In vitro SILAC is a well-established approach for quantification of proteins in cell culture. The same concept can also be applied to living organisms (in-vivo SILAC). The latter approach allows determination of protein patterns of all tissues of an organism with respect to a reference organism. This approach has been successfully applied to the analysis of mouse organs.
Prerequisite for the application of the in-vivo SILAC approach is the availability of an appropriate SILAC diet in which all lysins are replaced by 13C-lysine.
Silantes provides SILAC diets for in-vivo labelling of mouse, but also for other model organisms such as fly and worm (see literature for Silantes SILAC diets for Fly and Worm). Contact us for more information.
Silantes SILAC Diets for in-vivo labelling of mice
Mice are fed with Silantes SILAC mouse feed according to the scheme in Figure 1 for about 90 days requiring about 800 g Silantes mice feed.
13C-lysine labelled mice feed has been developed in cooperation with the group of Prof. Matthias Mann, Max-Planck-Institute of Biochemistry as a kit containing labelled and unlabelled mouse feed. The kit consists of 13C-lysine-labelled “heavy” diet (B) and unlabelled “light” diet (A). The feed is an artificial, amino acid-based feed using Harlan components.
After the metabolic labelling (feeding) of the mice according to the scheme in Figure 1, the mice are sacrificed. Differences in the protein patterns are determined in analogy to the established SILAC approach in cell culture (see literature for Silantes in vitro SILAC). Instead of 13C-lysine labelled feed, other isotopic formulations are also available on request, e.g. 13C,15N-labelled feed.
A variation of the in-vivo SILAC-approach is the “spike-in” approach.
SILAC spike-in using Silantes 13C6-Lysine-labelled mouse tissue
Figure 1 shows the SILAC spike-in workflow: Differences in protein patterns of unlabelled tissue (A) with respect to unlabelled tissue (B) can be quantified by “spiking-in” a 13C-labelled reference tissue (R). This 13C-lysine reference tissue can be obtained from Silantes directly, as frozen material or lyophilized.
Short outline of the procedure:
The isotopically labelled “heavy” reference tissue (R) is mixed with the unlabelled “light” tissues (A) and (B), respectively. The proteomes of the (A)(R)-mix and (B)(R)-mix are isolated, digested and subjected to LC-MS as shown in the workflow. Therefore, the two peptide amount ratios (A/R) and (B/R) can be determined.
Calculating the ratio (A/R) : (B/R) cancels out the reference amount (R) and yields the ratio of peptides (A/B). The stable isotopically labelled mouse tissue (R) is used as a standard, permitting normalizing (A) with respect to (B), accounting for differences in the isolation procedure in the mixtures (A/R) and (B/R) without affecting the peptide ratios (A/B). (Strain (R) must not necessarily be the same as strain (A) and (B).)
The customer is free in regard to the choice of strain used for the experiment but can use the Silantes 13C6-Lysine-labelled mouse tissue as a reference which is spiked into tissue (A) and (B).
 Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey A., and Mann, M. (2002). Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Mol.Cell. Proteomics 1, 376–386.
 Ong, S.E., and Mann, M. (2006). A practical recipe for stable isotope labelling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660.
 Krueger M., Moser M., Ussar S., Thievessen I., Luber Ch.A, Forner F., Schmidt S., Zanivan S., Faessler R. and Mann M.(2008). SILAC Mouse for Quantitative Proteomics Uncovers Kindlin-3 as an Essential Factor for Red Blood Cell Function, Cell 134, 353–364.