Identification of phosphorylation site of Npas4 by LC-MS/MS
Description
To identify the phosphorylation sites of Npas4, GST-Npas4-390-489 aa, GST-Npas4 490-597 aa or GST-Npas4 598-701 aa was expressed in COS7 cells with or without the coexpression of MAP2K1-CA. Cells were lysed in lysis buffer containing protease inhibitor cocktail and PhosStop, and sonicated 3 times for 5 sec. After centrifugation at 16,000×g at 4°C for 10 min, the soluble supernatant was incubated in 30 µl of glutathione–Sepharose 4B beads for 1 hr at 4°C with rotation. The beads were then washed three times with lysis buffer and an additional three times with wash buffer. The bound proteins were extracted from the beads using urea solution, reduced via incubation in 5 mM dithiothreitol for 30 min, and alkylated using 10 mM iodoacetamide for 1 h in the dark. The proteins were digested with Trypsin/Lys-C or Glu-C/Asp-N. Demineralization was performed using SPE c-tips (Nikkyo Technos) according to the manufacturer’s instructions. The peptides were analyzed by LC−MS using an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific Inc). Dual-phosphorylated peptide DLVCTPPYTPHQPGGCAFLFSLHEPFQTHLPPPSSSLQE, containing T423 and T427 phosphorylation sites, was identified from digested fragments of GST-Npas4-390-489 aa; LPPSPSSPGNGDCTLLALAQLR, containing S577 and S580 phosphorylation sites, was identified from digested fragments of GST-Npas4 490-597 aa; and GLLTPEASPVKQSFFHYTEKE, containing T611 and S615 phosphorylation sites, was identified from digested fragments of GST-Npas4 598-701 aa. Selected ion monitoring (SIM) analysis revealed that the amount of these dual-phosphorylation was significantly increased by coexpression with MAP2K1-CA.