Identification of phosphorylation site of Npas4 by LC-MS/MS

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 [20 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (PhosStop, Roche), pH 7.5], 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 (GE Healthcare) 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 (20 mM Tris/HCl, 1 mM EDTA, and 150 mM NaCl, pH 7.5). 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 (Promega) or Glu-C (Promega)/Asp-N (FUJIFILM Wako). Demineralization was performed using SPE c-tips 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. These results suggest that Npas4 is phosphorylated by MAPK at T423, T427, S577, S580, T611 and S615 sites.

为鉴定神经元PAS结构域蛋白4（Neuronal PAS domain protein 4, Npas4）的磷酸化位点，将携带谷胱甘肽S-转移酶（Glutathione S-transferase, GST）标签的Npas4截短体GST-Npas4-390-489位氨基酸、GST-Npas4 490-597位氨基酸或GST-Npas4 598-701位氨基酸，在是否共表达组成型激活型丝裂原活化蛋白激酶激酶1（MAP2K1-CA）的条件下，于COS7细胞中进行表达。细胞经裂解缓冲液[20 mM Tris/HCl、1 mM EDTA、150 mM NaCl、1% NP-40、蛋白酶抑制剂混合物（Roche公司）及磷酸酶抑制剂混合物（PhosStop, Roche公司），pH 7.5]裂解后，以超声处理3次，每次5秒。4℃下以16,000×g离心10分钟后，取可溶性上清液与30 μl谷胱甘肽琼脂糖4B磁珠（GE Healthcare公司）于4℃旋转孵育1小时。随后用裂解缓冲液洗涤磁珠3次，再用洗涤缓冲液（20 mM Tris/HCl、1 mM EDTA、150 mM NaCl，pH 7.5）额外洗涤3次。结合于磁珠的蛋白经尿素溶液洗脱后，以5 mM二硫苏糖醇孵育30分钟进行还原，再于暗处用10 mM碘乙酰胺烷基化1小时。蛋白分别用胰蛋白酶/赖氨酸C（Promega公司）或谷氨酸蛋白酶C（Glu-C, Promega公司）/天冬氨酸蛋白酶N（Asp-N, FUJIFILM Wako公司）进行酶解。根据制造商说明书，采用固相萃取C18小柱（SPE c-tips）进行脱盐处理。采用Orbitrap Fusion质谱仪（赛默飞世尔科技公司，Thermo Fisher Scientific Inc.）通过液相色谱-质谱联用法（LC-MS）对肽段进行分析。从GST-Npas4-390-489位氨基酸的酶解片段中，鉴定得到包含T423与T427磷酸化位点的双磷酸化肽段DLVCTPPYTPHQPGGCAFLFSLHEPFQTHLPPPSSSLQE；从GST-Npas4 490-597位氨基酸的酶解片段中，鉴定得到包含S577与S580磷酸化位点的肽段LPPSPSSPGNGDCTLLALAQLR；从GST-Npas4 598-701位氨基酸的酶解片段中，鉴定得到包含T611与S615磷酸化位点的肽段GLLTPEASPVKQSFFHYTEKE。选择离子监测（SIM）分析结果显示，与MAP2K1-CA共表达后，这些双磷酸化肽段的含量显著升高。上述结果表明，Npas4可被丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinase, MAPK）在T423、T427、S577、S580、T611及S615位点磷酸化。