Fuller Brian, Stevens Stanley M, Sehnke Paul C, Ferl Robert J
Plant Molecular and Cellular Biology Program, Horticultural Sciences Department, University of Florida, Gainesville, FL 32601, USA.
Proteomics. 2006 May;6(10):3050-9. doi: 10.1002/pmic.200500729.
In this study, various proteomics-based methods were utilized to examine the 14-3-3 protein family in Arabidopsis thaliana. A protein extract was prepared from an Arabidopsis hypocotyl suspension culture and analyzed by two-dimensional gel electrophoresis and immunoblotting with a 14-3-3 monoclonal antibody that recognizes multiple Arabidopsis isoforms. Protein spots that cross-reacted with the monoclonal antibody as well as the surrounding spots were analyzed by high performance liquid chromatography in conjunction with electrospray-tandem mass spectrometry. Nine separate spots contained 14-3-3s and each spot contained multiple 14-3-3 isoforms. Every isoform observed was verified by the identification of at least one isoform-specific peptide. Further analysis by mass spectrometry revealed that the isoforms Chi, Upsilon, Omega, Phi, and Lambda were acetylated on their N termini and no non-acetylated N termini were recovered. These data, together with the distribution of isoforms and the confirmation that 14-3-3s are not complexed during urea denaturing isoelectric focusing, supports the conclusion that Arabidopsis 14-3-3s are acetylated in vivo and are significantly affected by other post-translational modifications.
在本研究中，运用了多种基于蛋白质组学的方法来检测拟南芥中的14-3-3蛋白家族。从拟南芥下胚轴悬浮培养物中制备蛋白质提取物，并通过二维凝胶电泳和用识别多种拟南芥异构体的14-3-3单克隆抗体进行免疫印迹分析。与单克隆抗体发生交叉反应的蛋白斑点以及周围的斑点通过高效液相色谱结合电喷雾串联质谱进行分析。九个不同的斑点含有14-3-3蛋白，并且每个斑点包含多种14-3-3异构体。观察到的每种异构体都通过鉴定至少一种异构体特异性肽段得到验证。通过质谱进一步分析发现，异构体Chi、Upsilon、Omega、Phi和Lambda在其N端被乙酰化，未检测到未乙酰化的N端。这些数据，连同异构体的分布以及在尿素变性等电聚焦过程中14-3-3蛋白不形成复合物的确认结果，支持了拟南芥14-3-3蛋白在体内被乙酰化且受到其他翻译后修饰显著影响的结论。
