Figure 3.

Comparison of SILAC and pSILAC strategies to assess miRNA impact on protein levels. (Left) In the SILAC labeling strategy, cells are grown for several doublings in SILAC medium containing either light (L) or heavy (H) amino acids and then subjected to different treatments, e.g. transfected with either control (CTRL) or miRNA over-expressing (OE) vectors. Cells from both conditions are then harvested, mixed in equal ratio and analyzed by LC-MS/MS as a single sample. Each protein-derived peptide will appear as a peak-pair that reflects a mixture of both the pre-existing and newly synthesized proteins. The intensity ratio of the two peaks within each peak-pair is proportional to the amount of the original peptide in the CTRL and miRNA OE cells, allowing the quantification of proteins in the two conditions. A peptide ratio (H)/(L) ≅ to 1 indicates that the protein level is unchanged, while a peptide ratio (H)/(L) < 1 indicates that the protein level is reduced by miRNA OE, hence the protein is a putative miRNA target. (Right) In pSILAC, CTRL and miRNA OE cells are initially grown in SILAC media, containing light amino acids. Subsequently, both CTRL and miRNA OE cells are pulsed with distinct SILAC media containing either medium-heavy (M) or heavy (H) amino acids. After a short time interval, cells are harvested, mixed in 1:1 ratio and the extracted proteins are digested and LC-MS/MS analyzed. Each protein-derived peptide will appear as a peak-triplet, whereby the pre-existing proteins appear as (L) peaks, while newly synthesized ones display either (M) or (H) peaks and the ratio of (H) over (M) peaks is the result of the miRNA OE impact on protein neo-synthesis. Newly synthesized proteins with a SILAC ratio (H)/(M) < 1 are putative miRNA targets while non-targeted proteins show a SILAC ratio (H)/(M) ≅ to 1.