The environmental stress response regulates ribosome content in cell cycle-arrested S. cerevisiae

Digestion was performed using modified trypsin at an enzyme:substrate ration of 1:50 in (Promega) in 100mM ammonium bicarbonate (pH 8.9) overnight at 25ºC and reactions were quenched in 5% formic acid (Fluka). Samples were desalted with Pierce Peptide Desalting Spin Columns (Thermo Fisher Science, # 89852). TMT labeling (10plex, Thermo) was performed as described by the manufacturer: Samples were dissolved in 100 mM triethylammonium bicarbonate (pH 8.5) and mixed with the respective TMT reagent dissolved in 41µl anhydrous acetonitrile for 1h at RT.

Labelled samples were combined prior to drying via vacuum centrifugation. Reverse phase HPLC (Thero Easy nLC1000) was performed on a precolumn (6 cm of 10 µm C18) and a self-pack 5 µm tip analytical columnd (12 cm of 5 µm C18). Samples were injected on a QExactive HF-X mass spectrometer (Thermo) operated in data-dependent mode (Full scan parameters: resolution of 70,000 across 350-2000 m/z, AGC 3e6, maximum IT 50 ms).

The top 15 precursor ions of each cycle were further analyzed with MS/MS (NCE of 24, dynamic exclusion of 30 seconds). Raw data files were searched using Proteome Discoverer (Thermo) and Mascot (version 2.4.1, Matrix Science). TMT data was normalized to the median of each TMT channel and only peptides with Mascot scores of ≥ 25 and isolation interference of ≤ 30 were required included.

[RP] was the sum of the isotopic abundances for all 40S ribosomal proteins, 60S ribosomal proteins, and 60S acidic ribosomal proteins with Total Peptide (TotPep) values greater than 2 and Unique Peptide (UniPep) greater than 1. [Total Protein] was the sum of isotopic abundances for all proteins with Total Peptide (TotPep) values greater than 2 and Unique Peptide (UniPep) greater than 1. [RP]/[Protein] values were normalized to the 1-hour time point in each experiment and subsequently log2 transformed.

The TMT data is summarized in Table S1.