Proteomics has increasingly become an invaluable tool to characterize proteomes from various subcellular compartments. Here, researchers from Brown University describe a quantitative proteomics method using the technique of Stable Isotope Labeling by Amino acids in Cell culture (SILAC) to analyze the effects of HIV infection on host exosomal proteomes. The procedure involves differential isotope labeling of cells, exosome purification, mass spectrometric quantification, and various bioinformatic analyses/verifications. This protocol can easily be adapted to other subcellular compartments under different stress conditions.
Overall experimental schema for SILAC labeling. Ready to be labeled cells are divided into two batches. One batch is expanded in “heavy” medium for six doublings to achieve complete labeling. The other batch is independently maintained for the same periods. When the cells are completely labeled, the heavy cells are infected with HIV-1, while light cells receive no infection. At the end of infection, heavy (infected) and light (uninfected) are subjected to exosome isolation in parallel.