Sample preparation and phospho‐enrichment

Cell pellets were resuspended in lysis buffer containing 8 M urea, 0.1 M NH₄HCO₃, and 5 mM EDTA, and cells were disrupted by glass bead beating (five times for 5 min at 4°C, allowing the samples to cool down between cycles). The total protein amount from the pooled supernatants was determined by BCA Protein Assay Kit (Thermo, USA). Three milligrams of extracted yeast proteins was reduced with 5 mM TCEP at 37°C for 30 min and alkylated with 12 mM iodoacetamide at room temperature in the dark for 30 min. The samples were then diluted with 0.1 M NH₄HCO₃ to a final concentration of 1 M urea, and the proteins were digested with sequencing‐grade porcine trypsin (Promega, Switzerland) at a final enzyme:substrate ratio of 1:100 (w/w). Digestion was stopped by adding formic acid to a final concentration of 1%. Peptide mixtures were desalted using 3cc reverse‐phase cartridges (Sep‐Pak tC18, Waters, USA) and according to the following procedure: washing of column with one volume of 100% methanol, washing with one volume of 50% acetonitrile, washing with three volumes of 0.1% formic acid, loading acidified sample, reloading flow‐through, washing column with sample with three volumes of 0.1% formic acid, and eluting sample with two volumes of 50% acetonitrile in 0.1% formic acid. Peptides were dried using a vacuum centrifuge and resolubilized in 100 µl of 0.1% formic acid. Retention time standard peptides (iRT‐Kit, Biognosys, Switzerland) were spiked into the samples before they were analyzed by LC‐MS for total protein abundance (“non‐enriched samples”). The remaining 95 µl was supplemented with 300 µl of an overnight recrystallized and cleared‐up phthalic acid solution prepared by carefully dissolving 5 g of phthalic acid in 50 ml of 80% acetonitrile before adding 1.75 ml of trifluoroacetic acid. The samples were then enriched for phosphopeptides by incubating for 1 h under rotation with 1.25 mg of TiO₂ resin (GL Sciences, Japan) preequilibrated twice with 500 µl of methanol, and twice with 500 µl of phthalic acid solution. Peptides bound to the TiO₂ resin were then washed twice with 500 µl phthalic acid solution, then twice with 80% acetonitrile with 0.1% formic acid, and finally twice with 0.1% formic acid. The phosphopeptides were eluted from the beads twice with 150 µl of 0.3 M ammonium hydroxide at pH 10.5 and immediately acidified again with 50ul of 5% trifluoroacetic acid to reach about pH 2.0. The enriched phosphopeptides were desalted on microspin columns (The Nest Group, USA) with the protocol described above, dried using a vacuum centrifuge, and resolubilized in 10 µl of 0.1% formic acid. Again, retention time standard peptides (iRT‐Kit, Biognosys, Switzerland) were spiked into the samples before they were analyzed by LC‐MS for total peptide abundance (“phosphopeptides samples”).