Native SILAC: Metabolic Labeling of Proteins in Prototroph Microorganisms Based on Lysine Synthesis Regulation

Abstract

Mass spectrometry (MS)-based quantitative proteomics has matured into a methodology able to detect and quantitate essentially all proteins of model microorganisms, allowing for unprecedented depth in systematic protein analyses. The most accurate quantitation approaches currently require lysine auxotrophic strains, which precludes analysis of most existing mutants, strain collections, or commercially important strains (e.g. those used for brewing or for the biotechnological production of metabolites). Here, we used MS-based proteomics to determine the global response of prototrophic yeast and bacteria to exogenous lysine. Unexpectedly, down-regulation of lysine synthesis in the presence of exogenous lysine is achieved via different mechanisms in different yeast strains. In each case, however, lysine in the medium down-regulates its biosynthesis, allowing for metabolic proteome labeling with heavy-isotope-containing lysine. This strategy of native stable isotope labeling by amino acids in cell culture (nSILAC) overcomes the limitations of previous approaches and can be used for the efficient production of protein standards for absolute SILAC quantitation in model microorganisms. As proof of principle, we have used nSILAC to globally analyze yeast proteome changes during salt stress. Molecular & Cellular Proteomics 12:

Global Proteome Turnover Analyses of the Yeasts S. cerevisiae and S. pombe

“…Native SILAC protocol was followed for heavy pre-labeling using heavy [13C6/15N2] L-lysine (Cambridge Isotope Labs) (Frohlich et al, 2013). …”

“…Here, we overcome these limitations of globally determining protein turnover by capitalizing on recent advances in mass spectrometry-based proteomic technology (Bensimon et al, 2012; Frohlich et al, 2013; Mann et al, 2013; Michalski et al, 2011; Zhang et al, 2013). We report protein turnover rates of nearly all proteins expressed under standard conditions for two model organisms, S. cerevisiae and S. pombe.…”

Stromal cell–derived factor 2 is critical for Hsp90-dependent eNOS activation

“…Analysis of the peptide mixture was performed as described previously ( 37 ). Briefly, peptides were separated on 15-cm columns (New Objectives) with a 75-μm inner diameter, packed in-house with 1.9 μm C18 resin (Dr. Maisch GmbH).…”

Rom2-dependent Phosphorylation of Elo2 Controls the Abundance of Very Long-chain Fatty Acids

“…The samples were dried in a SpeedVac to a final volume of 2 ml and buffer A (0.1% formic acid in water) was added to a final volume of 6 ml. 5 ml were analyzed by online nanoflow liquid chromatography tandem mass spectrometry on a Q-Exactive Orbitrap (Thermo) as described previously (31). Raw data were processed by the MaxQuant software package as described previously with the addition of STY phosphorylation as a variable modification (31).…”