posted on 2021-02-12, 01:14authored byNicolas Sénécaut, Gelio Alves, Hendrik Weisser, Laurent Lignières, Samuel Terrier, Lilian Yang-Crosson, Pierre Poulain, Gaëlle Lelandais, Yi-Kuo Yu, Jean-Michel Camadro
Simple light isotope
metabolic labeling (SLIM labeling) is an innovative
method to quantify variations in the proteome based on an original in vivo labeling strategy. Heterotrophic cells grown in
U-[12C] as the sole source of carbon synthesize U-[12C]-amino acids, which are incorporated into proteins, giving
rise to U-[12C]-proteins. This results in a large increase
in the intensity of the monoisotope ion of peptides and proteins,
thus allowing higher identification scores and protein sequence coverage
in mass spectrometry experiments. This method, initially developed
for signal processing and quantification of the incorporation rate
of 12C into peptides, was based on a multistep process
that was difficult to implement for many laboratories. To overcome
these limitations, we developed a new theoretical background to analyze
bottom-up proteomics data using SLIM-labeling (bSLIM) and established
simple procedures based on open-source software, using dedicated OpenMS
modules, and embedded R scripts to process the bSLIM experimental
data. These new tools allow computation of both the 12C
abundance in peptides to follow the kinetics of protein labeling and
the molar fraction of unlabeled and 12C-labeled peptides
in multiplexing experiments to determine the relative abundance of
proteins extracted under different biological conditions. They also
make it possible to consider incomplete 12C labeling, such
as that observed in cells with nutritional requirements for nonlabeled
amino acids. These tools were validated on an experimental dataset
produced using various yeast strains of Saccharomyces
cerevisiae and growth conditions. The workflows are
built on the implementation of appropriate calculation modules in
a KNIME working environment. These new integrated tools provide a
convenient framework for the wider use of the SLIM-labeling strategy.