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Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) is a widespread method for metabolic labeling of cells and tissues in quantitative proteomics; however, incomplete incorporation of the label has so far restricted its wider use in plants. Here, we argue that differential labeling by two different versions of the labeled amino acids renders SILAC fully applicable to dark-grown plant cell lines. By comparing Arabidopsis thaliana cell cultures labeled with two versions of heavy Lys (Lys-4 and Lys-8), we show that this simple modification of the SILAC protocol enables similar quantitation accuracy, precision, and reproducibility as conventional SILAC in animal cells.
INTRODUCTION
In a typical Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) experiment (Ong et al., 2002; Ong and Mann, 2006), cells are grown in a defined medium complemented with certain essential amino acids, usually Arg and Lys, containing naturally occurring atoms (the light culture) or a specific number of their stable isotope counterparts (the heavy culture). After full incorporation of the labeled amino acid, the light and heavy cultures are subjected to different perturbations, harvested, and combined. Peptides resulting from enzymatic digest of the combined cell lysate are then detected by mass spectrometry (MS) in form of ion pairs (doublets), and their ratios reflect the relative changes in protein abundance between the light and the heavy culture (Figure 1A).
Mixing of the light- and heavy-labeled cells for relative quantitation requires full incorporation of the labeled amino acid into the proteome and removal of free amino acids from the medium. In addition, the cell line of interest must be auxotrophic for the amino acid used for labeling. Since plants are autotrophic organisms, these two fundamental requirements have been a serious obstacle to a wider application of SILAC in plants (Thelen and Peck, 2007; Gouw et al., 2010; Schulze and Usadel, 2010). In the only systematic SILAC study of the Arabidopsis thaliana cells, a labeled amino acid incorporation of 80% was reported (Gruhler et al., 2005), which is high but requires elaborate experimental control for accurate quantitation.
Here, we reasoned that the quantification bias introduced by incomplete labeling could be eliminated by a simple modification of the labeling and quantitation strategy. Instead of contrasting two cultures with light (Lys-0) and heavy...