|Thannhauser, Theodore - Ted|
Submitted to: Journal of Biomolecular Techniques
Publication Type: Abstract Only
Publication Acceptance Date: 1/25/2008
Publication Date: 2/16/2008
Citation: Hucko, S., Catala, C., Howe, K.J., Rose, J., Thannhauser, T.W. 2008. Characterization of Extracellular Proteins in Tomato Fruit using Lectin Affinity Chromatography and LC-MALDI-MS/MS analysis. Journal of Biomolecular Techniques. Vol. 19:57
Technical Abstract: The large-scale isolation and analysis of glycoproteins by lectin affinity chromatography coupled with mass spectrometry has become a powerful tool to monitor changes in the “glycoproteome” of mammalian cells. Thus far, however, this approach has not been used extensively for the analysis of plant glycoproteins. As with all eukaryotes, N-glycosylation in plant cells is a common post-translational modification for proteins traveling through the secretory pathway. Many such proteins are destined for the cell wall, or apoplast, where they play important roles in processes such as modifying cell wall structure, sugar metabolism, signaling and defense against microbes. We describe a strategy to isolate and identify secreted plant proteins based on lectin affinity chromatography and 2DLC-MALDI MS/MS analysis, and demonstrate how this approach can be used to study fruit ripening, a plant developmental process that is fundamentally linked with major qualitative and quantitative changes in the secretome. Total soluble protein extracts of tomato (Solanum lycopersicum) fruit pericarp were applied to a Concanavalin A Sepharose column. After proteolysis of the selected proteins, the peptide mixtures were separated using strong cation-exchange (SCX) chromatography. The SCX fractions were then separated with a nano scale reverse phase liquid chromatograph coupled to a MALDI plate spotting robot. These spots were analyzed offline by MALDI MS/MS. Proteins were identified by using the mass spectral data to search both a tomato protein database (ftp.sgn.cornell.edu/proteins/) and a decoy database using MASCOT version 2.2. We show that this strategy not only provides information about the complement of tomato fruit extracellular proteins, but also constitutes an excellent approach to isolate apoplastic/cell wall localized proteins with minimal contamination by cytosolic proteins. We are using this methodology in conjunction with iTRAQ labeling for relative quantification of protein expression to study the tomato fruit secretome dynamics during fruit ripening.