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.

Fig. 1.

Proteome changes of prototrophic yeast in medium containing or lacking lysine. A, experimental design for the proteome analysis of S. cerevisiae grown in the presence or absence of lysine. FASP, filter aided sample preparation; WT, wild-type. B, S. cerevisiae down-regulates the abundance of lysine biosynthetic enzymes in response to external lysine. In the volcano plot, the protein abundance ratios in the presence or absence of lysine are plotted against the negative log₁₀ of the p value of the t test for each protein. C, the lysine biosynthesis pathway of S. cerevisiae is down-regulated (denoted by red boxes).

Fig. 2.

Prototrophic S. cerevisiae efficiently incorporates exogenous lysine. A, experimental strategy to determine incorporation of heavy labeled lysine into S. cerevisiae, analyzed via proteomics. FASP, filter aided sample preparation; WT, wild-type. B, density function of the rates of heavy lysine incorporation of all lysine-containing peptides for the lysine prototroph S. cerevisiae W303 strain. Maximum of the density function representing the most abundant incorporation rate is given above the graph. C, density function of the heavy lysine incorporation rate in the lysine auxotroph W303 lys2Δ strain. Maximum of the density function representing the most abundant incorporation rate is given above the graph. D, example MS spectrum plotted against chromatography runtime of Ss a1 peptide NQIESIAYSIK from the lysine prototroph strain W303.

Fig. 3.

Native SILAC (nSILAC) detects proteome changes in cells grown with or without lysine. A, experimental design for the proteome analysis of S. cerevisiae grown in the presence or absence of heavy-isotope-containing lysine. FASP, filter aided sample preparation; WT, wild-type. B, plot of protein intensities against normalized H/L SILAC ratios of WT W303. S. cerevisiae cells grown in absence (light lysine, L) or presence of non-radioactive stable-isotope-containing lysine (heavy lysine 8, H). Significant outliers are colored in red (p < 1^e−11), orange (p < 1^e−4), or light blue (p < 0.05); other proteins are shown in dark blue. C, proteome comparison of lysine prototroph and lysine auxotroph S. cerevisiae strains. Protein intensities are plotted against normalized SILAC ratios of heavy (lysine prototroph strain labeled with lysine 8, H) to light (lysine auxotroph strain labeled with light lysine, L). Significant outliers are colored in red (p < 1^e−11), orange (p < 1^e−4). or light blue (p < 0.05); other proteins are shown in dark blue.

Fig. 4.

Abundance of lysine biosynthesis of S. pombe is not regulated in response to external lysine. A, lysine prototrophic S. pombe incorporates heavy lysine 8 from the medium. A density function for lysine incorporation in S. pombe is shown. B, example MS spectrum plotted against chromatography runtime for the Thi3 peptide IQLDDLCSK from the lysine prototroph S. pombe strain grown in the presence of lysine 8. C, abundances of lysine biosynthetic enzymes are not changed in response to external lysine. Protein intensities are plotted against normalized SILAC ratios of heavy (heavy lysine 8 as external source) to light (no exogenous lysine). Significant outliers are colored in red (p < 1^e−11), orange (p < 1^e−4), or light blue (p < 0.05); other proteins are shown in dark blue. The enzymes of the lysine biosynthesis pathway are colored in green.

Fig. 5.

Regulation of lysine biosynthesis in E. coli allows for efficient proteome labeling. A, E. coli incorporates heavy lysine from the medium efficiently. A density function for lysine incorporation in E. coli is shown. B, example MS spectrum plotted against chromatography runtime for the GroL peptide AVAAGMNPMDIK from the lysine prototroph E. coli BL21 (DE3) strain is shown. C, enzymes of the diaminopimelate pathway are color coded according to the statistical significance of their abundance change. D, E. coli down-regulates lysine biosynthetic enzymes in response to external lysine. Protein intensities are plotted against SILAC ratios of heavy (lysine 8 as external source) to light (no external lysine). Significant outliers are colored in red (p < 1^e−11), orange (p < 1^e−4), or light blue (p < 0.05); other proteins are shown in dark blue. E, E. coli can be used to produce heavy-labeled recombinant proteins. An E. coli BL21 (DE3) strain carrying the plasmid pGex6P1 encoding the protein glutathione S-transferase (GST) was labeled with light lysine. A strain carrying the plasmid pGex6P1-PIL1 encoding a fusion protein of GST to the S. cerevisiae protein Pil1 was labeled with heavy lysine 8. After 1 h of recombinant protein induction, cells were analyzed via MS. Protein intensities are plotted against normalized heavy/light SILAC ratios. Significant outliers are colored in red (p < 1^e−11), orange (p < 1^e−4), or light blue (p < 0.05); other proteins are shown in dark blue.

Fig. 6.

Lysine-labeled prototroph yeast can be used for SILAC-based quantitative proteomics. Lysine auxotrophic (A) or prototrophic (B) S. cerevisiae strains were labeled with either light or heavy lysine. Heavy-labeled strains were exposed to a hyperosmotic shock (0.4 m NaCl for 20 min). Protein intensities are plotted against heavy/light SILAC ratios. Significant outliers are colored in red (p < 1^e−11), orange (p < 1^e−4), or light blue (p < 0.05); other proteins are shown in dark blue. Ratio scatter plots show protein abundance changes between 0 and 20 min of salt stress as measured in a lysine auxotroph (C) or prototroph (D) strain plotted versus a combined ratio (22). The Pearson correlation coefficient is shown in the top left corner of each plot. Osmotic shock regulates glycolysis and glycerol synthesis. Enzymes are color coded according to their fold abundance change in response to a hyperosmotic shock in a lysine auxotroph (E) or prototroph (F) strain.