Identification of CBP-interacting proteins by LC-MS/MS

To concentrate and identify transcriptional factors that are associated with reward-related learning and memory, we injected cocaine into mice and placed them into the conditioned place preference (CPP) test chamber for 30 min. Brains from 10 mice were rapidly removed and coronal slices (1 mm) were produced using a mouse brain slicer matrix (Brain Science Idea). The striatum and NAc (approximately +1.54 mm from Bregma) were collected using 3 mm diameter biopsy punch (Miltex). Collected tissues were homogenized with Hypotonic buffer (Nuclear Extract Kit, Active Motif) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (PhosStop, Roche) using potter homogenizer and incubated on ice for 15 min. The lysate was centrifuged at 800×g for 10 min at 4°C. The pellet was resuspended with Hypotonic buffer and incubated on ice for 15 min. After addition of a detergent and vortexing for 10 sec, the lysate was centrifuged at 16,000×g for 2 min. The nuclear pellet was extracted in lysis buffer [20 mM Tris/HCl, 1 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail and PhosStop, pH 7.5], and then sonicated 3 times for 10 sec. The lysate was centrifuged at 16,000×g for 10 min at 4°C. The supernatant was used as the nuclear extracts. A total of 250 pmol of GST or GST-CREB binding protein (CBP)-N-terminal transactivation domain (N-TAD), immobilized on glutathione Sepharose 4B beads, was incubated with the nuclear extracts 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) to remove the detergent from the beads. 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 hr in the dark. The proteins were digested with Trypsin/Lys-C (Promega). 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). To select proteins that specifically interact with CBP, the proteins from the control GST column were removed from the data. High-abundance proteins (such as keratin and ribosomal protein) were also excluded. We identified more than 400 proteins that specifically interact with GST-CBP-N-TAD but not with GST.

为筛选并鉴定与奖赏相关学习记忆相关的转录因子，我们向小鼠体内注射可卡因，并将其置于条件位置偏好（conditioned place preference, CPP）实验箱中放置30分钟。快速摘取10只小鼠的脑组织，使用小鼠脑切片模具（Brain Science Idea）制作厚度为1mm的冠状脑片。通过直径3mm的活检打孔器（Miltex）采集距前囟（Bregma）约+1.54mm的纹状体与伏隔核（NAc）。将采集的组织置于含有蛋白酶抑制剂混合物（Roche）与磷酸酶抑制剂混合物（PhosStop, Roche）的低渗缓冲液（细胞核提取试剂盒，Active Motif）中，使用波特氏匀浆器进行匀浆，冰浴15分钟。将裂解液于4℃、800×g条件下离心10分钟，沉淀重悬于低渗缓冲液中并再次冰浴15分钟。加入去污剂并涡旋振荡10秒后，将裂解液于4℃、16,000×g条件下离心2分钟。将得到的细胞核沉淀用裂解液[20 mM Tris/HCl、1 mM EDTA、150 mM NaCl、1% NP-40、蛋白酶抑制剂混合物及PhosStop，pH 7.5]重悬，随后超声破碎3次，每次10秒。将裂解液于4℃、16,000×g条件下离心10分钟，上清液即为细胞核提取物。将固定于谷胱甘肽琼脂糖4B磁珠上的总计250 pmol的谷胱甘肽S-转移酶（glutathione S-transferase, GST）或GST-CREB结合蛋白（CBP）N端反式激活结构域（N-TAD），与细胞核提取物于4℃下旋转孵育1小时。随后用裂解液洗涤磁珠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次以去除磁珠上的去污剂。使用尿素溶液从磁珠上洗脱结合的蛋白质，将其置于5 mM二硫苏糖醇中孵育30分钟以还原，随后于暗处用10 mM碘乙酰胺烷基化1小时。使用胰蛋白酶/赖氨酸C（Promega）对蛋白质进行酶解。按照制造商说明书，使用SPE c-tips（Nikkyo Technos）进行脱盐处理。采用Orbitrap Fusion质谱仪（Thermo Fisher Scientific Inc）通过液相色谱-质谱联用（LC−MS）对肽段进行分析。为筛选与CBP特异性结合的蛋白质，我们从数据中移除了对照GST磁珠结合的蛋白质，并排除了高丰度蛋白质（如角蛋白与核糖体蛋白）。最终我们鉴定出超过400种可与GST-CBP-N-TAD特异性结合、但不与GST结合的蛋白质。