Figure 1
From: SILAC–based quantitative MS approach for real-time recording protein-mediated cell-cell interactions
The strategies of ‘triple-SILAC’ (A) and ‘spike-in SILAC’ (B) approaches. In triple-SILAC, we combined the CMs from none labeled (L) co-cultured system, 13C6-Lys (H) labeled mono-cultured CT26 and 2H3-Leu (M) labeled Ana-1, then simultaneously analyzed by LC-MS/MS. The final ratio (Ratio T) of co-cultured system versus mono-cultured ones can be calculated directly by comparing the intensities of peaks with the certain molecular weight shifts in mass spectrum. In spike-in SILAC, CMs from 13C6-Lys (H) labeled Ana-1 and CT26 were combined separately with co-cultured one (L). To compare the peaks intensity with heavy (H) and light (L) labeled, Ratio1 and 2 were identified. The Ratio S is the inverse of the sum of. Ratio1 and Ratio2. Light: non-labeled. Medium: 2H3-Leu labeled. Heavy: 13C6-lysine labeled. Ratio T and Ratio S: Final ratio of a protein for co-cultured system versus mono-cultured in triple and spike-in SILAC. Ratio 1: the protein ratio of 13C6-Lys-labeling mono-cultured CT26 versus co-cultured cells. Ratio 2: the protein ratio of 13C6-Lys-labeling mono-cultured Ana-1versus co-cultured cells.
