Poster: Proteomics
Abs #
965: Proteomic analysis of the tomato fruit cell wall
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Presenter: |
R. S., Saravanan , rss39@cornell.edu |
Authors | R. S., Saravanan (A) Lee, Sang-Jik (A) Dikler, Sergei (B) Rose, Jocelyn K C (A) | | Affiliations: |
(A): Cornell University
(B): Bruker Daltonics
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Tomato fruit development is an excellent model system in which to study molecular mechanisms associated with cell wall assembly, reorganization and disassembly. While several families of wall-modifying proteins have been identified to date that contribute to these processes, numerous new classes remain to be discovered and preliminary studies suggest that most extracellular proteins cannot yet be assigned a biochemical function. We are using several strategies to characterize the cell wall proteome of tomato fruit to identify novel proteins and to reveal the suites of proteins that are co-expressed at specific developmental stages. A critical examination is described here of one common method to isolate wall-associated proteins using sequential extraction of wall material with a variety of solvents, including buffers of varying ionic concentrations, phenol and SDS. Importantly, to validate this approach, the potential contamination of the extracts with cytosolic proteins was carefully assessed. Western analyses with antibodies to a range of proteins suggest that, contrary to published reports, both wall-localized and cytosolic proteins can show remarkably high affinity binding to the wall matrix during extraction. Proteins from different wall-bound fractions were analyzed by two-dimensional gel electrophoresis, followed by peptide mass fingerprinting and peptide sequencing using MALDI TOF and MALDI TOF/TOF mass spectrometry, respectively. The immunological analysis combined with this particular proteomic approach suggests that this represents a useful method to identify new cell wall proteins and to provide an integrated view of cell wall proteome of tomato fruit, but that this technique should ideally be complemented with additional non-destructive approaches.
