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Molecular & Cellular Proteomics 7:2176-2187, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Comparative Phosphoproteomics of Zebrafish Fyn/Yes Morpholino Knockdown Embryos^*,S

Simone Lemeer^,, Chris Jopling^,¶, Joost Gouw, Shabaz Mohammed, Albert J. R. Heck, Monique Slijper and Jeroen den Hertog^,||

From the Hubrecht Institute-Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands and Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, the Netherlands

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO₂-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

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