Categories Products for Proteomics Products for in-vivo SILAM
Products for in-vivo SILAM
|
|
SILAM Mouse Tissue
|
Stable isotopic labelling is the most reliable and accurate method for quantitative proteomics and metabolomics. Proteomics studies on uniformly 15N-labelled rats have been previously reported using 15N-labelled algae as protein source for Stable Isotope Metabolic Labelling in Mammals (SILAM).[1]
Silantes provides SILAM diets for in-vivo labelling of mouse, but also for other model organisms such as fly and worm. Contact us for more information.
Silantes 15N-SILAM Diets for in-vivo labelling of mice
Silantes has developed a 15N-diet in cooperation with the group of Professor Chris Turck, Max-Planck-Institute of Psychiatry as a kit containing a 15N-labelled “heavy” diet (B) and unlabelled “light” diet (A).[2] It is an artificial feed using Harlan components and a hydrolysate of the bacteria Ralstonia eutropha as 15N-protein source. Feeding experiments showed that the mice accepted bacterial-based protein better than algae-based protein.
After the metabolic labelling (feeding) of the mice according to the scheme in figure 2 or figure 3, 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).
A variation of the in-vivo SILAM-approach is the “spike-in” approach.
SILAM spike-in using Silantes 15N-labelled mouse tissue
Figure 2 shows the SILAM spike-in workflow: Differences in protein patterns of unlabelled case tissue with respect to unlabelled control tissue can be quantified by “spiking-in” a 15N-labelled reference tissue.
This 15N reference tissue can be obtained from Silantes directly, as frozen material or lyophilized.
Short outline of the procedure:
The isotopically labelled “heavy” reference tissue is mixed with the unlabelled “light” case and control tissue, respectively. The proteomes of the case/reference-mix and control/reference-mix are isolated, digested and subjected to LC-MS. Therefore, the two peptide amount ratios can be determined in analogy to the in-vivo SILAC-workflow to determine the case/control ratio (see Silantes in-vivo SILAC literature).
In addition to spiking-in the 15N-tissue as reference material for relative protein quantification by mass spectrometry, the SILAM approach is used in further applications such as
- Partially labelled mouse specimens for global protein turnover analysis by mass spectrometry (see figure 2)[3]
- Labelled metabolite standards for molecular structure confirmation
- Biomarker discovery and verification
[1] Daniel B. McClatchy, Meng-Qiu Dong, Christine C. Wu, John D. Venable, and John R. Yates (2007). 15N Metabolic Labeling of Mammalian Tissue with Slow Protein, Journal of Proteome Research, 6(5), 2005-2010.
[2] Zhang, M.D. Filiou, G. Chen, H. Heumann, Chris W. Turck (2008). Biomarker discovery in a mouse model of trait anxiety using 15N metabolic labeling, Poster HUPO Amsterdam.
[3] Zhang, Reckow S, Webhofer C, Boehme M, Gormanns P, Egge-Jacobsen WM, Turck CW. (2011) Proteome scale turnover analysis in live animals using stable isotope metabolic labeling. Anal Chem 83:1665-1672.
