A Simple Light Isotope Metabolic Labeling (SLIM-labeling) Strategy: A Powerful Tool to Address the Dynamics of Proteome Variations In Vivo

Thibaut Léger,Camille Garcia,Laetitia Collomb,Jean-Michel Camadro

doi:10.1074/mcp.m117.066936

Abstract

Many quantitative proteomics strategies rely on in vivo metabolic incorporation of amino acids with modified stable isotope profiles into proteins. These methods give rise to multiple ions for each peptide, with possible distortion of the isotopolog distribution, making the overall analytical process complex. We validated an alternative strategy, simple light isotope metabolic labeling (SLIM-labeling), which alleviates many of these problems. SLIM-labeling is based on the in vivo reduction of the isotopic composition of proteins using metabolic precursors with a unique light isotope composition to label all amino acids. This brings a new dimension to in-depth, high resolution MS-based quantitative proteomics. Here, we describe a 12C-based SLIM-labeling strategy using U-[12C]-glucose as the metabolic precursor of all amino acids in the pathogenic yeast Candida albicans Monoisotopic ion intensity increased exponentially following 12C enrichment, substantially improving peptide identification scores and protein sequence coverage in bottom-up analyses. Multiplexing samples of 12C composition varying from natural abundance (98.93%) to 100% makes it possible to address relative quantification issues, keeping all the critical information for each peptide within a single isotopolog cluster. We applied this method to measure, for the first time, protein turnover at the proteome scale in Candida albicans and its modulation by inhibitors of the proteasome and vacuolar protein degradation systems.

Highlights

From the ‡Mass Spectrometry Laboratory, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Sorbonne Paris Cite, F-75205 Paris Cedex 13, France; §Mitochondria, Metals, and Oxidative Stress Group, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Sorbonne Paris Cite, F-75205 Paris Cedex 13, France
Matching the labeled and unlabeled peaks for 15N or 13C labeling methods can be challenging, because the mass shift is variable and depends on the total number of atoms (N or C, respectively) in the selected peptides. This problem does not occur in SILAC experiments, in which incorporation of labeled arginine and/or lysine combined with the use of trypsin, Lys-C or Lys-N [8] for digestion, is associated with a fixed mass shift between labeled and unlabeled peptides
The proposed SLIM-labeling strategy allows direct evaluation of the contribution of labeled and unlabeled peptides within a single isotope cluster instead of the two observed in other metabolic labeling methods. This is a major advantage over methods in which the mass shift between labeled and unlabeled peptides is variable and depends on the total number of labeled atoms

Summary

Introduction

From the ‡Mass Spectrometry Laboratory, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Sorbonne Paris Cite , F-75205 Paris Cedex 13, France; §Mitochondria, Metals, and Oxidative Stress Group, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Sorbonne Paris Cite , F-75205 Paris Cedex 13, France. Comparison of the datasets showed a remarkable improvement in protein identifications, scores, and sequence coverages when the isotopic complexity was reduced in vivo Mixing samples from both growth conditions opens the way to a new quantitative proteomics method. We show that the experimental isotope clusters fit remarkably well with the theoretical clusters computed using the MIDAS webserver at the NCBI (http://www.ncbi.nlm.nih.gov/CBBresearch/qmbp/ midas/index.html) [17], making it possible to generate high quality calibration standards for 12C enrichment measurements. We believe that this strategy for in vivo protein labeling may contribute significantly to the development of new approaches to explore key features of proteome dynamics

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Conclusion