人骨骼肌双向电泳条件优化及部分蛋白质鉴定  

Optimizing protein solubility for two-dimensional gel electrophoresis analysis of human skeletal muscle

作　　者：贾林芝[1] 蒋明[2] 徐雷[3] 顾玉东[3] 张志鸿[2] 

机构地区：[1]华东师范大学生命科学学院,上海200062 [2]复旦大学生命科学学院,上海200433 [3]复旦大学附属华山医院手外科研究所,上海200040

出　　处：《西北大学学报（自然科学版）》2007年第2期248-252,共5页Journal of Northwest University（Natural Science Edition）

基　　金：国家重点基础研究发展规划基金资助项目(2001CB510202);国家自然科学基金资助项目(30370380;30070205)

摘　　要：目的建立和优化人骨骼肌组织双向电泳模型,为深入探讨骨骼肌萎缩的分子机制提供方法上的保障。方法使用两性离子去垢剂CHAPS和SB3-10,并配合不同浓度的离液剂抽提骨骼肌蛋白质,并通过双向电泳对蛋白质进行分离,用基质辅助激光解析电离飞行时间质谱(MALDI-TOF)对选取的蛋白质进行鉴定。结果优化过的双向电泳模型可在一张凝胶上分离800多个蛋白质点,通过MALDI-TOF鉴定出HUMANserine/threonine protein kinase KKIALRE等4种低丰度蛋白质。结论多种去垢剂和离液剂的组合使用才能获得对组织中蛋白质较好的抽提效果。获得的人骨骼肌双向电泳图谱是对人类骨骼肌蛋白质组学数据库的有益补充,为使用比较蛋白质组学方法筛选人肌病关键蛋白质提供了实践和理论基础。Aim To provide technological basis for investigating the molecular mechanism of muscle atrophy, it is necessary to establish the model of two - dimensinal gel electrophoresis （2-DE） of human skeletal muscle and optimize the method of extracting proteins. Methods Human skeletal muscle proteins were extracted by using two zwitterionic detergents （3-[ （3-cholamidopropyl） dimethylamino 1-1-propane sulfonate, CHAPS; N-decyl-N-N＇- dimethyl-3-ammonio-1-propane sulfonate, SB3-10） in different concentration of chaotrops. Proteins separated by 2- DE were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry. Results More than 800 protein spots were detected on each 2-D gel. 4 low abundant proteins were excised and characterized as HUMAN serine/threonine protein kinase KKIALRE and so on. Conclusion Rational concentrations of detergent and chaotrope result in superior solubility of protein. The initial two-dimensional gel reference maps for normal skeletal muscle of the patient suffering from brachial plexus root avulsion were established. This data should be a valuable resource for screening key protein of skeletal muscle pathologies.

关 键 词：人骨骼肌 双向电泳 MALDI-TOF 去垢剂 离液剂 

分 类 号：Q503[生物学—生物化学]