Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow.

Stéphane Roudeau, Benjamin G Trist, Asuncion Carmona, Katherine M Davies, Glenda M Halliday, Yann Rufin, Stéphane Claverol, Stijn J M Van Malderen, Gerald Falkenberg, Kay L Double, Richard Ortega

Analytical chemistry 2021 Aug 17

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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.

Citation

Stéphane Roudeau, Benjamin G Trist, Asuncion Carmona, Katherine M Davies, Glenda M Halliday, Yann Rufin, Stéphane Claverol, Stijn J M Van Malderen, Gerald Falkenberg, Kay L Double, Richard Ortega. Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow. Analytical chemistry. 2021 Aug 17;93(32):11108-11115