Mutations in the metalloenzyme copper-zinc superoxide dismutase (SOD1) cause one form of familial amyotrophic lateral sclerosis (ALS), and metals are suspected to play a pivotal role in ALS pathology. To learn more about metals in ALS, we determined the metallation states of human wild-type or mutant (G37R, G93A, and H46R/H48Q) SOD1 proteins from SOD1-ALS transgenic mice spinal cords. SOD1 was gently extracted from spinal cord and separated into insoluble (aggregated) and soluble (supernatant) fractions, and then metallation states were determined by HPLC inductively coupled plasma MS. Insoluble SOD1-rich fractions were not enriched in copper and zinc. However, the soluble mutant and WT SOD1s were highly metallated except for the metal-bindingregion mutant H46R/H48Q, which did not bind any copper. Due to the stability conferred by high metallation of G37R and G93A, it is unlikely that these soluble SOD1s are prone to aggregation in vivo, supporting the hypothesis that immature nascent SOD1 is the substrate for aggregation. We also investigated the effect of SOD1 overexpression and disease on metal homeostasis in spinal cord cross-sections of SOD1-ALS mice using synchrotron-based x-ray fluorescence microscopy. In each mouse genotype, except for the H46R/H48Q mouse, we found a redistribution of copper between gray and white matters correlated to areas of high SOD1. Interestingly, a diseasespecific increase of zinc was observed in the white matter for all mutant SOD1 mice. Together these data provide a picture of copper and zinc in the cell as well as highlight the importance of these metals in understanding SOD1-ALS pathology.First described in 1869 by French neurologist Jean-Martin Charcot (1), amyotrophic lateral sclerosis (ALS) 4 is defined by the progressive demise of lower and upper motor neurons leading to the degeneration of muscle tissue and eventual paralysis (2). Familial ALS was linked in 1993 to mutations in the gene that encodes the metalloenzyme copper-zinc, superoxide dismutase (SOD1) (3), and since that time more than 140 different disease-causing mutations have been identified (4). Before the discovery linking SOD1 and ALS, epidemiological studies on ALS suggested that exposure to metallic trace elements was a significant factor among many other environmental factors such as viruses, strenuous exercise, and excitotoxic chemicals that may have a role in ALS etiology (4 -6). Studies on exposure to and tissue accumulation of metallic elements in ALS patients show a correlative yet unspecified role for many metals including copper and zinc in SOD1-related ALS (7-19). Over the years, a number of hypotheses relating metal toxicity to ALS have been proposed. For example, abnormal accumulations of redox-active metal ions such as iron or copper in ALS patients were generally believed to be detrimental due to their ability to mediate oxidative damage through participation in reactions that lead to formation of reactive oxygen species. A more specific hypothesis linking metallation levels of mutant SOD1 to...

Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALSmutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.aggregation | mass spectrometry | protein misfolding | neurodegeneration A myotrophic lateral sclerosis (ALS) or Lou Gehrig's disease is a devastating motor neuron disease characterized by the formation of abnormal protein aggregates in neuronal cells. More than 100 different mutations in superoxide dismutase-1 (sod1) have been identified and linked to familial ALS (fALS). Although the precise mechanism(s) by which this diverse group of mutations causes fALS remains unclear, it generally is agreed that the ALSmutant SOD1 proteins are prone to misfold and that they acquire toxic properties as a consequence (1-3). Abnormal protein deposits are seen frequently in protein misfolding diseases, and SOD1-containing aggregates have been found consistently in the spinal cords of ALS transgenic mice and fALS patients (4, 5). Moreover, in ALS transgenic mice, these proteinaceous deposits frequently have been shown to have amyloid-like properties such as filamentous structures and the ability to bind thioflavin-S (6-8).A number of other proteins that have been linked to neurodegenerative diseases form amyloid fibrils. Such fibrils are elongated, unbranched, and highly ordered protein aggregates composed mainly of cross-β-sheets, with parallel or anti-parallel β-strands stacking perpendicular to the axis of fibril growth (9-13).Detergent-resistant aggregates isolated from the spinal cords of ALS transgenic mice contain full-length and metal-free human SOD1 (hSOD1) proteins, suggesting that it is full-length metal-free (apo) hSOD1 that acquires toxic properties in the disease mechanism (14). Moreover, as esta...

Determining the composition of aggregated superoxide dismutase 1 (SOD1) species associated with amyotrophic lateral sclerosis (ALS), especially with respect to co-aggregated proteins and post-translational modifications, could identify cellular or biochemical factors involved in the formation of these aggregates and explain their apparent neurotoxicity. The results of mass spectrometric and shotgun-proteomic analyses of SOD1-containing aggregates isolated from spinal cords of symptomatic transgenic ALS mice using two different isolation strategies are presented, including 1) resistance to detergent extraction and 2) size exclusion-coupled anti-SOD1 immunoaffinity chromatography. Forty-eight spinal cords from three different ALS-SOD1 mutant mice were analyzed, namely G93A, G37R, and the unnatural double mutant H46R/H48Q. The analysis consistently revealed that the most abundant proteins recovered from aggregate species were full-length unmodified SOD1 polypeptides. Although aggregates from some spinal cord samples contained trace levels of highly abundant proteins, such as vimentin and neurofilament-3, no proteins were consistently found to co-purify with mutant SOD1 in stoichiometric quantities. The results demonstrate that the principal protein in the high molecular mass aggregates whose appearance correlates with symptoms of the disease is the unmodified, full-length SOD1 polypeptide.Mutations in the gene encoding superoxide dismutase 1 (SOD1) 7 induce familial amyotrophic lateral sclerosis (ALS), and aggregation of the mutant SOD1 protein is hypothesized to cause pathogenesis (1). Support for the aggregation hypothesis includes pathological, cell-culture, and biophysical data such as: 1) the appearance of fibrillar, spherical or irregularly shaped SOD1-containing aggregates in murine and human ALS spinal cord (2-8); 2) the formation of detergent-insoluble SOD1 aggregates with the onset of motor neuron degeneration in ALS mice (2, 9 -12); 3) the observation that cultured neural cells that form SOD1 aggregates die faster than cells that do not (13); and 4) various biophysical and structural perturbations to ALS variant SOD1 proteins (14 -18) that accelerate aggregation in vitro (1).Although the aggregation of SOD1 is a conspicuous signature of SOD1-linked ALS, the protein composition of SOD1 aggregates remains unclear. Identifying proteins that might be co-aggregated with SOD1 could help explain how the aggregates are formed and why they are apparently toxic. For example, the expression of ALS-SOD1 variants in mice and cell cultures can induce: the slowing of axonal transport (19), glutamate excitotoxicity (20,21), and proteasomal inhibition (22,23). Data also suggest that variant SOD1 proteins aggregate with other proteins and lower the concentration of these proteins below a threshold for normal viability (24 -27). Therefore, the identification of binding interactions or co-aggregation between SOD1 and specific proteins involved in any of these processes could provide clues about the cellular processes involv...

Multi-attribute methods (MAM), based on proteolytic digestion followed by liquid chromatography-mass spectrometry analysis of proteolytic peptides, have gained substantial attention in the biopharmaceutical industry for quantifying a variety of quality attributes for therapeutic proteins. Most MAM developed so far have been based on high-resolution mass spectrometers, due to their superb resolving power to distinguish analyte signals from interferences. Lower-resolution instruments, if demonstrated suitable, may further promote the adoption of the technology due to their low cost, small footprint, and ease of use. In this work, we compared the performance of a high-resolution instrument with a few low-resolution quadrupole-type instruments in quantifying a diverse set of quality attributes in a monoclonal antibody product. Different modes of operation for the quadrupole instruments, including scan mode, selected-ion monitoring and multiple-reaction monitoring, were evaluated. The high-resolution instrument has superb performance, with a quantitation limit of 0.002%. Single-quadrupole instruments in scan mode, on the other hand, provide a quantitation limit of about 1%, which may be fit-for-purpose for many routine MAM applications.

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