Novel insights into quantitative proteomics from an innovative bottom-up Simple Light Isotope Metabolic (bSLIM) labeling data processing strategy

ABSTRACT: The Simple Light Isotope Metabolic labeling (SLIM-labeling) is an inovative method to quantify proteome variations based on an original in vivo labeling strategy. Heterotrophic cells grown on a U-[12C] sole source of carbon synthesize U-[12C]-amino acids which are incorporated into proteins giving rise ultimately to U-[12C]-proteins. This results in a large increase of the intensity of the monoisotope ion of peptides and proteins, and therefore allows higher identification scores and protein sequence coverage in mass spectrometry experiments. The method initially developed for signal processing and quantification of the rate of incorporation of 12C into peptides was based on a multistep process that was found difficult to implement by many laboratories. To overcome these limitations, we developed a new theoretical background to analyze the bottom-up proteomics data using SLIM-labeling (bSLIM) and set 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 both the computation of the 12C abundance in peptides to follow kinetics of protein labeling, and of the molar fraction of unlabeled and 12C-labeled peptides in multiplexing experiments to determine the relative abun-dance of proteins extracted from different biological conditions. The tools also allow us to take into account incomplete 12C labeling as observed in cells with nutritional requirements for non-labeled amino acids. These tools were validated on experimental dataset produced using various yeast strains of Saccharomyces cerevisiae and growth conditions. The workflows are built on the implemen-tation of appropriate calculus modules in a KNIME working environment. These new integrated tools provide a convenient frame-work for a wider use of the SLIM-labeling strategy.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Nicolas SENECAUT  

LAB HEAD: Jean-Michel CAMADRO

PROVIDER: PXD021329 | Pride | 2022-02-15

REPOSITORIES: Pride