Targeted glycoprotein enrichment and identification in stromal cell secretomes using azido sugar metabolic labeling.

PURPOSE: Effectively identifying the proteins present in the cellular secretome is complicated due to the presence of cellular protein leakage and serum protein supplements in culture media. A metabolic labeling and click chemistry capture method is described that facilitates the detection of lower abundance glycoproteins in the secretome, even in the presence of serum. EXPERIMENTAL
DESIGN: Two stromal cell lines were incubated with tetraacetylated sugar-azide analogs for 48 h in serum-free and low-serum conditions. Sugar-azide labeled glycoproteins were covalently linked to alkyne-beads, followed by on-bead trypsin digestion and MS/MS. The resulting glycoproteins were compared between media conditions, cell lines, and azide-sugar labels.
RESULTS: Alkyne-bead capture of sugar-azide modified glycoproteins in stromal cell culture media significantly improved the detection of lower abundance secreted glycoproteins compared to standard serum-free secretome preparations. Over 100 secreted glycoproteins were detected in each stromal cell line and significantly enriched relative to a standard secretome preparation. CONCLUSION AND CLINICAL RELEVANCE: Sugar-azide metabolic labeling is an effective way to enrich for secreted glycoproteins present in cell line secretomes, even in culture media supplemented with serum. The method has utility for identifying secreted stromal proteins associated with cancer progression and the epithelial-to-mesenchymal transition.