Metabolic labeling and LC-MS/MS-based identification of interleukin-1α-induced secreted proteomes from epithelial cells in the presence or absence of serum

The unbiased identification of cytokine-induced, secreted proteins from cells cultured in serum-containing medium is challenging. Here, we describe an experimental and bioinformatics workflow to label interleukin-1α-regulated proteins in living cells with the methionine analogue L-homopropargylglycine. We detail their purification and identification by means of CLICK-chemistry-based biotinylation followed by nanoHPLC-MS/MS. A side-by-side comparison of enriched proteins and their ontologies to serum-free conditions demonstrates the sensitivity and specificity of this approach to study the inducible secreted proteomes of epithelial cells.