Combined use of RNAi and quantitative proteomics to study gene function in Drosophila
Combined use of RNAi and quantitative proteomics to study gene function in Drosophila
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DOI：
10.1016/j.molcel.2008.07.018
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发表时间：
2008-09-05
期刊：
影响因子：
16
通讯作者：
Mann, Matthias
中科院分区：
文献类型：
--
作者：
Bonaldi, Tiziana;Straub, Tobias;Mann, Matthias
RNA干扰是研究基因功能的有效方法，经常与微阵列分析相结合。在这里，我们介绍了一个类似的技术，在蛋白质水平上，同时应用稳定同位素标记的氨基酸在细胞培养（SILAC）和RNA干扰（RNAi）的果蝇SL 2细胞。在敲除ISWI（不同染色质重塑复合物的ATP水解马达）后，我们获得了超过4，000种蛋白质的定量蛋白质组。ISWI本身减少10倍，如SILAC所定量。数百种蛋白质被显著调节并聚集成不同的功能类别。Acf-1，ISWI的直接相互作用伙伴，在蛋白质水平上被严重耗尽，但不是转录水平;这最有可能是由于蛋白质稳定性降低。我们发现转录组和蛋白质组的变化之间几乎没有总体相关性，许多蛋白质的变化并不伴随着信息的变化。然而，相关性高，为那些基因表达谱显着改变。
RNA interference is a powerful way to study gene function and is frequently combined with microarray analysis. Here we introduce a similar technology at the protein level by simultaneously applying Stable Isotope Labeling by Amino acids in Cell culture (SILAC) and RNA interference (RNAi) to Drosophila SL2 cells. After knockdown of ISWI, an ATP-hydrolyzing motor of different chromatin remodeling complexes, we obtained a quantitative proteome of more than 4,000 proteins. ISWI itself was reduced 1 0-fold as quantified by SILAC. Several hundred proteins were significantly regulated and clustered into distinct functional categories. Acf-1, a direct interaction partner of ISWI, is severely depleted at the protein, but not the transcript, level; this most likely results from reduced protein stability. We found little overall correlation between changes in the transcriptome and proteome with many protein changes unaccompanied by message changes. However, correlation was high forthose mRNAs that changed significantly by microarray.