Abstract
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.
Highlights
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 or a specific number of their stable isotope counterparts
Peptides resulting from enzymatic digest of the combined cell lysate are detected by mass spectrometry (MS) in form of ion pairs, and their ratios reflect the relative changes in protein abundance between the light and the heavy culture (Figure 1A)
(A) In a conventional SILAC experiment, light- and heavy-labeled cultures are mixed upon treatment and the relative protein changes are determined from the ratio of heavy and light peptide signals in MS. (B) Arabidopsis liquid suspension cell cultures were labeled with medium-heavy (Lys-4) and heavy label (Lys-8)
Summary
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). Peptides resulting from enzymatic digest of the combined cell lysate are 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).
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