Figure 4. Overview of SILAC workflow.

Astrocytes were grown in “light” or “heavy” amino acid containing media. Proteins from the wild-type (“light” labeled) and GFAP-Nrf2 (“heavy” labeled) cells were mixed and digested using trypsin. Tryptic peptides were then separated offline by high pH reverse phase HPLC. Fractions were collected and then individually analyzed by LC-MS/MS. The differences in expression were quantified by calculating the area under the curve for the “light” and “heavy” peptide pairs for each protein.