Sarry Jean-Emmanuel, Kuhn Lauriane, Le Lay Pascaline, Garin Jérôme, Bourguignon Jacques
Laboratoire de Physiologie Cellulaire Végétale, Department Reponse & Dynamique Cellulaires, CEA, Grenoble, France.
Electrophoresis. 2006 Feb;27(2):495-507. doi: 10.1002/elps.200500561.
In an effort to determine the best extraction procedure compatible with the high-reproducible 2-DE, different methods of soluble protein extraction from Arabidopsis cell culture suspensions grown in Gamborg B5 medium were tested. A reference 2-DE map was established for this soluble extract revealing 1184 spots. The most abundant protein spots were excised, trypsin-digested, and mass spectra obtained via MALDI-TOF and/or LC coupled to ESI-MS. Three hundred and thirty one proteins were identified and their functions were defined based on sequence comparisons and classified in different protein families. In order to analyze the impact of culture medium on the Arabidopsis proteome, we performed the 2-DE map from Arabidopsis cell suspensions cultured in another growth medium Murashige and Skoog (M-S) and 327 major spots were identified. Using PDQuest imaging analysis, significant increases in the amount of several housekeeping enzymes, stress/defense proteins, and heat shock proteins were found in M-S medium. Modified expression of certain proteins and detection of new isoforms involved in nitrate assimilation, nitrogen, and sulfur metabolism were also observed in the M-S medium. This study provides the first 2-DE maps of the soluble proteome of Arabidopsis cell suspensions. The comparative analysis of the Arabidopsis proteome in respect to different nutrient supplies shows that the culture medium may significantly influence the expression pattern of major soluble proteins in Arabidopsis cells. This work also constitutes an important step for further proteomic analysis concerning cell responses to abiotic or biotic stresses.
为了确定与高重复性双向电泳兼容的最佳提取方法，我们测试了从在甘博格B5培养基中生长的拟南芥细胞培养悬浮液中提取可溶性蛋白质的不同方法。为这种可溶性提取物建立了一张参考双向电泳图谱，显示出1184个斑点。切除最丰富的蛋白质斑点，用胰蛋白酶消化，然后通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）和/或液相色谱与电喷雾电离质谱（ESI-MS）联用获得质谱。鉴定出331种蛋白质，并根据序列比较确定其功能，将它们归类到不同的蛋白质家族中。为了分析培养基对拟南芥蛋白质组的影响，我们对在另一种生长培养基——穆拉希格和斯科格培养基（M-S）中培养的拟南芥细胞悬浮液进行了双向电泳图谱分析，鉴定出327个主要斑点。使用PDQuest图像分析发现，在M-S培养基中几种管家酶、应激/防御蛋白和热休克蛋白的含量显著增加。在M-S培养基中还观察到某些蛋白质的表达发生改变以及参与硝酸盐同化、氮和硫代谢的新同工型的检测。本研究提供了拟南芥细胞悬浮液可溶性蛋白质组的首张双向电泳图谱。关于不同营养供应对拟南芥蛋白质组的比较分析表明，培养基可能会显著影响拟南芥细胞中主要可溶性蛋白质的表达模式。这项工作也为进一步开展关于细胞对非生物或生物胁迫反应的蛋白质组学分析迈出了重要一步。
