Chapter title |
Preparation of heteroelement-incorporated and stable isotope-labeled protein standards for quantitative proteomics.
|
---|---|
Chapter number | 23 |
Book title |
Shotgun Proteomics
|
Published in |
Methods in molecular biology, April 2014
|
DOI | 10.1007/978-1-4939-0685-7_23 |
Pubmed ID | |
Book ISBNs |
978-1-4939-0684-0, 978-1-4939-0685-7
|
Authors |
Konopka A, Zinn N, Wild C, Lehmann WD, Anna Konopka, Nico Zinn, Christina Wild, Wolf D. Lehmann, Konopka, Anna, Zinn, Nico, Wild, Christina, Lehmann, Wolf D. |
Editors |
Daniel Martins-de-Souza |
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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Members of the public | 1 | 100% |
Mendeley readers
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Unknown | 8 | 100% |
Demographic breakdown
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Researcher | 4 | 50% |
Student > Ph. D. Student | 2 | 25% |
Professor > Associate Professor | 1 | 13% |
Unspecified | 1 | 13% |
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Biochemistry, Genetics and Molecular Biology | 3 | 38% |
Chemistry | 3 | 38% |
Computer Science | 1 | 13% |
Unspecified | 1 | 13% |