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