standards-for-quantitative-metalloproteomic-analysis-using-size

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

The Florey Institute of Neuroscience and Mental Health

Trace elements are required for a variety of normal biological functions. As our understanding of neurodegenerative disease advances we are identifying a number of metalloenzymes involved in disease process. Thus, the future of metals in neurobiology will rely more on detailed information regarding what metalloenzymes are present and how they are involved in the pathophysiology of disease. To gain this detailed information, we will rely less on bulk measures of the amount of a trace elements in a particular tissue and turn to metalloproteomic techniques to help elucidate both metalloprotein structure and function. Recent advances in metalloproteomics will translate to a richer understanding of the mechanism and precise role of metalloenzymes and proteins in the brain.

Metalloproteomics: principles, challenges and applications to neurodegeneration.

Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method, which was applied to profiling the metal-binding proteins found in primary cultures of neurons and astrocytes. This method can be conducted using similar amounts of sample to those used for Western blotting (20-150 μg protein), and has a turnaround time of <15 minutes. Metalloprotein standards for Fe (as ferritin), Cu and Zn (as superoxide dismutase-1) were used to construct multi-point calibration curves for online quantification of metalloproteins by SEC-ICP-MS. Homogenates of primary neuron and astrocyte cultures were analysed by SEC-ICP-MS. Online quantification by external calibration with metalloprotein standards determined the mass of metal eluting from the column relative to time (as pg s(-1)). Total on-column Fe, Cu and Zn detection limits ranged from 0.825 ± 0.005 ng to 13.6 ± 0.7 pg. Neurons and astrocytes exhibited distinct metalloprotein profiles, featuring both ubiquitous and unique metalloprotein species. Separation and detection by SEC-ICP-MS allows appraisal of these metalloproteins in their native state, and online quantification was achieved using this relatively simple external calibration process.

Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion that encodes a polyglutamine tract in huntingtin (htt) protein. Dysregulation of brain iron homeostasis, oxidative stress and neurodegeneration are consistent features of the HD phenotype. Therefore, environmental factors that exacerbate oxidative stress and iron dysregulation may potentiate HD. Iron supplementation in the human population is common during infant and adult-life stages. In this study, iron supplementation in neonatal HD mice resulted in deterioration of spontaneous motor running activity, elevated levels of brain lactate and oxidized glutathione consistent with increased energetic dysfunction and oxidative stress, and increased striatal and motor cortical neuronal atrophy, collectively demonstrating potentiation of the disease phenotype. Oxidative stress, energetic, and anatomic markers of degeneration were not affected in wild-type littermate iron-supplemented mice. Further, there was no effect of elevated iron intake on disease outcomes in adult HD mice. We have demonstrated an interaction between the mutant huntingtin gene and iron supplementation in neonatal HD mice. Findings indicate that elevated neonatal iron intake potentiates mouse HD and promotes oxidative stress and energetic dysfunction in brain. Neonatal-infant dietary iron intake level may be an environmental modifier of human HD.

Neonatal iron supplementation potentiates oxidative stress, energetic dysfunction and neurodegeneration in the R6/2 mouse model of Huntington's disease.

Amber Lothian

Metalloproteomics Laboratory,
Neuroproteomics Facility,
Metalloproteomics Laboratory, Neuroproteomics Facility

The Florey Institute of Neuroscience and Mental Health

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Amber Lothian

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The Florey Institute of Neuroscience and Mental Health

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metalloproteomics Laboratory,
Neuroproteomics Facility,
Metalloproteomics Laboratory, Neuroproteomics Facility