The purpose of this essay is to propose that metallomic studies in the area of extracellular copper transport are incomplete without the explicit consideration of kinetics of Cu2+ion binding and exchange reactions. The kinetic data should be interpreted in the context of time constraints imposed by specific physiological processes. Examples from experimental studies of Cu2+ ion interactions with amino-terminal copper and nickel binding site/N-terminal site motifs are used to demonstrate that duration and periodicity of such processes as bloodstream transport or neurotransmission promote the reaction intermediates to the role of physiological effectors. The unexpectedly long lifetimes of intermediate complexes lead to their accumulation and novel reactivities. The emerging ideas are discussed in the context of other research areas in metallomics.

Copper is a highly reactive element involved in a myriad of biological reactions. Thus, while essential for mammalian cells, its concentrations must be kept in check in order to avoid toxicity. This metal participates in redox reactions and may exacerbate oxidative stress in aerobic organisms. Nonetheless, the actual driving force of copper-induced cell death is yet unknown. Likely, free copper ions may target different biomolecules that are crucial for the proper functioning of an organism. In this work, we show that free copper induces protein aggregation in serum. The wide set of proteins present in these biological samples are not equally prone to copper-induced aggregation and some, such as albumin, are highly resistant, whereas γ-globulins are highly sensitive. The identity of the proteins in the aggregates becomes fairly homogeneous as metal concentrations go as low as 20 μM. The identification of the proteins by mass spectrometry indicates a preponderance of IgG and a minor presence of other different proteins. Therefore, free copper in blood may contribute to the formation of circulating protein aggregates with a core of IgG. This may impact health not only due to the activity of aggregated IgG but also due to the many proteins co-aggregated. Understanding whether the γ-globulin core and the heterogeneous subgroup of proteins elicit differential responses in the organisms requires further research.

Synchrotron X-ray fluorescence microscopy (SXRF) presents a valuable opportunity to study the metallome of single cells because it simultaneously provides high-resolution subcellular distribution and quantitative cellular content of multiple elements. Different sample preparation techniques have been used to preserve cells for observations with SXRF, with a goal to maintain fidelity of the cellular metallome. In this case study, mouse pancreatic beta-cells have been preserved with optimized chemical fixation. We show that cell-to-cell variability is normal in the metallome of beta-cells due to heterogeneity and should be considered when interpreting SXRF data. In addition, we determined the impact of several immunofluorescence (IF) protocols on metal distribution and quantification in chemically fixed beta-cells and found that the metallome of beta-cells was not well preserved for quantitative analysis. However, zinc and iron qualitative analysis could be performed after IF with certain limitations. To help minimize metal loss using samples that require IF, we describe a novel IF protocol that can be used with chemically fixed cells after the completion of SXRF.