Ubiquitination proteome profiling of Candida albicans

Protein extraction The two groups (C, CS) were treated in same way, sample was first grinded with liquid nitrogen, then the powder was transferred to a 5-mL centrifuge tube and sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer (including 1% TritonX-100, 10 mM dithiothreitol, and 1% protease inhibitor cocktail, 50 μM PR-619, 3 μM TSA, 50 mM NAM, 2 mM EDTA, and 1% phosphatase inhibitor for phosphorylation.). An equal volume of Tris-saturated phenol (pH 8.0) was added. Then, the mixture was further vortexed for 5 min. After centrifugation (4 °C, 10 min, 5 000g), the upper phenol phase was transferred to a new centrifuge tube. Proteins were precipitated by adding at least four volumes of ammonium sulfate-saturated methanol and incubated at -20 °C for at least 6 h. After centrifugation at 4 °C for 10 min, the supernatant was discarded. The remaining precipitate was washed with ice-cold methanol, followed by ice-cold acetone for three times. The protein was redissolved in 8 M urea and the protein concentration was determined with BCA kit according to the manufacturer’s instructions.