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201218_ChemRXiv_Roudeau_et_al.pdf (1.9 MB)

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

preprint
submitted on 18.12.2020, 14:37 and posted on 21.12.2020, 07:42 by 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
We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and high sensitivity X-ray-based metal detection within electrophoresis gels to analyze the metal content of single proteins purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. An important metalloprotein in the human central nervous system is copper-zinc superoxide dismutase (SOD1), an antioxidant enzyme linked to the aetiology of both amyotrophic lateral sclerosis and Parkinson’s disease. Abnormal SOD1 metallation is suspected to play a role in the pathogenic aggregation of SOD1 in both disorders, although data describing SOD1 metal occupancy in human tissues has not previously been reported. Validating our novel approach we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein vs confounding metalloproteins. We found Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28 in human central nervous system tissues from healthy individuals, a ratio close to the expected value of 1. Our methodological workflow can be adapted to study a range of metalloproteins from human tissues and other sources.

Funding

project CALIPSOplus under the Grant Agreement 730872 from the EU Framework Programme for Research and Innovation HORIZON 2020

AIFIRA facility is financially supported by CNRS/IN2P3, University of Bordeaux and the Région Nouvelle Aquitaine (France)

National Health and Medical Research Council of Australia (1181864)

Parkinson’s NSW (Australia)

the University of Sydney (Biomedical Science)

The Motor Neurone Disease Research Institute of Australia

The Michael J Fox Foundation for Parkinson’s Research in partnership with the Shake It Up Australia Foundation

National Health and Medical Research Council of Australia Program Grant (1132524)

Dementia Research Team Grant (1095127)

CogSleep Centre of Research Excellence (1152945)

Flemish Research Foundation FWO (grant number 12S5718N)

History

Email Address of Submitting Author

roudeau@cenbg.in2p3.fr

Institution

Univ. Bordeaux, CNRS, CENBG, UMR-5797, F-33170 Gradignan

Country

France

ORCID For Submitting Author

0000-0002-4539-9380

Declaration of Conflict of Interest

The authors declare no conflict of interest

Version Notes

version 1, uploaded 18 December 2020

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