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

ABSTRACT Extracellular chaperones engage in extensive protein–protein interactions, complicating their selective enrichment and characterisation in complex biological fluids such as human plasma. Consequently, affinity purification workflows are often employed for their analysis. However, the extensive interaction networks of chaperones make purification conditions particularly important, as they can strongly influence the balance between target selectivity and preservation of biologically relevant interactions. In the present study, we systematically evaluated how purification conditions shape affinity purification outcomes for extracellular chaperones using clusterin as a model system. Affinity purification was performed under conditions ranging from interaction-preserving to interaction-disruptive, followed by bottom-up proteomic analysis to assess enrichment and co-purification profiles. Under interaction-preserving conditions, clusterin was enriched together with a broad range of associated proteins, enabling characterisation of its plasma interactome and providing insight into its biological context. Increasing purification stringency progressively reduced co-purification while maintaining clusterin enrichment. Under the most stringent conditions, co-purifying proteins were minimised, enabling more selective isolation of clusterin. Together, these findings demonstrate that affinity purification outcomes can be systematically directed toward either interactome characterisation or selective target enrichment through controlled adjustment of purification conditions. This work establishes a framework for tailoring affinity purification strategies for extracellular chaperones and other highly interactive proteins.