Metabolic power

User:
Matthias Mann, professor, Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany

Project:
Measuring proteome changes in cultured Drosophila cells from RNAi-induced knockdown of specific gene products

Problem:
Mann wanted to cut down on side reactions that can plague chemical and enzymatic quantitation techniques.

Solution:
Rather than using enzymatic or chemical reactions, Mann prefers to let the cells do the work with a metabolic labeling strategy called "stable-isotope labeling with amino acids in cell culture" (SILAC). The process: Grow two batches of cells, one in the presence of 13C-arginine and 13C-lysine to label newly synthesized proteins. Then mix the protein extracts, digest with protease, and analyze. Because trypsin cleaves at basic residues, each peptide will be labeled only once. In the analysis, each protein fragment will appear as a doublet, with one of expected mass, and the other offset by 6 or 8 Da (per labeled arginine and lysine, respectively). The ratio of labeled to unlabeled peaks reveals a given peptide's relative abundance.

As with proteolytic 18O labeling, SILAC labels every peptide. SILAC is designed for cultured cells, not clinical or animal samples, though Mann notes a recent fix. "Let's say I want to quantify something like mouse liver for toxicology," he says. By mixing the labeled cell line into liver tissue from knockout and normal mice, "we can still quantify the proteins in both." The reagents are expensive, however.

The Equipment:
Though not technically required, a tandem mass spec is best, says Mann, so that peptides can be both quantified and sequenced. His choice: an electrospray-coupled hybrid linear ion trap-Orbitrap instrument from Thermo.

Cost:
Applied Biosystems SILAC kits cost $1,195.