SILAC analysis of exosome secretion in response to centrosome amplification

We performed quantitative proteomics analysis by Stable Isotope Labelling by Amino Acids in Cell culture (SILAC) to understand how centrosome amplification changes the composition of human small extracellular vesicles. We carried out SILAC labelling with medium and heavy isotopes, as it enables the exclusion of contaminant serum proteins, which would be unlabeled (equivalent of light labeling), as well as allowing for simultaneous processing of purification steps to decrease sample-to-sample variability. We isolated small extracellular vesicles by ultracentifugation followed by size exclusion chromatography (SEC). All experiments were performed in duplicate with switched SILAC labelling.