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bioRxiv · 10.1101/145086

Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg

Presler, M. S.·Van Itallie, E.·Klein, A. M.·Kunz, R.·Coughlin, P.·Peshkin, L.·Gygi, S.·Wühr, M.·Kirschner, M.
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Abstract

Fertilization triggers release from meiotic arrest and initiates events that prepare for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phospho-regulation is still unknown. We examined absolute protein and phospho-site dynamics after fertilization by mass spectrometry-based proteomics. To do this, we developed a new approach for calculating the stoichiometry of phospho-sites from multiplexed proteomics that is compatible with dynamic, stable and multi-site phosphorylation. Overall, the data suggest that degradation is limited to a few low abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium activated kinases. We identify putative degradation targets as well as new components of the block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.\n\nSignificance StatementProtein phosphorylation and degradation drive critical events in early embryogenesis and the cell cycle; however, comprehensive and accurate analysis of these changes is currently difficult. Using a mass spectrometric approach, we present a quantitative view of the protein and posttranslational economy of the fertilization response in the frog egg. Protein degradation affects a small but very important class of proteins, while regulatory phosphorylation and protein release occur on a far larger scale. We have developed new, broadly applicable analytical methods for phosphorylation that provide absolute quantification with confidence intervals for improved interpretability of post-translational modification analysis.

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Presler, M. S., Van Itallie, E., Klein, A. M., Kunz, R., Coughlin, P., Peshkin, L., Gygi, S., Wühr, M., Kirschner, M.. 2017-06-04. Proteomics of phosphorylation and protein dynamics during fertilization and meiotic exit in the Xenopus egg. https://doi.org/10.1101/145086

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