| Note |
For dephosphorylation, 100 μg of proteins were reacted with 1 μL of TSAP (1 MBU/μL) at 37 °C for 1 h, and TSAP was inactivated by heating to 75 °C for 30 min. For in vitro kinase reaction, each 100 μg of dephosphorylated proteins (1 μg/μL) from the supernatant or the pellet fraction was reacted with 1 μL of each recombinant kinase (0.5 μg/μL) or distilled water as a control at 37 °C in kinase reaction buffer (40 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 1 mM ATP) for 3 h. The reaction was stopped by heating to 95 °C for 5 min. The protein aliquot was diluted with 8 M urea in 0.2 mM Tris-HCl (pH 9.0). After protein reduction/alkylation, Lys-C/trypsin digestion (1/100 w/w) was performed. Phosphopeptides were enriched by TiO2-based hydroxy acid-modified metal oxide chromatography (HAMMOC) with elution by 0.5% piperidine. Phosphopeptides were desalted by StageTips and suspended in the loading buffer (0.5% TFA and 4% ACN) for subsequent nanoLC−MS/MS analyses. The experimental process with either supernatant or pellet fraction was repeated five times for each kinase. |