Glycosylation of proteins is central to cell signaling, immune function, and pathogen interactions, yet existing methods for monitoring glycan changes require specialized instrumentation and primarily report on membrane-anchored, rather than secreted, glycoproteins, with a slow turnover. Here, we present the Enzyme-Linked Cycloaddition Assay (ELCA), a click chemistry-based platform for ultra-sensitive detection and semi-quantitative analysis of secreted sialoglycoproteins. By metabolically incorporating an azide-modified sialic acid into newly synthesized glycoproteins and capturing labeled material via strain-promoted cycloaddition, ELCA quantifies aggregate sialylation using a microplate reader-compatible, ELISA-like workflow. We demonstrate that the secreted glycoproteome responds rapidly to pharmacological perturbation, with changes detectable within hours. Benchmarking against common glycosylation inhibitors and profiling cytokine-driven macrophage polarization further establishes ELCA's sensitivity and temporal resolution. Compatible with serum-containing conditions and requiring no specialized instrumentation, ELCA provides a broadly accessible tool for rapid, cost-effective monitoring of secreted glycoprotein dynamics.
Lundstrom, J., Yang, J., Bojar, D.
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