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

  1. Marc W. Kirschnera,1
  1. aDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115;
  2. bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115;
  3. cDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544;
  4. dThe Lewis–Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544
  1. Contributed by Marc W. Kirschner, October 27, 2017 (sent for review June 6, 2017; reviewed by William M. Bement, James E. Ferrell, and Matthias Mann)


Protein 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 broadly applicable analytical methods for phosphorylation that provide absolute quantification with confidence intervals for improved interpretability of posttranslational modification analysis.


Fertilization releases the meiotic arrest and initiates the events that prepare the egg 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 phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite 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 and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.


  • ?1To whom correspondence may be addressed. Email: wuhr{at}princeton.edu or marc{at}hms.harvard.edu.
  • Author contributions: M.P., M.W., and M.W.K. designed research; M.P., E.V.I., R.K., M.L.C., and M.W. performed research; S.P.G. contributed new reagents/analytic tools; M.P., E.V.I., A.M.K., and M.W. contributed the stoichiometry method; M.P., E.V.I., L.P., and M.W. analyzed data; and M.P., E.V.I., A.M.K., M.W., and M.W.K. wrote the paper.

  • Reviewers: W.M.B., University of Wisconsin–Madison; J.E.F., Stanford University; and M.M., Max Planck Institute of Biochemistry.

  • The authors declare no conflict of interest.

  • Data deposition: The data reported in this paper have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD006639).

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1709207114/-/DCSupplemental.

Published under the PNAS license.

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