title: Monitoring protein expression in whole-cell extracts by targeted label- and standard-free LC-MS/MS.
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.
authors.affiliation: Mass Spectrometry Unit, Max Planck Institute for Molecular Genetics, Berlin, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Bluemlein
authors.forename: Markus
authors.initials: M
authors.lastname: Ralser
mesh_terms: D002457:Cell Extracts / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods*; D014176:Protein Biosynthesis; D040901:Proteomics / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D002457:Cell Extracts; D029701:Saccharomyces cerevisiae Proteins