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Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons50106

Bluemlein,  K.
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

http://pubman.mpdl.mpg.de/cone/persons/resource/persons50483

Ralser,  M.
Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Zitation

Bluemlein, K., & Ralser, M. (2011). Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS. Nature Protocols, 6(6), 859-69. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21637204 http://www.nature.com/nprot/journal/v6/n6/pdf/nprot.2011.333.pdf.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0010-78CE-2
Zusammenfassung
Targeted quantification of proteins is a daily task in biological research but often relies on techniques such as western blotting that are only barely quantitative. Here we present a broadly applicable workflow for protein quantification from unpurified whole-cell extracts that can be completed in less than 3 d. Without prefractionation or affinity enrichment, a whole-cell extract is trypsin-digested in an acetonitrile-containing ammonium carbonate buffer and high-molecular-weight compounds are removed by filtration. A normalization strategy, which involves endogenous reference proteins, facilitates the determination of relative changes in protein expression without requiring isotope labeling or standard addition. On a triple-quadrupole mass spectrometer, we demonstrate standard-free quantification of yeast proteins present over five orders of magnitude and present at >/=500 copies per cell. Liquid chromatography/multiple reaction monitoring (LC-MRM)-based proteomics is therefore a next-generation alternative to western blotting, as it allows simultaneous and reliable quantification of multiple endogenous proteins without the need for enrichment, isotope labeling or use of antibodies.