Three conditions (WT, WT + rapamycin, tor1) were run with three biological replicates each. For each of these runs, three data acquisitions methods were run (20 m/z overlap, 10 m/z, and DDA).
Three conditions were tested in this experiment: wild type yeast, wild type yeast grown in rapamycin, and tor1 deletion mutant yeast. The wild type and tor1 haploid strains of S. cerevisiae were from an ORF deletion collection33 and had the parental background BY4742 -- MATα hist3∆1 leu2∆0 lys2∆0 ura3∆0. Both strains were grown in YPD media in three biological replicates. The wild type strain was grown either in the presence or absence of 10 nM rapamycin. Strains were grown from OD600 ~0.15 to OD600 0.6 prior to lysis and digestion. The samples without rapamycin grew to this density in about 6 hours, while the rapamycin treated strains required 8 hours. Cells were lysed by bead beating using a lysis buffer of 50 mM ammonium bicarbonate at pH 7.8 with phosphatase inhibitors (Pierce – Halt phosphatase inhibitor cocktail). Bead beating was for one minute, followed by one minute of cooling the sample on ice, repeated three times. The digestion was performed for one hour using sequencing-grade trypsin (Promega, Madison, WI) as in Hoopmann et. al.34 except using 0.1% PPS Silent Surfactant (Protein Discoveries) instead of RapiGest SF (Waters Corporation, Milford, Ma). Experiment Design