2 research outputs found
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Systematic evaluation of Copper(II)-loaded immobilized metal affinity chromatography for selective enrichment of copper-binding species in human serum and plasma.
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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Elucidation of a Copper Binding Site in Proinsulin C‑peptide and Its Implications for Metal-Modulated Activity
The connecting peptide (C-peptide) is a hormone with promising health benefits in ameliorating diabetes-related complications, yet mechanisms remain elusive. Emerging studies point to a possible dependence of peptide activity on bioavailable metals, particularly Cu(II) and Zn(II). However, little is known about the chemical nature of the interactions, hindering advances in its therapeutic applications. This work uncovers the Cu(II)-binding site in C-peptide that may be key to understanding its metal-dependent function. A combination of spectroscopic studies reveal that Cu(II) and Zn(II) bind to C-peptide at specific residues in the N-terminal region of the peptide and that Cu(II) is able to displace Zn(II) for C-peptide binding. The data point to a Cu(II)-binding site consisting of 1N3O square-planar coordination that is entropically driven. Furthermore, the entire random coil peptide sequence is needed for specific metal binding as mutations and truncations reshuffle the coordinating residues. These results expand our understanding of how metals influence hormone activity and facilitate the discovery and validation of both new and established paradigms in peptide biology