title: 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.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Florian
authors.initials: F
authors.lastname: Fröhlich
authors.forename: Romain
authors.initials: R
authors.lastname: Christiano
authors.forename: Tobias C
authors.initials: TC
authors.lastname: Walther
mesh_terms: D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007553:Isotope Labeling; D008239:Lysine / Q000378:metabolism*; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D008239:Lysine