An Affinity-Based Workflow for Clusterin Purification and Interactome Characterisation in Human Plasma

Clusterin is an extracellular chaperone protein involved in maintaining proteostasis by binding to unfolded or misfolded proteins to prevent their aggregation. Its chaperone activity plays a significant role in human health, as seen in Alzheimers disease, where clusterin co-localises with amyloid-{beta} plaques to facilitate their clearance. However, its strong tendency to "cluster" with a wide range of client proteins and its low abundance in biological matrices complicate its characterisation. To enable comprehensive analysis of clusterin in human plasma, we established two affinity-based purification workflows, one for targeted clusterin purification and a second for interactome analysis, combining affinity enrichment with bottom-up proteomics. Systematic optimisation of the purification workflow revealed that disrupting ionic and hydrophobic protein-protein interactions was essential to improve clusterin enrichment while minimising co-purification. In contrast, preserving native protein-protein interactions enabled clusterin interactome analysis using affinity purification-mass spectrometry (AP-MS). Bottom-up proteomics profiling identified proteins within the clusterin interactome involved in immune response, hemostasis, and high-density lipoprotein (HDL)-related pathways. Together, these complementary workflows enable targeted clusterin purification and systematic characterisation of its interactome, offering new insights into clusterins biological roles in human plasma.