Abstract
Since gene expression is controlled on many different levels in a cell, capturing a comprehensive snapshot of all regulatory processes is a difficult task. One possibility to monitor effective changes within a cell is to directly quantify changes in protein synthesis, which reflects the accumulative impact of regulatory mechanisms on gene expression. Pulsed stable isotope labeling by amino acids in cell culture (pSILAC) has been shown to be a viable method to investigate de novo protein synthesis on a proteome-wide scale (Schwanhausser et al., Proteomics 9:205–209, 2009; Selbach et al., Nature 455:58–63, 2008). One application of pSILAC is to study the regulation of protein expression by microRNAs. Here, we describe how pSILAC in conjunction with shotgun mass spectrometry can assess differences in the protein profile between cells transfected with a microRNA and non-transfected cells.
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References
Aebersold, R., and Mann, M. (2003) Mass spectrometry-based proteomics, Nature 422, 198–207.
Bantscheff, M., Schirle, M., Sweetman, G., Rick, J., and Kuster, B. (2007) Quantitative mass spectrometry in proteomics: a critical review, Anal Bioanal Chem 389, 1017–1031.
Ong, S. E., and Mann, M. (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC), Nat Protoc 1, 2650–2660.
Schwanhausser, B., Gossen, M., Dittmar, G., and Selbach, M. (2009) Global analysis of cellular protein translation by pulsed SILAC, Proteomics 9, 205–209.
Selbach, M., Schwanhausser, B., Thierfelder, N., Fang, Z., Khanin, R., and Rajewsky, N. (2008) Widespread changes in protein synthesis induced by microRNAs, Nature 455, 58–63.
Cox, J., Matic, I., Hilger, M., Nagaraj, N., Selbach, M., Olsen, J. V., and Mann, M. (2009) A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics, Nat Protoc 4, 698–705.
Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat Protoc 1, 2856–2860.
Rappsilber, J., Mann, M., and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips, Nat Protoc 2, 1896–1906.
Acknowledgments
The authors would like to thank Björn Schwanhäusser for providing data and templates for the shown figures.
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Ebner, O.A., Selbach, M. (2011). Whole Cell Proteome Regulation by MicroRNAs Captured in a Pulsed SILAC Mass Spectrometry Approach. In: Hobman, T., Duchaine, T. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 725. Humana Press. https://doi.org/10.1007/978-1-61779-046-1_20
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DOI: https://doi.org/10.1007/978-1-61779-046-1_20
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