期刊
MOLECULAR SYSTEMS BIOLOGY
卷 8, 期 -, 页码 -出版社
WILEY
DOI: 10.1038/msb.2012.63
关键词
nuclear export; ribosome assembly; selected reaction monitoring mass spectrometry; targeted proteomics
资金
- Swiss National Science Foundation
- ETH Zurich
- Promedica Stiftung
- Marie Curie Reintegration Grant [ERG-277147]
- European Research Council [EURIBIO260676]
Construction and intracellular targeting of eukaryotic pre-ribosomal particles involve a multitude of diverse transiently associating trans-acting assembly factors, energy-consuming enzymes, and transport factors. The ability to rapidly and reliably measure co-enrichment of multiple factors with maturing pre-ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise contribution of >50 energy-consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity-capture, and selected reaction monitoring mass spectrometry (SRM-MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre-60S particles after nuclear export. We uncovered assembly factors that travel with pre-60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre-60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport. Molecular Systems Biology 8: 628; published online 4 December 2012; doi:10.1038/msb.2012.63
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