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Title: Systematic evaluation of Copper(II)-loaded immobilized metal affinity chromatography for selective enrichment of copper-binding species in human serum and plasma
Abstract

Copper is essential in a host of biological processes, and disruption of its homeostasis is associated with diseases including neurodegeneration and metabolic disorders. Extracellular copper shifts in its speciation between healthy and disease states, and identifying molecular components involved in these perturbations could widen the panel of biomarkers for copper status. While there have been exciting advances in approaches for studying the extracellular proteome with mass spectrometry–based methods, the typical workflows disrupt metal–protein interactions due to the lability of these bonds either during sample preparation or in gas-phase environments. We sought to develop and apply a workflow to enrich for and identify protein populations with copper-binding propensities in extracellular fluids using an immobilized metal affinity chromatography (IMAC) resin. The strategy was optimized using human serum to allow for maximum quantity and diversity of protein enrichment. Protein populations could be differentiated based on protein load on the resin, likely on account of differences in abundance and affinity. The enrichment workflow was applied to plasma samples from patients with Wilson’s disease and protein IDs and differential abundancies relative to healthy subjects were compared to those yielded from a traditional proteomic workflow. While the IMAC workflow preserved differential abundance and protein ID information from the traditional workflow, it identified several additional proteins being differentially abundant including those involved in lipid metabolism, immune system, and antioxidant pathways. Our results suggest the potential for this IMAC workflow to identify new proteins as potential biomarkers in copper-associated disease states.

 
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NSF-PAR ID:
10370604
Author(s) / Creator(s):
; ; ; ;
Publisher / Repository:
Oxford University Press
Date Published:
Journal Name:
Metallomics
Volume:
14
Issue:
9
ISSN:
1756-591X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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