Abstract
A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Both label types can be placed within a single protein RISQ standard (recombinant isotope-labeled and selenium quantified) or can be distributed over two types of related RSQ and RIQ standards for the same target protein (recombinant selenium quantified and recombinant isotope-labeled and quantified). The combination of cell-free synthesis as production method with ICP-MS and ESI-MS/MS as detection methods results in protein standards, which are quantified at an outstanding level of accuracy.
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Acknowledgements
We are thankful to L. Adler, M. Schilling, and U. Klingmüller for numerous valuable discussions and to M. Boehm and D. Winter for support in nanoUPLC-ESI-MS analysis. We thank also W. Konopka for training and support in molecular biology techniques. We gratefully acknowledge funding by the SBCancer network in the Helmholtz Alliance on Systems Biology and the German Federal Ministry of Education and Research (BMBF)-funded MedSys-Network LungSys and the Virtual Liver Network. This research was undertaken within the project HLT05 (EMRP Researcher Grant). The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.
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Konopka, A., Zinn, N., Wild, C., Lehmann, W.D. (2014). Preparation of Heteroelement-Incorporated and Stable Isotope-Labeled Protein Standards for Quantitative Proteomics. In: Martins-de-Souza, D. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 1156. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0685-7_23
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DOI: https://doi.org/10.1007/978-1-4939-0685-7_23
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