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A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans


Jovanovic, M; Reiter, L; Picotti, P; Lange, V; Bogan, E; Hurschler, B A; Blenkiron, C; Lehrbach, N J; Ding, X C; Weiss, M; Schrimpf, S P; Miska, E A; Grosshans, H; Aebersold, R; Hengartner, M O (2010). A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans. Nature Methods, 7(10):837-842.

Abstract

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.

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2010
Deposited On:05 Nov 2010 09:49
Last Modified:05 Apr 2016 14:18
Publisher:Nature Publishing Group
ISSN:1548-7091
Publisher DOI:10.1038/nmeth.1504
PubMed ID:20835247
Permanent URL: http://doi.org/10.5167/uzh-36726

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