Population cages of wild-type, fertilized females were maintained at 25oC. Embryos were collected on apple juice agar plates in 2 hr windows, aged at 25oC for the required time, then dechorionated. Early larval collections were obtained from 2 hr embryo collections that were appropriately aged. Later larval and pupal stages were collected directly from flasks at the indicated time points. Adult flies were collected either within 4 hr of eclosion, or as one week old flies. Samples were snap-frozen. For each time point, samples were collected in quadruplicate.
Snap-frozen samples were mechanically lysed by bead milling in lysis buffer with protease inhibitor. Homogenates were collected by centrifugation and proteins were acetone precipitated. Protein pellets were resuspended in sample loading buffer containing DTT, boiled 10 min, sonicated 10 min, then separated on 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.