Journal article
Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click–SILAC approach
Journal of proteomics, Vol.77, pp.e1-e10
12/21/2012
DOI: 10.1016/j.jprot.2012.08.019
Abstract
Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography–tandem mass spectrometry. This combined Click–SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click–SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.
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► Click–SILAC is a method for identifying newly synthesized proteins. ► Click–SILAC identifies newly synthesized proteins after short-term pulse-labelling. ► Click–SILAC identified 72 newly synthesized proteins. ► YB-1 regulates the translation of proteins involved in diverse biological pathways. ► Down‐regulation of YB-1 increases overall mRNA translation in TC32 cells.
Details
- Title: Subtitle
- Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click–SILAC approach
- Creators
- Syam Prakash Somasekharan - Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada BC V6T 1Z4Nikolay Stoynov - Department of Biochemistry and Molecular Biology and Centre for High-Throughput Biology, University of British Columbia, Vancouver, Canada BC V6T 1Z4Barak Rotblat - Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada BC V6T 1Z4Gabriel Leprivier - Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada BC V6T 1Z4Jason D Galpin - Department of Anesthesiology, Pharmacology and Therapeutics, Life Science Centre, University of British Columbia, Vancouver, Canada BC V6T 1Z3Christopher A Ahern - Department of Anesthesiology, Pharmacology and Therapeutics, Life Science Centre, University of British Columbia, Vancouver, Canada BC V6T 1Z3Leonard J Foster - Department of Biochemistry and Molecular Biology and Centre for High-Throughput Biology, University of British Columbia, Vancouver, Canada BC V6T 1Z4Poul H.B Sorensen - Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada BC V6T 1Z4
- Resource Type
- Journal article
- Publication Details
- Journal of proteomics, Vol.77, pp.e1-e10
- Publisher
- Elsevier B.V
- DOI
- 10.1016/j.jprot.2012.08.019
- ISSN
- 1874-3919
- eISSN
- 1876-7737
- Language
- English
- Date published
- 12/21/2012
- Academic Unit
- Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984070279802771
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