Cell Type Specific SILAC

The SILAC method (Stable Isotope Labeling of Amino Acids in Cell Culture) is a powerful tool in quantitative proteomics of cell cultures.[1] Find out more about the SILAC method. In multicellular contexts, however, the SILAC method has limited applicability as cultures usually have to be grown in isolation. A new technique for the investigation of cell-cell interactions is the cell type-specific labeling using amino acid precursors (CTAP).[2] Instead of the essential amino acid itself, a stable isotope labeled substrate or precursor is used. By overexpressing an enzyme that synthesizes the desired amino acid from its precursor, a metabolic incorporation of the label into a specific cell type is achieved.[3]

Silantes High Quality Components for Cell Type-Specific Labeling using Amino Acid Precursors

Silantes provides stable isotope labeled diaminopimelic acid (DAP), which is a precursor for the SILAC amino acid lysine. By transfecting distinct cell populations with the gene encoding the enzyme diaminopimelate decarboxylase (DDC), these cells gain the ability to grow on DAP instead of lysine.

Silantes offers ¹³C-,  ¹⁵N-,  and ¹³C¹⁵N-labeled DAP in powder form, which is produced by fermentation. We guarantee an isotopic enrichment of > 97 atom % and a chemical purity of > 95 %. The isotopic and chemical purities are confirmed by mass spectrometry and HPLC, respectively.

Applications of the CTAP Approach using stable isotope-labeled DAP

In cell-of-origin secretome studies with lysine-auxotrophic cells, biomarker and cell-cell communication analysis can be performed in co-culture by metabolic labeling with DAP as a substrate (Figure 1).

Figure 2 shows an example of a cell-of-origin secretome study using the method of Gauthier et al..3 A mouse cell line modified to express DDC for growth on DAP together with a human cell line capable of growing on D-lysine (expressing lysine racemase) were grown in co-culture on labeled D-lysine and unlabeled DAP. Nearly all proteins secreted from both cell lines could be distinguished by the difference in label illustrating the aptitude of CTAP for multicellular analysis. Additional applications of these studies are possible with the new availability of SI-labeled DAP.

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[1] Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., 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(5):376-386.

[2] Gauthier, N., Sander, C., Miller, M., (2014), Cell selective proteome labeling, WO 2014039643.

[3] Gauthier, N. P., Soufi, B., Walkowicz, W. E., Pedicord, V. A., Mavrakis, K. J., Macek, B., Gin, D. Y., Sander, C. and Miller, M. L. (2013). Cell-selective labeling with amino acid precursors for proteomic studies of multicellular environments. Nat Methods 10(8), 768-773.