A Genetic Engineering Solution to the “Arginine Conversion Problem” in Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Abstract

Stable isotope labeling by amino acids in cell culture (SILAC) provides a straightforward tool for quantitation in proteomics. However, one problem associated with SILAC is the in vivo conversion of labeled arginine to other amino acids, typically proline. We found that arginine conversion in the fission yeast Schizosaccharomyces pombe occurred at extremely high levels, such that labeling cells with heavy arginine led to undesired incorporation of label into essentially all of the proline pool as well as a substantial portion of glutamate, glutamine, and lysine pools. We found that this can be prevented by deleting genes involved in arginine catabolism using methods that are highly robust yet simple to implement. Deletion of both fission yeast arginase genes or of the single ornithine transaminase gene, together with a small modification to growth medium that improves arginine uptake in mutant strains, was sufficient to abolish essentially all arginine conversion. We demonstrated the usefulness of our approach in a large scale quantitative analysis of proteins before and after cell division; both up- and down-regulated proteins, including a novel protein involved in septation, were successfully identified. This strategy for addressing the “arginine conversion problem” may be more broadly applicable to organisms amenable to genetic manipulation.

Examining post‐translational modification‐mediated protein–protein interactions using a chemical proteomics approach

“…As shown in Figure 2(c), GST-HP1 CD and GFP-Swi6 were selectively captured by probe 2, whereas Cdc4, an abundant yeast protein that does not bind H3K9Me 3 , was not captured by the probe. We believe that these chemical probes, when combined with recently developed fission yeast SILAC technology, 19 can be used to comprehensively profile H3K9Me 3 ''readers'' in yeast lysates. However, rather than comprehensively profiling H3K9Me 3 -binding proteins, we shifted our focus to phosphorylation, a more dynamic PTM.…”

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Technology in Fission Yeast

“…In mammalian cells and budding yeast, arginine conversion is minor, and several approaches have been developed to minimize the problem, including titration of arginine and proline concentrations and computational methods (Gruhler et al 2005;Blagoev and Mann 2006;Ong and Mann 2006;Bendall et al 2008;Graumann et al 2008;Mousson et al 2008;Park et al 2009;Prokhorova et al 2009). In fission yeast, arginine conversion is much more extensive (Bicho et al 2010) and thus must be dealt with more decisively. Two general approaches are possible, and both have been used successfully (Bicho et al 2010;Koch et al 2011).…”

“…In fission yeast, arginine conversion is much more extensive (Bicho et al 2010) and thus must be dealt with more decisively. Two general approaches are possible, and both have been used successfully (Bicho et al 2010;Koch et al 2011). The first approach (Koch et al 2011) avoids heavy arginine altogether.…”

“…We describe construction of fission yeast strains suitable for SILAC MS, as well as conditions for robust growth, harvesting, and processing of cells (Bicho et al 2010;Koch et al 2011) ). This strategy has been used in several large-scale phosphoproteomics studies in eukaryotes (Olsen et al 2006 and prokaryotes (Maček et al 2007(Maček et al , 2008.…”

“…The second approach (Bicho et al 2010) takes advantage of yeast genetics and deletes genes that encode enzymes involved in arginine catabolism, thereby preventing or at least decreasing arginine conversion. Deletion of the predicted ornithine transaminase gene car2 + or simultaneous double deletion of the redundant predicted arginase genes car1 + and aru1 + has been shown to decrease conversion to acceptably low levels (Bicho et al 2010). Because constructing singledeletion strains is easier, deletion of the car2 + gene has generally been the preferred method.…”