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

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.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-78CE-2 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-78CF-F
Genre: Journal Article

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Bluemlein, K.1, Author              
Ralser, M.1, Author              
Affiliations:
1Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1433550              

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Free keywords: Cell Extracts/*chemistry; Chromatography, Liquid/*methods; *Protein Biosynthesis; Proteomics/methods; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/biosynthesis/*chemistry; Tandem Mass Spectrometry/instrumentation/*methods
 Abstract: 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.

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 Dates: 2011
 Publication Status: Published in print
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Title: Nature Protocols
Source Genre: Journal
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Pages: - Volume / Issue: 6 (6) Sequence Number: - Start / End Page: 859 - 69 Identifier: ISSN: 1750-2799 (Electronic) 1750-2799 (Linking)