4.8 Article

Complete Native Stable Isotope Labeling by Amino Acids of Saccharomyces cerevisiae for Global Proteomic Analysis

Journal

ANALYTICAL CHEMISTRY
Volume 90, Issue 17, Pages 10501-10509

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.8b02557

Keywords

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Funding

  1. European Research Council (ERC) [648235]
  2. Excellence Initiative of the German Federal and State Governments [EXC 294]
  3. Deutsche Forschungsgemeinschaft [WA 1598/S-1, RTG 2202, FOR 1905, SFB 746, SFB 1140]

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Knowledge about the functions of individual proteins on a system-wide level is crucial to fully understand molecular mechanisms underlying cellular processes. A considerable part of the proteome across all organisms is still poorly characterized. Mass spectrometry is an efficient technology for the global study of proteins. One of the most prominent methods for accurate proteome-wide comparative quantification is stable isotope labeling by amino acids in cell culture (SILAC). However, application of SILAC to prototrophic organisms such as Saccharomyces cerevisiae, also known as baker's yeast, is compromised since they are able to synthesize all amino acids on their own. Here, we describe an advanced strategy, termed 2nSILAC, that allows for in vivo labeling of prototrophic baker's yeast using heavy arginine and lysine under fermentable and respiratory growth conditions, making it a suitable tool for the global study of protein functions. This generic 2nSILAC strategy allows for directly using and systematically screening yeast mutant strain collections available to the scientific community. We exemplarily demonstrate its high potential by analyzing the effects of mitochondria] gene deletions in mitochondrial fractions using quantitative mass spectrometry revealing the role of Coil for the assembly of cytochrome c oxidase (respiratory chain complex IV).

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