Systematic evaluation of Copper(II)-loaded immobilized metal affinity chromatography for selective enrichment of copper-binding species in human serum and plasma.
Systematic evaluation of Copper(II)-loaded immobilized metal affinity chromatography for selective enrichment of copper-binding species in human serum and plasma.
复制标题
系统评价负载铜 (II) 的固定金属亲和色谱法选择性富集人血清和血浆中的铜结合物质。
DOI：
10.1093/mtomcs/mfac059
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Heffern,MarieC
中科院分区：
文献类型：
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作者：
Janisse,SamuelE;Sharma,VibhaA;Caceres,Amanda;Medici,Valentina;Heffern,MarieC
铜在许多生物过程中是必不可少的，其稳态的破坏与包括神经变性和代谢紊乱在内的疾病有关。细胞外铜在健康和疾病状态之间的形态变化，识别参与这些扰动的分子组分可以拓宽铜状态的生物标志物。虽然用基于质谱的方法研究细胞外蛋白质组的方法已经取得了令人兴奋的进展，但由于这些键在样品制备过程中或气相环境中的不稳定性，典型的工作流程会破坏金属-蛋白质相互作用。我们试图开发和应用的工作流程，以丰富和确定蛋白质群体与铜结合的倾向，在细胞外液中使用固定化金属亲和层析（IMAC）树脂。使用人血清优化该策略以允许最大量和蛋白富集的多样性。蛋白质群体可以基于树脂上的蛋白质负载来区分，可能是由于丰度和亲和力的差异。将富集工作流程应用于来自威尔逊病患者的血浆样品，并将相对于健康受试者的蛋白质ID和差异丰度与从传统蛋白质组学工作流程产生的那些进行比较。虽然IMAC工作流程保留了传统工作流程的差异丰度和蛋白质ID信息，但它鉴定了几种额外的差异丰度蛋白质，包括参与脂质代谢、免疫系统和抗氧化剂途径的蛋白质。我们的研究结果表明，这种IMAC工作流程有可能在铜相关疾病状态中识别新蛋白质作为潜在的生物标志物。
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.