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.
Raw measurement files were analyzed with MaxQuant 18.104.22.168 standard settings (Cox and Mann 2008, PMID:19029910), except for LFQ quantitation, and match between run option was activated. Peptides were identified by comparison to the translated Ensembl transcript database (release 79) of D. melanogaster and the S. cerevisiae protein database. Protein abundance was measured using the MaxLFQ method of label-free quantitation (Cox et al., 2014, PMID:24942700). Known contaminants, protein groups only identified by site, and reverse hits of the MaxQuant results were removed. Only unique peptide intensities for each protein group were used for protein quantification.
For the purposes of analysis, imputation was performed to replace missing normalized intensity (LFQ) values for a given protein, using one of two approaches. If the protein completely lacked a raw intensity value, then the smallest measured value was assigned (log2 of LFQ = 21.8797761147). If a protein had a measurable intensity, but a normalized LFQ intensity value was not calculated, then the value was imputed from a normal distribution with a mean value shifted by -0.6 from the mean value of all measured LFQ intensities and half of the standard deviation.
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