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.
Raw measurement files were analyzed with MaxQuant 220.127.116.11 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 values for a given protein. Missing values were drawn from a distribution calculated with the logspline R package (https://cran.r-project.org/package=logspline). For cases where three or more replicates were measured, the mean of the measured replicates was used as the mean parameter of the distribution. Otherwise, the average of the two neighboring time points was used. In cases of no measured values in neighboring time points or for proteins measured only in one replicate with no surrounding values, a fixed value of 22.5 close to the limit of detection (LOD) was used.
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