Bead-Based Enrichment Methods for Unbiased Secretome Profiling Enable Unprecedented Protein Coverage in an Immune Cell Model.

Bead-based enrichment strategies for low-abundance proteins in biofluids, followed by LC-MS/MS analysis, have dramatically increased the coverage of the serum and plasma proteome. These technologies effectively mitigate the dynamic range limitations imposed by high-abundance proteins like albumin, thereby facilitating the detection of low-abundance proteins. Here, we demonstrate that bead-based enrichment strategies are also disrupting the field of secretomics by enabling easy access to thousands of proteins released into the cell-conditioned media, even in the presence of physiologically relevant growth supplements, such as fetal bovine serum. Using lipopolysaccharide-stimulated THP-1 human acute monocytic leukemia cells as a model of inflammatory activation, we demonstrate that two independent bead-based enrichment platforms, ENRICH-iST and Proteograph, enable comprehensive characterization of secreted proteins in the conditioned media. Application of these approaches provided novel insights into the regulation of cytokine and chemokine signaling pathways that remained inaccessible without enrichment, even when employing labor-intensive and time-consuming deep fractionation workflows. Taken together, these findings underscore the potential of bead-based enrichment methods to overcome long-standing limitations in secretome analysis, offering a scalable and efficient route to uncover complex signaling networks with direct relevance to both fundamental biology and translational research.