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

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 mitochondrial gene
deletions in mitochondrial fractions using quantitative mass spectrometry
revealing the role of Coi1 for the assembly of cytochrome c oxidase (respiratory chain complex IV).