Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
Combining RNA Interference Mutants and Comparative Proteomics to Identify Protein Components and Dependences in a Eukaryotic Flagellum
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DOI：
10.1074/jbc.m808859200
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发表时间：
2009-02-27
影响因子：
4.8
通讯作者：
Gull, Keith
中科院分区：
文献类型：
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作者：
Portman, Neil;Lacomble, Sylvain;Gull, Keith
来自生物体如布氏锥虫的真核鞭毛可以被分离，并且它们的蛋白质组分可以通过质谱法鉴定。在这里，我们使用了一种比较的方法，利用二维差异凝胶电泳和同量异位素标签的相对和绝对定量揭示鞭毛结构的蛋白质组分通过诱导RNA干扰突变消融。通过这种方法，我们确定了旁鞭毛杆（PFR）的20种新成分。使用表位标记，我们通过免疫荧光验证了这些表位的一个子集存在于PFR中。PFR队列的生物信息学分析揭示了一些组分之间可能的钙/钙调蛋白调节/信号传导联系。我们将RNA干扰突变体/比较蛋白质组学分析扩展到PFR蛋白质组的单个新组分，表明该方法具有揭示队列内亚组之间依赖性的能力。
Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.