On-line automated zebrafish phosphoproteomics : from large-scale analysis down-to a single embryo (THESIS VERSION)

Abstract

In the developing embryo, as in many other biological processes, complex signaling pathways are under tight control of reversible phosphorylation, guiding cell proliferation, differentiation and growth. Therefore the large-scale identification of signaling proteins and their post-translational modifications is crucial to understand the proteome biology of the developing zebrafish embryo.Here we used an automated, robust and sensitive on-line TiO2 based LC MS/MS setup to enrich for phosphorylated peptides from 1 day old zebrafish embryos. We identified, with high confidence, 1067 endogenous phosphorylation sites in a sample taken from 60 embryos (approx. 180 ug), 321 from 10 embryos and 47 phosphorylation sites from a single embryo, illustrating the sensitivity of the method. This dataset, representing by far the largest for zebrafish, was further exploited by searching for serine/threonine or tyrosine kinase motifs using Scansite. For one-third of the identified phosphopeptides a potential kinase motif could be predicted, where it appeared that Cdk5 kinase, p38MAPK, PKA and Casein Kinase 2 substrates were the most predominant motifs present, underpinning the importance of these kinases in signaling pathways in embryonic development. The phosphopeptide dataset was further interrogated using alignments with phosphopeptides identified in recent large-scale phosphoproteomics screens in human and mouse samples. These alignments revealed conservation of phosphorylation sites in several prominent proteins suggesting preserved functions.