Figure 4.

Workflow for stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic profiling of exosomal proteins. Cell lines are cultured in SILAC media that has been supplemented with arginine and lysine containing ¹³C and ¹⁵N (Lys8, Arg10; “heavy”) or the naturally occurring ¹²C and ¹⁴N isotopes (Lys0, Arg0; “light”). After exposure to hypoxic or normoxic conditions, the exosomes are isolated from each cell line and are mixed at a 1:1 ratio followed by enzymatic protein digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.