Solid phase extraction cartridges

Yeast pellets were removed from −80 °C conditions and resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM Tris). The samples were then boiled at 100°C for 5 minutes and sonicated in a bath sonicator (Qsonica) with a 5-minute-long program of 20 seconds on, 10 seconds off. Methanol was added to each sample to 90% concentration, and the samples were centrifuged at 10,000 × g for 5 minutes to precipitate proteins. After precipitation, the supernatant was discarded from each sample, and the protein pellets were allowed to air dry for 7 minutes. The dried pellets were resuspended in digestion solution (8 M urea, 10 mM TCEP, 40 mM CAA, 100 mM Tris), and the samples were sonicated in the bath sonicator with the same program as above to facilitate resolubilization. LysC (Wako Chemicals) was added to each resolubilized sample in an estimated 50:1 protein/enzyme ratio. The samples were incubated on a rocker at room temperature for 4 hours before being diluted fourfold with 100 mM Tris. Trypsin (Promega) was added to each sample in an estimated 50:1 protein/enzyme ratio before they were incubated on a rocker at room temperature for 14 hours. Each sample was finally acidified with TFA to pH of 2, desalted by solid phase extraction cartridges (Phenomenex), and dried under vacuum (Thermo Scientific).