來自瑞士蘇黎世大學(xué)分子生命科學(xué)研究所,、蘇黎世理工學(xué)院分子系統(tǒng)生物學(xué)研究所,、蘇黎世系統(tǒng)生理學(xué)和代謝疾病合作中心、瑞士弗雷德里希米歇爾生物醫(yī)藥研究所,、英國劍橋大學(xué)生物化學(xué)系,、英國劍橋大學(xué)威康信托/英國格登癌癥研究所及蘇黎世大學(xué)理學(xué)院等多家研究單位發(fā)明的一種大尺度鑒定miRNA靶點(diǎn)新方法,。Nature Methods網(wǎng)絡(luò)版9月12日在線出版了該新方法。
在過去幾年中,，隨著實(shí)驗(yàn)和計(jì)算方法的發(fā)展和完善,，miRNA靶點(diǎn)的鑒定和預(yù)測研究取得很大進(jìn)展。然而,，全面正確地鑒定miRNA的生物學(xué)相關(guān)靶點(diǎn)依然是個(gè)難點(diǎn),。恩加特納研究組發(fā)明了一種叫做“選擇性反應(yīng)監(jiān)測”（selected reaction monitoring，SRM）的方法,，該方法利用有針對性的質(zhì)譜技術(shù)實(shí)現(xiàn)了對miRNA-靶點(diǎn)互作的大尺度蛋白組學(xué)分析,，進(jìn)而對預(yù)測的靶點(diǎn)進(jìn)行驗(yàn)證。
恩加特納等人利用線蟲let-7突變體及其野生型為實(shí)驗(yàn)材料,。為了測定由于let-7突變體數(shù)量減少導(dǎo)致的蛋白含量的下降,，恩加特納等人對感興趣的let-7突變體和野生型的蛋白提取物進(jìn)行化學(xué)標(biāo)記，之后對其進(jìn)行SRM質(zhì)譜分析,，獲得了可靠的目標(biāo)蛋白的定量數(shù)據(jù),。該實(shí)驗(yàn)使用了一套let-7可能的靶點(diǎn)基因及一套對照基因。結(jié)果發(fā)現(xiàn),，161個(gè)蛋白中的29個(gè)蛋白的表達(dá)含量在突變體中發(fā)生變化,，這可能是由于let-7基因和不同的靶點(diǎn)互作結(jié)果——該結(jié)果得到了獨(dú)立下游實(shí)驗(yàn)包括遺傳互作、多聚核糖體分析和熒光素酶檢測的驗(yàn)證,。
SRM技術(shù)可以作為一個(gè)驗(yàn)證工具來進(jìn)一步探討對于靶點(diǎn)的理解,；此外，SRM也可能用來精細(xì)描述目標(biāo)蛋白質(zhì)組亞群,，促進(jìn)miRNA生物功能模型的研究,。（生物谷Bioon.com）
生物谷推薦英文摘要：
Nature Methods doi:10.1038/nmeth.1504
A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans
Marko Jovanovic1,2,3,11, Lukas Reiter1,2,3,10,11, Paola Picotti4, Vinzenz Lange4,5,10, Erica Bogan1,2, Benjamin A Hurschler6, Cherie Blenkiron7,8,10, Nicolas J Lehrbach7,8, Xavier C Ding6, Manuel Weiss1,2,3, Sabine P Schrimpf1,3, Eric A Miska7,8, Helge Gro?hans6, Ruedi Aebersold4,5,9 & Michael O Hengartner1,3
Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.
