Yeast proteomics microflow 23 min gradient DIA-MS

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.