Bayer Roman G, Stael Simon, Teige Markus
Department of Ecogenomics and Systems Biology, University of Vienna, Althanstr. 14, A-1090, Vienna, Austria.
Methods Mol Biol. 2015;1295:211-23. doi: 10.1007/978-1-4939-2550-6_16.
Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.
蛋白质组的详细知识对于推动生物科学发展至关重要。低丰度蛋白质受到许多生物学家的特别关注，因为它们包括例如那些参与信号转导的蛋白质。最近的技术进步使得通过质谱（MS）鉴定蛋白质的灵敏度大幅提高。然而，蛋白质丰度的动态范围仍然是一个基本问题，限制了在复杂蛋白质组中检测低丰度蛋白质。由于存在其他丰度高出几个数量级的蛋白质，这些蛋白质通常会在鸟枪法质谱实验中逃脱检测。因此，需要特定的富集策略来克服基于质谱的蛋白质发现的这一技术限制。我们一直在寻找新型信号转导蛋白，更具体地说是激酶和钙结合蛋白，在此我们描述了从豌豆和拟南芥分离的叶绿体中富集这些低丰度蛋白质的不同方法，以便随后通过质谱进行蛋白质组分析。这些方法可以扩展到包括其他信号转导蛋白，并针对不同的细胞器。
