神经元蛋白质磷酸化修饰分析方法的初步研究

A New Approach to Protein Phosphorylation Modification Analysis for Neuron

目的 建立一种对神经细胞中的磷酸化蛋白质进行分离分析的方法。方法 培养新生乳小鼠神经细胞 ,加入 32 P- Na H2 PO4 2 .78× 10 6 Bq/ ml,37℃孵育 1.5 h。刺激组分别加入表皮生长因子 (EGF) 2 0 ng/ m l或胰岛素10 0 nm ol/ L,对照组加入等量的培养基 ,作用不同时间后液氮终止反应。裂解细胞 ,用 Bio- Rad DC Protein Assaykit测定细胞蛋白质含量 ,双向电泳分离细胞蛋白。放射自显影。结果 1双向电泳自显影图谱显示近数十个蛋白质斑点 ,绝大部分集中于 p H偏酸性和中性区域 ,p H4 .6～ 6 .5 ,其相对分子质量主要介于 2 0× 10 3～ 130× 10 3之间。2 EGF、胰岛素刺激不同时间后虽出现有磷酸化蛋白质斑点的增加或消失 ,但更多表现为某些磷酸化蛋白质斑点灰度的增强或减弱。 3对比分析发现 ,放射自显影图谱中只有少数斑点出现在双向电泳 Coom assie Brilliant BlueR- 2 5 0染色图谱中 ,提示神经细胞中的大多数磷酸化蛋白质为低丰度蛋白。结论 采用本研究建立的双向电泳和放射自显影技术可对神经细胞中的蛋白质可逆磷酸化修饰状态进行较为全面、动态的分析 ,方法灵敏、特异性强 。

Objective To develop a new method for analysis of protein phosphorylation modification in cultured neuron. Methods Cultured neurons were pre-incubated in DMEM without sodium phosphate for 15 min to deplete the metabolic pools. Neurons were then labeled with orthophosphate (2.78×10 6 Bq/ml) for 1.5 h and stimulated by either insulin(100 nmol/L), EGF(20 nm/L) or saline for 0, 5, 20, 60, 120 min. Reactions were terminated by freezing neurons in liquid nitrogen prior to the solubilizing of them in a lysis buffer containing 8 mol/L urea, 4% CHAPS, 2% Bio-lyte,pH 3-10, 2 mmol/L TBP. Protein concentrations were determined with Bio-Rad DC Protein Assay kit. The 32P-labled lysates isoelectrically focused on IPG Drystrip pH 3-10 or pH 4-7 Linear gels were subsequently separated in second-dimensional SDS-PAGE. The dried gel was autoradiographed for 5days at -70 ℃ with an intensifying screen. Alternatively, the separated proteins were visualized by Coomassie Brilliant Blue(CBB) R250 straining. Results Autoradiography of the 2-DE-separated 32P-laebled neuron lysates revealed around 100 phosphoproteins. This phosphoprotein pattern was stable at 1.5 h after radiolabelling and did not vary significantly for up to 4 h further incubation in the absence of hormone. Most of the major proteins which are phosphorylated in response to insulin or EGF migrated with pH 4.6-6.5 and MW 20 000-130 000. Insulin and EGF induced similar but not identical patterns of protein phosphoryltion in neurons. Only a few phosphoproteins were abundant enough to be visualized by CBB straining, suggesting that abundance of these phosphoproteins is extremely low. Responses to both isulin and EGF are marked by more increased labeling of the constitutive phosphoproteins, compared with the appearance of new phosphoproteins. Conclusion This approach co-application of 32P-labeled with 2-DE separation and autoradiography has proven to be specific and sensitive in phosphoprotein analysis for neuron. It was valuable in functional proteomic analysis for protein phosphorylation modification during cellular signal transduction.