Protemoic analysis of Yeast during retenostat cultivation

Yeast was cultured in 2 parallel retentostats reaching near-zero growth. 5 time points were sampled from each reactor and labeled with 6-plex TMT. Pooled samples were fractionated with SCX. 2+ and 3+ fractions were run individually on a 3 h gradient of RP-UHPLC and sprayed into a Q-Exactive mass spectrometer. The raw data obtained, were initially processed with proteome discoverer 1.3 (Thermo Fisher, Bremen, Germany). The created peak lists were searched with Mascot (Matrix Science, Version 2.3) using the SGD database (containing 5779 entries) and the following parameters: 50 p.p.m. precursor mass tolerance and 0.05 Da fragment ion tolerance. Up to two missed cleavages were accepted, oxidation of methionine was set up as variable modification whereas cysteine carbamidomethylation and the TMT label on lysines and the N-terminus as fixed modifications. The resulting .dat files were exported and filtered for <1% false discovery rate at peptide level using in-house developed software Rockerbox (version 2.0.1) utilizing the percolator algorithm (van den Toorn et al, 2011). The filtering for significant changing proteins was done with the isobar software and a significance threshold of p ≤ 0.05. Isobar employs robust statistics that captures spectra and sample variability into a single statistical framework which is described in (Breitwieser et al, 2011).