期刊
ANALYTICAL CHEMISTRY
卷 93, 期 32, 页码 11108-11115出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c01128
关键词
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资金
- Neuroscience Research Australia
- University of New South Wales
- Medical Research Council
- Brains for Dementia Research program by Alzheimer's Research UK
- LABEX BRAIN [ANR-10-LABX-43]
- project CALIPSOplus under EU [730872]
- CNRS/IN2P3, University of Bordeaux
- National Health and Medical Research Council of Australia [1181864, 1132524]
- Parkinson's NSW (Australia)
- University of Sydney (Biomedical Science)
- Motor Neurone Disease Research Institute of Australia
- Dementia Research Team grant [1095127]
- CogSleep Centre of Research Excellence [1152945]
- Brains for Dementia Research program by Alzheimer's Society
- Region Nouvelle Aquitaine
- Shake It Up Australia Foundation
- Flemish Research Foundation FWO [12S5718N]
- ForeFront
- Michael J Fox Foundation for Parkinson's Research
- National Health and Medical Research Council of Australia [1181864] Funding Source: NHMRC
A new methodology was developed to analyze the metal content of copper-zinc superoxide dismutase (SOD1) in tissues from the human central nervous system, demonstrating successful preservation of metal, activity, and enrichment of the protein. Results showed Cu and Zn bound to SOD1 in a ratio very close to the expected value of 1, in tissues from healthy individuals. This approach can be adapted to study other metalloproteins in human tissues and other sources.
Studies of the metal content of metalloproteins in tissues from the human central nervous system (CNS) can be compromised by preparative techniques which alter levels of, or interactions between, metals and the protein of interest within a complex mixture. We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and either proton or synchrotron X-ray fluorescence within electrophoresis gels to analyze the endogenous metal content of copper-zinc superoxide dismutase (SOD1) purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis and Parkinson's disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. Validating our novel approach, we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein versus confounding metalloproteins. We analyzed tissues from nine healthy individuals and five CNS regions (occipital cortex, substantia nigra, locus coeruleus, dorsal spinal cord, and ventral spinal cord). We found that Cu and Zn were bound to SOD1 in a ratio of 1.12 +/- 0.28, a ratio very close to the expected value of 1. Our methodological workflow can be applied to the study of endogenous native SOD1 in a pathological context and adapted to a range of metalloproteins from human tissues and other sources.
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