Stable Isotope Labeling of Arabidopsis thaliana Cells and Quantitative Proteomics by Mass Spectrometry

Quantitative mass spectrometry in proteomics: a critical review

“…Initially described for total labeling of bacteria using 15 N-enriched cell culture medium [2], it has gained wider popularity in the form of the stable isotope labeling by amino acids in cell culture (SILAC) approach introduced by Mann and co-workers in 2002 [12]. In the most commonly used implementation of the method, the medium contains 13 C 6 -arginine and 13 C 6 -lysine which ensures that all tryptic cleavage products of a protein (except for the very C-terminal peptide) carry at least one labeled amino acid resulting in a constant mass increment over the non-labeled counterpart.…”

“…Protein identification is based on fragmentation spectra of at least one of the coeluting 'heavy' and 'light' peptides and relative quantitation is performed by comparing the intensities of isotope clusters of the intact peptide in the survey spectrum. In contrast to full metabolic protein labeling by 15 N, the number of incorporated labels in SILAC is defined and not dependent on the peptide sequence thus facilitating data analysis. The main advantage of all metabolic labeling strategies is that the differentially treated samples can be combined at the level of intact cells.…”

“…This excludes all sources of quantification error introduced by biochemical and mass spectrometric procedures as these will affect both protein populations in the same way. Despite a number of cases that demonstrate the feasibility of total 15 N metabolic protein labeling of higher organisms in vivo such as C. elegans, Drosophila melanogaster [13], rat [14], or plants [15], it is neither possible nor practical to apply this strategy routinely. The cost and time required for creating and [65] maintaining these systems is often incommensurate with the value of the information provided.…”

Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance

“…This is particularly appealing within plant proteomics, because the most conventional labeling strategy, Stable Isotope Labeling by Amino Acids in Cell Culture, is not easily suited for quantitative plant proteomic studies. The average labeling efficiency achieved using exogenous amino acid supply to Arabidopsis (Arabidopsis thaliana) cell cultures was found to be only 70% to 80% (Gruhler et al, 2005). Quantitative strategies with 15 N metabolic labeling have been described for plant proteome analysis; however, care should be taken to ensure complete 15 N incorporation, because even small amounts of 14 N in the labeled sample can have significant detrimental effects on the number of peptide identifications (Nelson et al, 2007;Guo and Li, 2011;Arsova et al, 2012).…”

Proteomic analysis of post translational modifications in cyanobacteria

“…However, it works well for mammalian cell lines [113][114][115], but not so effective in autotrophic organisms such as cyanobacteria due to their ability to synthesize all amino acids [116,117]. The second primary method in stable isotope metabolic labeling relies on the introduction of a heavy isotope such as 15 N, often in the form of Na 15 NO 3 or K 15 NO 3 as a nitrogen source, is a powerful tool for the quantitative analysis in plant and bacteria [118,119].…”