Saccharomyces cerevisiae (BY4743 rendered prototrophic with a plasmid encoding for HIS3, LEU2 and URA3 [25]) were grown to exponential phase in minimal synthetic nutrient media. Proteins were extracted by bead beating for 5min at 1500rpm in 8M urea/0.1M ammonium bicarbonate. Proteins were reduced with 5mM dithiothreitol, alkylated with 10mM iodoacetamide. The sample was diluted to 1.5M urea/0.1M ammonium bicarbonate before the proteins were digested overnight with Trypsin (1:30 Trypsin to total protein ratio). Peptides were cleaned-up with 96-well MacroSpin plates (Nest Group) and iRT peptides (Biognosys AG) were spiked in. The digested peptides were analysed on a nanoAcquity (Waters) coupled to a TripleTOF 6600 (Sciex). Peptides were separated with a 23 minute non-linear gradient (4% Acetonitrile/0.1 % formic acid to 36% Acetonitrile/0.1% formic acid) on a Waters HSS T3 column (150mm x 300μm, 1.8μm Particles) with a 5μl/min flow rate. The DIA method consisted of an MS1 scan from m/z 400 to m/z 1250 (50ms accumulation time) and 40 MS2 scans (35ms accumulation time) with variable precursor isolation width covering the mass range from m/z 400 to m/z 1250.

This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001134), the UK Medical Research Council (FC001134), and the Wellcome Trust (FC001134), and received specific funding from the BBSRC (BB/N015215/1 and BB/N015282/1).