Proteomic analysis of the 14-3-3 family in Arabidopsis
Proteomic analysis of the 14-3-3 family in Arabidopsis
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DOI：
10.1002/pmic.200500729
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发表时间：
2006-05-01
期刊：
影响因子：
3.4
通讯作者：
Ferl, Robert J.
中科院分区：
文献类型：
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作者：
Fuller, Brian;Stevens, Stanley M., Jr.;Ferl, Robert J.
本研究利用多种蛋白质组学方法对拟南芥14-3-3蛋白家族进行了研究。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免疫印迹法用识别多种拟南芥同种型的14-3-3单克隆抗体进行分析。通过高效液相色谱结合电喷雾串联质谱法分析与单克隆抗体交叉反应的蛋白质斑点以及周围斑点。9个单独的斑点含有14-3-3s，每个斑点含有多个14-3-3同种型。通过鉴定至少一种亚型特异性肽来验证观察到的每种亚型。通过质谱法的进一步分析显示，同种型Chi、Uppi、Omega、Phi和Lambda在其N末端上被乙酰化，并且没有回收到非乙酰化的N末端。这些数据，连同异构体的分布和尿素变性等电聚焦过程中14-3-3s不复合的确认，支持拟南芥14-3-3s在体内乙酰化并受到其他翻译后修饰的显著影响的结论。
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.