Ms approved trypsin

Phosphoproteome method was performed as described previously (Borisova et al., 2017 (link)). Proteins were precipitated in fourfold excess of ice-cold acetone and subsequently re-dissolved in denaturation buffer (6 M urea, 2 M thiourea in 10 mM HEPES pH 8.0). Cysteines were reduced with 1 mM dithiothreitol (DTT) and alkylated with 5.5 mM chloroacetamide. Proteins were digested with endoproteinase Lys-C (Wako Chemicals) and MS-approved trypsin (Serva). Protease digestion was stopped by addition of TFA to 0.5% and precipitates were removed by centrifugation. Peptides were purified using reversed-phase Sep-Pak C18 cartridges (Waters) and eluted in 50% acetonitrile, 0.1% TFA. Phosphopepetides were enriched by incubation with titanium dioxide spheres (GL Sciences) for 2 × 1h with rotation. They were eluted sequentially with 5% NH4OH and 10% NH4OH 25% ACN, and vacuum concentrated to remove NH4OH. Peptides were separated into ten fractions using micro-column-based SCX and desalted on reversed phase C18 StageTips.

Aliquots containing 50 µg of dissolved protein were taken from each sample and diluted with 100 mM ABC to a final concentration of 0.42% DOC. The obtained samples were digested with 1 µg of MS approved trypsin (Serva Electrophoresis GmbH, Heidelberg, Germany) at 30 °C for 12 h. The DOC was then precipitated by acidification with trifluoroacetic acid (TFA) and subsequent centrifugation (15,000 rpm, 20 min, 4 °C).