Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro, and in transgenic animal models of amyotrophic lateral sclerosis (ALS). Detailed examination of the protein in disease-affected tissues from ALS patients, however, remains scarce. We employed histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from ALS cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally-disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial ALS cases, and sporadic ALS cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically-active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in ALS cases, and clearly differentiated all forms of ALS from controls. Substantial heterogeneity in the presence of these changes was also observed between ALS cases. Our data demonstrates that varying forms of SOD1 proteinopathy are a common feature of all forms of ALS, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in ALS. The majority of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.

Aims: Cysteine (Cys) is a major target for redox post-translational modifications (PTMs) that occur in response to changes in the cellular redox environment. We describe multiplexed, peptide-based enrichment and quantitative mass spectrometry (MS) applied to globally profile reversible redox Cys PTM in rat hearts during ischemia/ reperfusion (I/R) in the presence or absence of an aminothiol antioxidant, N-2-mercaptopropionylglycine (MPG). Parallel fractionation also allowed identification of irreversibly oxidized Cys peptides (Cys-SO 2 H/SO 3 H). Results: We identified 4505 reversibly oxidized Cys peptides of which 1372 were significantly regulated by ischemia and/or I/R. An additional 219 peptides (247 sites) contained Cys-SO 2 H/Cys-SO 3 H modifications, and these were predominantly identified from hearts subjected to I/R (n = 168 peptides). Parallel reaction monitoring MS (PRM-MS) enabled relative quantitation of 34 irreversibly oxidized Cys peptides. MPG attenuated a large cluster of I/R-associated reversibly oxidized Cys peptides and irreversible Cys oxidation to less than nonischemic controls (n = 24 and 34 peptides, respectively). PRM-MS showed that Cys sites oxidized during ischemia and/or I/R and ''protected'' by MPG were largely mitochondrial, and were associated with antioxidant functions (peroxiredoxins 5 and 6) and metabolic processes, including glycolysis. Metabolomics revealed I/R induced changes in glycolytic intermediates that were reversed in the presence of MPG, which were consistent with irreversible PTM of triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), altered GAPDH enzyme activity, and reduced I/R glycolytic payoff as evidenced by adenosine triphosphate and NADH levels. Innovation: Novel enrichment and PRM-MS approaches developed here enabled large-scale relative quantitation of Cys redox sites modified by reversible and irreversible PTM during I/R and antioxidant remediation. Conclusions: Cys sites identified here are targets of reactive oxygen species that can contribute to protein dysfunction and the pathogenesis of I/R. Antioxid. Redox Signal. 34,

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