4.7 Article

Novel Insights into Quantitative Proteomics from an Innovative Bottom-Up Simple Light Isotope Metabolic (bSLIM) Labeling Data Processing Strategy

期刊

JOURNAL OF PROTEOME RESEARCH
卷 20, 期 3, 页码 1476-1487

出版社

AMER CHEMICAL SOC
DOI: 10.1021/acs.jproteome.0c00478

关键词

In vivo metabolic labeling; light carbon isotope; C-12; quantitative proteomics; data processing workflow; OpenMS; KNIME; yeast

资金

  1. Agence Nationale de la Recherche [ANR-18-CE44-0014]
  2. Center for Interdisciplinary Research (CRI-Paris)
  3. Agence Nationale de la Recherche (ANR) [ANR-18-CE44-0014] Funding Source: Agence Nationale de la Recherche (ANR)

向作者/读者索取更多资源

The SLIM labeling strategy is a novel method for quantifying proteome variations, using simple procedures and open-source software to facilitate the analysis of bottom-up proteomics data.
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-[C-12] as the sole source of carbon synthesize U-[C-12]-amino acids, which are incorporated into proteins, giving rise to U-[C-12]-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 C-12 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 C-12 abundance in peptides to follow the kinetics of protein labeling and the molar fraction of unlabeled and C-12-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 C-12 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.

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