Population cages of wild-type, fertilized females were maintained at 25oC. Embryos were collected on apple juice agar plates in 30m windows, aged at 25oC for the required time, then dechorionated. For each sample, about 20 ul embryo pellets were transferred to PBS buffer for lysis. To assess the homogeneity of embryonic stages of each collection, about 10% of the sample was fixed and staged. The remainder of the sample was snap-frozen. For each time point, samples were collected in quadruplicate.

Snap-frozen samples were mechanically lysed in PBS using a microtube pestle. Cells were pelleted, resuspended in sample loading buffer containing DTT, boiled 10 min, then separated on a 4-12% Bis/Tris gel. Proteins were digested in-gel and mass spec performed as described in Kappei et al., 2013 (PMID:23685356).

Peptides were separated by nanoflow liquid chromatography coupled to a Q Exactive Plus mass spectrometer (Thermo). The instrument was operated in the data-dependent mode.