding so many proteins that traditional antibody-based methods for studying protein level expression become nearly impossible. Since information about protein content with respect to developmental stage, subcellular localization and environmental conditions can give important clues regarding function, a method for identifying proteins of a multi-gene family and measuring their relative levels in plant tissues would be valuable. The approach to be developed in this proof of concept project makes use of antibodies that recognize common epitopes in a gene family as a tool to isolate family members for proteomic analysis. This project is justified for support as a Small Grant for Exploratory Research because it involves preliminary work on untested and novel ideas.
The procedure starts with peptide digests and differential labeling of two sets of peptides with coded mass tags. After mixing the two sets, immunoaffinity chromatography is employed to isolate the epitope-bearing peptides. These are resolved by reverse phase chromatography in-line to a tandem mass spectrometer where relative peak intensities of the peptide from different sets are determined and parent proteins are identified. To test the method, we will start with known mixtures of peptides prepared from expressed clones. Situations in which mixtures contain peptides of varying affinity to antibody or where a few are highly abundant will be simulated. This work will be carried out using protein extracts prepared from Arabidopsis thaliana and rice seedling leaves.
Although the proof of concept work will focus on the subtilisin protease gene family, this proteomics approach will be directly applicable to many other multi-gene families. Homology within multi-gene families extends across plant species. Therefore, this new approach can be used to uncover orthologous genes in crop plants that share the same tissue specificity and subcellular localization. Those orthologs that also show parallel changes in protein level with respect to developmental stage and responses to hormonal cues and environmental stresses are likely to share similar functions. Thus, understanding of the function of an individual protein in a well-studied model species can be multiplied rapidly toward understanding the function of orthologs in other plant species. Conversely, an ortholog can be identified for deeper study in a model species, and the results applied back to other plant species to increase crop productivity or to develop new sources of fiber and specialty chemicals. The project will provide research opportunities for graduate and undergraduate students. It will also provide data for discussions and problem-solving sessions in biochemistry and proteomics courses taught by the Principal Investigators.
