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Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons97138

Engelsberger,  W. R.
Signalling Proteomics, Department Stitt, Max Planck Institute of Molecular Plant Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons97140

Erban,  A.
Applied Metabolome Analysis, Department Willmitzer, Max Planck Institute of Molecular Plant Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons97239

Kopka,  J.
Applied Metabolome Analysis, Department Willmitzer, Max Planck Institute of Molecular Plant Physiology, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons97400

Schulze,  W. X.
Signalling Proteomics, Department Stitt, Max Planck Institute of Molecular Plant Physiology, Max Planck Society;

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Engelsberger-2006-Metabolic labeling o.pdf
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Zitation

Engelsberger, W. R., Erban, A., Kopka, J., & Schulze, W. X. (2006). Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites. Plant Methods, 2, 14. doi:10.1186/1746-4811-2-14.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0014-2AA0-4
Zusammenfassung
Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of N-15-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized N-15-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic N-15-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.