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Multiple sclerosis (MS) is a debilitating affliction of the central nervous system (CNS) that involves demyelination of neuronal axons and neurodegeneration resulting in disability that becomes more pronounced in progressive forms of the disease. The involvement of neurodegeneration in MS underscores the need for effective neuroprotective approaches necessitating identification of new therapeutic targets. Herein, we applied an integrated elemental analysis workflow to human MS-affected spinal cord tissue utilising multiple inductively coupled plasma-mass spectrometry methodologies. These analyses revealed shifts in atomic copper as a notable aspect of disease. Complementary gene expression and biochemical analyses demonstrated that changes in copper levels coincided with altered expression of copper handling genes and downstream functionality of cuproenzymes. Copper-related problems observed in the human MS spinal cord were largely reproduced in the experimental autoimmune encephalomyelitis (EAE) mouse model during the acute phase of disease characterised by axonal demyelination, lesion formation, and motor neuron loss. Treatment of EAE mice with the CNS-permeant copper modulating compound Cu II (atsm) resulted in recovery of cuproenzyme function, improved myelination and lesion volume, and neuroprotection. These findings support targeting copper perturbations as a therapeutic strategy for MS with Cu II (atsm) showing initial promise., Competing Interests: Declaration of competing interest Collaborative Medicinal Development has licensed intellectual property pertaining to Cu(II)(atsm) from the University of Melbourne where the inventors include ARW and PSD. AIB is a paid consultant for Collaborative Medicinal Development LLC and has a profit share interest in Collaborative Medicinal Development Pty Ltd. PJC and JSB are unpaid consultants for Collaborative Medicinal Development LLC. DJH received research and material support from Agilent Technologies and ESI Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The copper compound Cu II (atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Cu II (atsm) is neuroprotective in mutant SOD1 mouse models of ALS where its activity is ascribed in part to improving availability of essential copper. However, SOD1 mutations cause only ~ 2% of ALS cases and therapeutic relevance of copper availability in sporadic ALS is unresolved. Herein we assessed spinal cord tissue from human cases of sporadic ALS for copper-related changes. We found that when compared to control cases the natural distribution of spinal cord copper was disrupted in sporadic ALS. A standout feature was decreased copper levels in the ventral grey matter, the primary anatomical site of neuronal loss in ALS. Altered expression of genes involved in copper handling indicated disrupted copper availability, and this was evident in decreased copper-dependent ferroxidase activity despite increased abundance of the ferroxidases ceruloplasmin and hephaestin. Mice expressing mutant SOD1 recapitulate salient features of ALS and the unsatiated requirement for copper in these mice is a biochemical target for Cu II (atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for Cu II (atsm) involving copper availability may also be pertinent to sporadic cases of ALS., (© 2024. The Author(s).)

Background: Ferroptosis is a form of regulated cell death characterised by lipid peroxidation as the terminal endpoint and a requirement for iron. Although it protects against cancer and infection, ferroptosis is also implicated in causing neuronal death in degenerative diseases of the central nervous system (CNS). The precise role for ferroptosis in causing neuronal death is yet to be fully resolved., Methods: To elucidate the role of ferroptosis in neuronal death we utilised co-culture and conditioned medium transfer experiments involving microglia, astrocytes and neurones. We ratified clinical significance of our cell culture findings via assessment of human CNS tissue from cases of the fatal, paralysing neurodegenerative condition of amyotrophic lateral sclerosis (ALS). We utilised the SOD1 G37R mouse model of ALS and a CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo., Results: We found that sublethal ferroptotic stress selectively affecting microglia triggers an inflammatory cascade that results in non-cell autonomous neuronal death. Central to this cascade is the conversion of astrocytes to a neurotoxic state. We show that spinal cord tissue from human cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Further, we show the molecular correlation between ferroptosis and neurotoxic astrocytes evident in human ALS-affected spinal cord is recapitulated in the SOD1 G37R mouse model where treatment with a CNS-permeant ferroptosis inhibitor, Cu II (atsm), ameliorated these markers and was neuroprotective., Conclusions: By showing that microglia responding to sublethal ferroptotic stress culminates in non-cell autonomous neuronal death, our results implicate microglial ferroptotic stress as a rectifiable cause of neuronal death in neurodegenerative disease. As ferroptosis is currently primarily regarded as an intrinsic cell death phenomenon, these results introduce an entirely new pathophysiological role for ferroptosis in disease., (© 2024. The Author(s).)

14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH 2 , site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3β phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric-but not dimeric-14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Tandem mass spectrometry of denatured, multiply charged high mass protein precursor ions yield extremely dense spectra with hundreds of broad and overlapping product ion isotopic distributions of differing charge states that yield an elevated baseline of unresolved "noise" centered about the precursor ion. Development of mass analyzers and signal processing methods to increase mass resolving power and manipulation of precursor and product ion charge through solution additives or ion-ion reactions have been thoroughly explored as solutions to spectral congestion. Here, we demonstrate the utility of electron capture dissociation (ECD) coupled with high-resolution cyclic ion mobility spectrometry (cIMS) to greatly increase top-down protein characterization capabilities. Congestion of protein ECD spectra was reduced using cIMS of the ECD product ions and "mobility fractions", that is, extracted mass spectra for segments of the 2D mobiligram ( m / z versus drift time). For small proteins, such as ubiquitin (8.6 kDa), where mass resolving power was not the limiting factor for characterization, pre-IMS ECD and mobility fractions did not significantly increase protein sequence coverage, but an increase in the number of identified product ions was observed. However, a dramatic increase in performance, measured by protein sequence coverage, was observed for larger and more highly charged species, such as the +35 charge state of carbonic anhydrase (29 kDa). Pre-IMS ECD combined with mobility fractions yielded a 135% increase in the number of annotated isotope clusters and a 75% increase in unique product ions compared to processing without using the IMS dimension. These results yielded 89% sequence coverage for carbonic anhydrase.

Mitochondria serve vital roles critical for overall cellular function outside of energy transduction. Thus, mitochondrial decay is postulated to be a key factor in aging and in age-related diseases. Mitochondria may be targets of their own decay through oxidative damage. However, treating animals with antioxidants has been met with only limited success in rejuvenating mitochondrial function or in increasing lifespan. A host of nutritional strategies outside of using traditional antioxidants have been devised to promote mitochondrial function. Dietary compounds are under study that induce gene expression, enhance mitochondrial biogenesis, mitophagy, or replenish key metabolites that decline with age. Moreover, redox-active compounds may now be targeted to mitochondria which improve their effectiveness. Herein we review the evidence that representative dietary effectors modulate mitochondrial function by stimulating their renewal or reversing the age-related loss of key metabolites. While in vitro evidence continues to accumulate that many of these compounds benefit mitochondrial function and/or prevent their decay, the results using animal models and, in some instances human clinical trials, are more mixed and sometimes even contraindicated. Thus, further research on optimal dosage and age of intervention are warranted before recommending potential mitochondrial rejuvenating compounds for human use., (Published by Elsevier Inc.)

Localizing metal binding to specific sites in proteins remains a challenging analytical problem in vitro and in vivo. Although metal binding can be maintained by "native" electrospray ionization with intact proteins for quantitation by mass spectrometry, subsequent fragmentation of proteins with slow-heating methods like collision-induced dissociation (CID) can scramble and detach metals. In contrast, electron capture dissociation (ECD) fragmentation produces highly localized bond cleavage that is well known to preserve posttranslational modifications. We show how a newly available ECD tool that can be retrofitted on standard QTOF mass spectrometers allows the sites of copper and zinc binding to be localized in the antioxidant enzyme Cu, Zn superoxide dismutase (SOD1). The loss of zinc from Cu, Zn SOD1 has been shown to induce motor neuron death and could have a causal role in the fatal neurodegenerative disease, amyotrophic lateral sclerosis (ALS). The methods described enable copper loss to be distinguished from zinc using distinct ECD fragments of SOD1 and are broadly applicable to other metalloproteins., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Electron-based dissociation (ExD) produces uncluttered mass spectra of intact proteins while preserving labile post-translational modifications. However, technical challenges have limited this option to only a few high-end mass spectrometers. We have developed an efficient ExD cell that can be retrofitted in less than an hour into current LC/Q-TOF instruments. Supporting software has been developed to acquire, process, and annotate peptide and protein ExD fragmentation spectra. In addition to producing complementary fragmentation, ExD spectra enable many isobaric leucine/isoleucine and isoaspartate/aspartate pairs to be distinguished by side-chain fragmentation. The ExD cell preserves phosphorylation and glycosylation modifications. It also fragments longer peptides more efficiently to reveal signaling cross-talk between multiple post-translational modifications on the same protein chain and cleaves disulfide bonds in cystine knotted proteins and intact antibodies. The ability of the ExD cell to combine collisional activation with electron fragmentation enables more complete sequence coverage by disrupting intramolecular electrostatic interactions that can hold fragments of large peptides and proteins together. These enhanced capabilities made possible by the ExD cell expand the size of peptides and proteins that can be analyzed as well as the analytical certainty of characterizing their post-translational modifications.

Mass spectrometry (MS)-based denaturing top-down proteomics (dTDP) requires high-capacity separation and extensive gas-phase fragmentation of proteoforms. Herein, we coupled capillary zone electrophoresis (CZE) to electron-capture collision-induced dissociation (ECciD) on an Agilent 6545 XT quadrupole time-of-flight (Q-TOF) mass spectrometer for dTDP for the first time. During ECciD, the protein ions were first fragmented using ECD, followed by further activation and fragmentation by applying a CID potential. In this pilot study, we optimized the CZE-ECciD method for small proteins (lower than 20 kDa) regarding the charge state of protein parent ions for fragmentation and the CID potential applied to maximize the protein backbone cleavage coverage and the number of sequence-informative fragment ions. The CZE-ECciD Q-TOF platform provided extensive backbone cleavage coverage for three standard proteins lower than 20 kDa from only single charge states in a single CZE-MS/MS run in the targeted MS/MS mode, including ubiquitin (97%, +7, 8.6 kDa), superoxide dismutase (SOD, 87%, +17, 16 kDa), and myoglobin (90%, +16, 17 kDa). The CZE-ECciD method produced comparable cleavage coverage of small proteins (i.e., myoglobin) with direct-infusion MS studies using electron transfer dissociation (ETD), activated ion-ETD, and combinations of ETD and collision-based fragmentation on high-end orbitrap mass spectrometers. The results render CZE-ECciD a new tool for dTDP to enhance both separation and gas-phase fragmentation of proteoforms.

Distal axonopathy is a recognized pathological feature of amyotrophic lateral sclerosis (ALS). In the peripheral nerves of ALS patients, motor axon loss elicits a Wallerian-like degeneration characterized by denervated Schwann cells (SCs) together with immune cell infiltration. However, the pathogenic significance of denervated SCs accumulating following impaired axonal growth in ALS remains unclear. Here, we analyze SC phenotypes in sciatic nerves of ALS patients and paralytic SOD1 G93A rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100β, GFAP, isolectin and/or p75 NTR immunoreactivity. Different subsets of reactive SCs expressed colony-stimulating factor-1 (CSF1) and Interleukin-34 (IL-34) and closely interacted with numerous endoneurial CSF-1R-expressing monocyte/macrophages, suggesting a paracrine mechanism of myeloid cell expansion and activation. SCs bearing phagocytic phenotypes as well as endoneurial macrophages expressed stem cell factor (SCF), a trophic factor that attracts and activates mast cells through the c-Kit receptor. Notably, a subpopulation of Ki67+ SCs expressed c-Kit in the sciatic nerves of SOD1 G93A rats, suggesting a signaling pathway that fuels SC proliferation in ALS. c-Kit+ mast cells were also abundant in the sciatic nerve from ALS donors but not in controls. Pharmacological inhibition of CSF-1R and c-Kit with masitinib in SOD1 G93A rats potently reduced SC reactivity and immune cell infiltration in the sciatic nerve and ventral roots, suggesting a mechanism by which the drug ameliorates peripheral nerve pathology. These findings provide strong evidence for a previously unknown inflammatory mechanism triggered by SCs in ALS peripheral nerves that has broad application in developing novel therapies., (© 2019 Wiley Periodicals, Inc.)

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multilevel experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the Thermoplasma acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast ( Saccharomyces cerevisiae ) and mammals (rat - Rattus norvegicus , rabbit - Oryctolagus cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native MS structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution, three-dimensional structures, highly resembled that of the human complex. Using cryoelectron microscopy single-particle analysis, we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument's existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.

One challenge associated with the discovery and development of monoclonal antibody (mAb) therapeutics is the determination of heavy chain and light chain pairing. Advances in MS instrumentation and MS/MS methods have greatly enhanced capabilities for the analysis of large intact proteins yielding much more detailed and accurate proteoform characterization. Consequently, direct interrogation of intact antibodies or F(ab')2 and Fab fragments has the potential to significantly streamline therapeutic mAb discovery processes. Here, we demonstrate for the first time the ability to efficiently cleave disulfide bonds linking heavy and light chains of mAbs using electron capture dissociation (ECD) and 157 nm ultraviolet photodissociation (UVPD). The combination of intact mAb, Fab, or F(ab')2 mass, intact LC and Fd masses, and CDR3 sequence coverage enabled determination of heavy chain and light chain pairing from a single experiment and experimental condition. These results demonstrate the potential of top-down and middle-down proteomics to significantly streamline therapeutic antibody discovery.

CuATSM is a PET-imaging agent that has recently received attention for its success in extending the lifespan in animals in several neurodegenerative disease models. In the SOD1 G93A model of ALS, CuATSM prolonged mouse longevity far longer than any previously tested therapeutic agents. The mechanism underlying this outcome has not been fully understood, but studies suggest that this copper complex contributes to maintaining copper homeostasis in mitochondria. More specifically for the SOD1 model, the molecule supplies copper back to the SOD1 protein. Additionally, CuATSM demonstrated similar protective effects in various in vivo Parkinson's disease mouse models. In the current pilot study, we utilized a neurodegenerative mouse model of motor neuron degeneration induced by the neurotoxin β-sitosterol β-D-glucoside. In this model, slow but distinct and progressive features of sporadic ALS occur. Treatment with CuATSM kept animal behavioural performance on par with the controls and prevented the extensive motor neuron degeneration and microglia activation seen in the untreated animals. These outcomes support a broader neuroprotective role for CuATSM beyond mutant SOD models of ALS., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of upper and lower motor neurons accompanied by proliferation of reactive microglia in affected regions. However, it is unknown whether the hematopoietic marker CD34 can identify a subpopulation of proliferating microglial cells in the ALS degenerating spinal cord. Immunohistochemistry for CD34 and microglia markers was performed in lumbar spinal cords of ALS rats bearing the SOD1 G93A mutation and autopsied ALS and control human subjects. Characterization of CD34-positive cells was also performed in primary cell cultures of the rat spinal cords. CD34 was expressed in a large number of cells that closely interacted with degenerating lumbar spinal cord motor neurons in symptomatic SOD1 G93A rats, but not in controls. Most CD34 + cells co-expressed the myeloid marker CD11b, while only a subpopulation was stained for Iba1 or CD68. Notably, CD34 + cells actively proliferated and formed clusters adjacent to damaged motor neurons bearing misfolded SOD1. CD34 + cells were identified in the proximity of motor neurons in autopsied spinal cord from sporadic ALS subjects but not in controls. Cell culture of symptomatic SOD1 G93A rat spinal cords yielded a large number of CD34 + cells exclusively in the non-adherent phase, which generated microglia after successive passaging. A yet unrecognized CD34 + cells, expressing or not the microglial marker Iba1, proliferate and accumulate adjacent to degenerating spinal motor neurons, representing an intriguing cell target for approaching ALS pathogenesis and therapeutics.

Age is a recognized risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive loss of motor neurons and neuroinflammation. A hallmark of aging is the accumulation of senescent cells. Yet, the pathogenic role of cellular senescence in ALS remains poorly understood. In rats bearing the ALS-linked SOD1 G93A mutation, microgliosis contribute to motor neuron death, and its pharmacologic downregulation results in increased survival. Here, we have explored whether gliosis and motor neuron loss were associated with cellular senescence in the spinal cord during paralysis progression. In the lumbar spinal cord of symptomatic SOD1 G93A rats, numerous cells displayed nuclear p16 INK4a as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16 INK4a -positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16 INK4a -positive nuclei and Lamin B1-negative nuclei were typically localized in a subset of hypertrophic Iba1-positive microglia, occasionally exhibiting nuclear giant multinucleated cell aggregates and abnormal nuclear morphology. Next, we analyzed senescence markers in cell cultures of microglia obtained from the spinal cord of symptomatic SOD1 G93A rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days in vitro and subsequent passages. Senescent SOD1 G93A microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-β-Gal) activity as well as positive labeling for p16 INK4a , p53, matrix metalloproteinase-1 (MMP-1) and nitrotyrosine, suggesting a senescent-associated secretory phenotype (SASP). Remarkably, in the degenerating lumbar spinal cord other cell types, including ChAT-positive motor neurons and GFAP-expressing astrocytes, also displayed nuclear p16 INK4a staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1 G93A rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS.

Neuroinflammation is a recognized pathogenic mechanism underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS), but the inflammatory mechanisms influencing peripheral motor axon degeneration remain largely unknown. A recent report showed a pathogenic role for c-Kit-expressing mast cells mediating inflammation and neuromuscular junction denervation in muscles from SOD1G93A rats. Here, we have explored whether mast cells infiltrate skeletal muscles in autopsied muscles from ALS patients. We report that degranulating mast cells were abundant in the quadriceps muscles from ALS subjects but not in controls. Mast cells were associated with myofibers and motor endplates and, remarkably, interacted with neutrophils forming large extracellular traps. Mast cells and neutrophils were also abundant around motor axons in the extensor digitorum longus muscle, sciatic nerve, and ventral roots of symptomatic SOD1G93A rats, indicating that immune cell infiltration extends along the entire peripheral motor pathway. Postparalysis treatment of SOD1G93A rats with the tyrosine kinase inhibitor drug masitinib prevented mast cell and neutrophil infiltration, axonal pathology, secondary demyelination, and the loss of type 2B myofibers, compared with vehicle-treated rats. These findings provide further evidence for a yet unrecognized contribution of immune cells in peripheral motor pathway degeneration that can be therapeutically targeted by tyrosine kinase inhibitors.

Compared to traditional collision induced dissociation methods, electron capture dissociation (ECD) provides more comprehensive characterization of large peptides and proteins as well as preserves labile post-translational modifications. However, ECD experiments are generally restricted to the high magnetic fields of FTICR-MS that enable the reaction of large polycations and electrons. Here, we demonstrate the use of an electromagnetostatic ECD cell to perform ECD and hybrid ECD methods utilizing 193 nm photons (ECuvPD) or collisional activation (EChcD) in a benchtop quadrupole-Orbitrap mass spectrometer. The electromagnetostatic ECD cell was designed to replace the transfer octapole between the quadrupole and C-trap. This implementation enabled facile installation of the ECD cell, and ions could be independently subjected to ECD, UVPD, HCD, or any combination. Initial benchmarking and characterization of fragmentation propensities for ECD, ECuvPD, and EChcD were performed using ubiquitin (8.6 kDa). ECD yielded extensive sequence coverage for low charge states of ubiquitin as well as for the larger protein carbonic anhydrase II (29 kDa), indicating pseudo-activated ion conditions. Additionally, relatively high numbers of d- and w-ions enable differentiation of isobaric isoleucine and leucine residues and suggest a distribution of electron energies yield hot-ECD type fragmentation. We report the most comprehensive characterization to date for model proteins up to 29 kDa and a monoclonal antibody at the subunit level. ECD, ECuvPD, and EChcD yielded 93, 95, and 91% sequence coverage, respectively, for carbonic anhydrase II (29 kDa), and targeted online analyses of monoclonal antibody subunits yielded 86% overall antibody sequence coverage.

Since 1981, Gordon Research Conferences have been held on the topic of Oxygen Radicals on a biennial basis, to highlight and discuss the latest cutting edge research in this area. Since the first meeting, one special feature of this conference has been the awarding of the so-called Iron Bolt, an award that started in jest but has gained increasing reputation over the years. Since no written documentation exists for this Iron Bolt award, this perspective serves to overview the history of this unusual award, and highlights various experiences of previous winners of this "prestigious" award and other interesting anecdotes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Intracellular delivery of therapeutic or analytic copper from copper bis-thiosemicabazonato complexes is generally described in terms of mechanisms involving one-electron reduction to the Cu(I) analogue by endogenous reductants, thereby rendering the metal ion labile and less strongly coordinating to the bis-thiosemicarbazone (btsc) ligand. However, electrochemical and spectroscopic studies described herein indicate that one-electron oxidation of Cu II (btsc) and Zn II ATSM (btsc = diacetyl-bis(4-methylthiosemicarbazonato)) complexes occurs within the range of physiological oxidants, leading to the likelihood that unrecognized oxidative pathways for copper release also exist. Oxidations of Cu II (btsc) by H 2 O 2 catalyzed by either myeloperoxidase or horseradish peroxidase, by HOCl and taurine chloramine (which are chlorinating agents generated primarily in activated neutrophils from MPO-catalyzed reactions), and by peroxynitrite species (ONOOH, ONOOCO 2 - ) that can form under certain conditions of oxidative stress are demonstrated. Unlike reduction, the oxidative reactions proceed by irreversible ligand oxidation, culminating in release of Cu(II). 2-Pyridylazoresorcinol complexation was used to demonstrate that Cu(II) release by reaction with peroxynitrite species involved rate-limiting homolysis of the peroxy O-O bond to generate secondary oxidizing radicals (NO 2 • , • OH, and CO 3 •- ). Because the potentials for Cu II (btsc) oxidation and reduction are ligand-dependent, varying by as much as 200 mV, it is clearly advantageous in designing therapeutic methodologies for specific treatments to identify the operative Cu-release pathway.

Introduction: Glycating stress can occur together with oxidative stress during neurodegeneration and contribute to the pathogenic mechanism. Nerve growth factor (NGF) accumulates in several neurodegenerative diseases. Besides promoting survival, NGF can paradoxically induce cell death by signaling through the p75 neurotrophin receptor (p75 NTR ). The ability of NGF to induce cell death is increased by nitration of its tyrosine residues under conditions associated with increased peroxynitrite formation., Aims: Here we investigated whether glycation also changes the ability of NGF to induce cell death and assessed the ability of post-translational modified NGF to signal through the receptor for advanced glycation end products (RAGEs). We also explored the potential role of RAGE-p75 NTR interaction in the motor neuron death occurring in amyotrophic lateral sclerosis (ALS) models., Results: Glycation promoted NGF oligomerization and ultimately allowed the modified neurotrophin to signal through RAGE and p75 NTR to induce motor neuron death at low physiological concentrations. A similar mechanism was observed for nitrated NGF. We provide evidence for the interaction of RAGE with p75 NTR at the cell surface. Moreover, we observed that post-translational modified NGF was present in the spinal cord of an ALS mouse model. In addition, NGF signaling through RAGE and p75 NTR was involved in astrocyte-mediated motor neuron toxicity, a pathogenic feature of ALS., Innovation: Oxidative modifications occurring under stress conditions can enhance the ability of mature NGF to induce neuronal death at physiologically relevant concentrations, and RAGE is a new p75 NTR coreceptor contributing to this pathway., Conclusion: Our results indicate that NGF-RAGE/p75 NTR signaling may be a therapeutic target in ALS. Antioxid. Redox Signal. 28, 1587-1602.

As the application of mass spectrometry intensifies in scope and diversity, the need for advanced instrumentation addressing a wide variety of analytical needs also increases. To this end, many modern, top-end mass spectrometers are designed or modified to include a wider range of fragmentation technologies, for example, ECD, ETD, EThcD, and UVPD. Still, the majority of instrument platforms are limited to more conventional methods, such as CID and HCD. While these latter methods have performed well, the less conventional fragmentation methods have been shown to lead to increased information in many applications including middle-down proteomics, top-down proteomics, glycoproteomics, and disulfide bond mapping. We describe the modification of the popular Q Exactive Orbitrap mass spectrometer to extend its fragmentation capabilities to include ECD. We show that this modification allows ≥85% matched ion intensity to originate from ECD fragment ion types as well as provides high sequence coverage (≥60%) of intact proteins and high fragment identification rates with ∼70% of ion signals matched. Finally, the ECD implementation promotes selective disulfide bond dissociation, facilitating the identification of disulfide-linked peptide conjugates. Collectively, this modification extends the capabilities of the Q Exactive Orbitrap mass spectrometer to a range of new applications.

Evidence indicates that neuroinflammation contributes to motor neuron degeneration in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease leading to progressive muscular paralysis. However, it remains elusive whether inflammatory cells can interact with degenerating distal motor axons, influencing the progressive denervation of neuromuscular junctions (NMJs). By analyzing the muscle extensor digitorum longus (EDL) following paralysis onset in the SOD1G93A rat model, we have observed a massive infiltration and degranulation of mast cells, starting after paralysis onset and correlating with progressive NMJ denervation. Remarkably, mast cells accumulated around degenerating motor axons and NMJs, and were also associated with macrophages. Mast cell accumulation and degranulation in paralytic EDL muscle was prevented by systemic treatment over 15 days with masitinib, a tyrosine kinase inhibitor currently in clinical trials for ALS exhibiting pharmacological activity affecting mast cells and microglia. Masitinib-induced mast cell reduction resulted in a 35% decrease in NMJ denervation and reduced motor deficits as compared with vehicle-treated rats. Masitinib also normalized macrophage infiltration, as well as regressive changes in Schwann cells and capillary networks observed in advanced paralysis. These findings provide evidence for mast cell contribution to distal axonopathy and paralysis progression in ALS, a mechanism that can be therapeutically targeted by masitinib.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become a powerful tool for proteins structure and interactions studies. Unlike structural analysis of proteins using chemical reagents specific for lysine or cysteine residues, identification of gas-phase fragmentation patterns of endogenous dityrosine cross-linked peptides have not been investigated. Dityrosine cross-linking in proteins and peptides are clinical markers of oxidative stress, aging, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. In this study, we investigated and characterized the fragmentation pattern of a synthetically prepared dityrosine cross-linked dimer of Aβ(1-16) using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked Aβ(1-16), using collision induced dissociation (CID), higher-energy collision induced dissociation (HCD), electron transfer dissociation (ETD), and electron capture dissociation (ECD). Application of these generic fragmentation rules of dityrosine cross-linked peptides allowed for the identification of dityrosine cross-links in peptides of Aβ and α-synuclein generated in vitro by enzymatic peroxidation. We report, for the first time, the dityrosine cross-linked residues in human hemoglobin and α-synuclein under oxidative conditions. Together these tools open up the potential for automated analysis of this naturally occurring post-translation modification in neurodegenerative diseases as well as other pathological conditions.

Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex Cu II (atsm) tested for therapeutic efficacy in mice expressing SOD1 G93A on a mixed genetic background. Oral administration of Cu II (atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1 G93A mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the Cu II (atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with Cu II (atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for Cu II (atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.

Objective: We aimed to determine the potential of aberrant glial cells (AbAs) isolated from the spinal cord of adult SOD1G93A symptomatic rats to induce gliosis and neuronal damage following focal transplantation into the lumbar spinal cord of wild-type rats., Methods: AbAs were obtained from the spinal cords of SOD1G93A symptomatic rats. One hundred thousand cells were injected using a glass micropipette into the lumbar spinal cords (L3-L5) of syngeneic wild-type adult rats. Equal volumes of culture medium or wild-type neonatal microglia were used as controls. Seven days after transplantation, immunohistochemistry analysis was carried out using astrocytic and microglia cell markers. Transplanted SOD1G93A AbAs were recognized by specific antibodies to human SOD1 (hSOD1) or misfolded human SOD1., Results: Seven days after transplantation, AbAs were mainly detected in the medial region of the lumbar ventral horn as a well-limited cell cluster formed at the site of injection by their immunoreactivity to either misfolded SOD1 or normally folded hSOD1. Compared with controls, transplanted AbAs were surrounded by marked microgliosis and reactive astrocytes. Marked microgliosis was observed to extend bilaterally up to the cervical cord. Motor neurons close to AbA transplants were surrounded by activated glial cells and displayed ubiquitin aggregation., Conclusions: AbAs bearing mutant SOD1G93A have the potential to induce neuroinflammation along the spinal cord and incipient damage to the motor neurons. The emergence of AbAs during amyotrophic lateral sclerosis pathogenesis may therefore be a mechanism to boost neuroinflammation and spread motor neuron damage along the neuroaxis., (© 2017 S. Karger AG, Basel.)

Background: In the SOD1(G93A) mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS., Methods: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1(G93A) rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset., Results: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1(G93A) spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1(G93A) rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %., Conclusions: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.

Over-expression of mutant copper, zinc superoxide dismutase (SOD) in mice induces ALS and has become the most widely used model of neurodegeneration. However, no pharmaceutical agent in 20 years has extended lifespan by more than a few weeks. The Copper-Chaperone-for-SOD (CCS) protein completes the maturation of SOD by inserting copper, but paradoxically human CCS causes mice co-expressing mutant SOD to die within two weeks of birth. Hypothesizing that co-expression of CCS created copper deficiency in spinal cord, we treated these pups with the PET-imaging agent CuATSM, which is known to deliver copper into the CNS within minutes. CuATSM prevented the early mortality of CCSxSOD mice, while markedly increasing Cu, Zn SOD protein in their ventral spinal cord. Remarkably, continued treatment with CuATSM extended the survival of these mice by an average of 18 months. When CuATSM treatment was stopped, these mice developed ALS-related symptoms and died within 3 months. Restoring CuATSM treatment could rescue these mice after they became symptomatic, providing a means to start and stop disease progression. All ALS patients also express human CCS, raising the hope that familial SOD ALS patients could respond to CuATSM treatment similarly to the CCSxSOD mice., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Electron capture dissociation (ECD), which generally preserves the position of labile post-translational modifications, can be a powerful method for de novo sequencing of proteins and peptides. In this report, ECD product-ion mass spectra of singly and doubly sodiated, nonphosphorylated, and phosphorylated peptides are presented and compared with the ECD mass spectra of their protonated counterparts. ECD of doubly charged, singly sodiated peptides yielded essentially the same sequence information as was produced by the corresponding doubly protonated peptides. The presence of several sodium binding sites on the polypeptide backbone, however, resulted in more complicated spectra. This situation is aggravated by the zwitterionic equilibrium of the free acid peptide precursors. The product-ion spectra of doubly and triply charged peptides possessing two sodium ions were further complicated by the existence of isomers created by the differential distribution of sodium binding sites. Triply charged, phosphorylated precursors containing one sodium, wherein the sodium is attached exclusively to the PO4 group, were found to be as useful for sequence analysis as the fully protonated species. Although sodium adducts are generally minimized during sample preparation, it appears that they can nonetheless provide useful sequence information. Additionally, they enable straightforward identification of a peptide's charge state, even on low-resolution instruments. The experiments were carried out using a radio frequency-free electromagnetostatic cell retrofitted into the collision-induced dissociation (CID) section of a hybrid quadrupole/time-of-flight tandem mass spectrometer. Graphical Abstract ᅟ.

Hypochlorous acid and simple chloramines (RNHCl) are stable biologically derived chlorinating agents. In general, the chlorination potential of HOCl is much greater than that of RNHCl, allowing it to oxidize or chlorinate a much wider variety of reaction partners. However, in this study we demonstrate by kinetic analysis that the reactivity of RNHCl can be dramatically promoted by imidazole and histidyl model compounds via intermediary formation of the corresponding imidazole chloramines. Two biologically relevant reactions were investigated--loss of imidazole-catalyzed chlorinating capacity and phenolic ring chlorination using fluorescein and the tyrosine analog, 4-hydroxyphenylacetic acid (HPA). HOCl reacted stoichiometrically with imidazole, N-acetylhistidine (NAH), or imidazoleacetic acid to generate the corresponding imidazole chloramines which subsequently decomposed. Chloramine (NH2Cl) also underwent a markedly accelerated loss in chlorinating capacity when NAH was present, although in this case N-α-acetylhistidine chloramine (NAHCl) did not accumulate, indicating that the catalytic intermediate must be highly reactive. Mixing HOCl with 1-methylimidazole (MeIm) led to very rapid loss in chlorinating capacity via formation of a highly reactive chlorinium ion (MeImCl(+)) intermediate; this behavior suggests that the reactive forms of the analogous imidazole chloramines are their conjugate acids, e.g., the imidazolechlorinium ion (HImCl(+)). HOCl-generated imidazole chloramine (ImCl) reacted rapidly with fluorescein in a specific acid-catalyzed second-order reaction to give 3'-monochloro and 3',5'-dichloro products. Equilibrium constants for the transchlorination reactions HOCl + HIm = H2O + ImCl and NH2Cl + HIm = NH3 + ImCl were estimated from the dependence of the rate constants on [HIm]/[HOCl] and literature data. Acid catalysis again suggests that the actual chlorinating agent is HImCl(+); consistent with this interpretation, MeIm markedly catalyzed fluorescein chlorination by HOCl. Time-dependent imidazole-catalyzed HPA chlorination by NH2Cl was also demonstrated by product analyses. Quantitative assessment of the data suggests that physiological levels of histidyl groups will react with primary chloramines to generate a flux of imidazole chloramine sufficient to catalyze biological chlorination via HImCl(+), particularly in environments that generate high concentrations of HOCl such as the neutrophil phagosome., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A radio frequency-free electromagnetostatic (EMS) cell devised for electron-capture dissociation (ECD) of ions has been retrofitted into the collision-induced dissociation (CID) section of a triple quadrupole mass spectrometer to enable recording of ECD product-ion mass spectra and simultaneous recording of ECD-CID product-ion mass spectra. This modified instrument can be used to produce easily interpretable ECD and ECD-CID product-ion mass spectra of tyrosine-phosphorylated peptides that cover over 50% of their respective amino-acid sequences and readily identify their respective sites of phosphorylation. ECD fragmentation of doubly protonated, tyrosine-phosphorylated peptides, which was difficult to observe with FT-ICR instruments, occurs efficiently in the EMS cell.

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1., (Copyright © 2014 the authors 0270-6474/14/348021-11$15.00/0.)

Microglia and reactive astrocytes accumulate in the spinal cord of rats expressing the Amyotrophic lateral sclerosis (ALS)-linked SOD1 (G93A) mutation. We previously reported that the rapid progression of paralysis in ALS rats is associated with the appearance of proliferative astrocyte-like cells that surround motor neurons. These cells, designated as Aberrant Astrocytes (AbA cells) because of their atypical astrocytic phenotype, exhibit high toxicity to motor neurons. However, the cellular origin of AbA cells remains unknown. Because AbA cells are labeled with the proliferation marker Ki67, we analyzed the phenotypic makers of proliferating glial cells that surround motor neurons by immunohistochemistry. The number of Ki67 (+)AbA cells sharply increased in symptomatic rats, displaying large cell bodies with processes embracing motor neurons. Most were co-labeled with astrocytic marker GFAP concurrently with the microglial markers Iba1 and CD163. Cultures of spinal cord prepared from symptomatic SOD1 (G93A) rats yielded large numbers of microglia expressing Iba1, CD11b, and CD68. Cells sorted for CD11b expression by flow cytometry transformed into AbA cells within two weeks. During these two weeks, the expression of microglial markers largely disappeared, while GFAP and S100β expression increased. The phenotypic transition to AbA cells was stimulated by forskolin. These findings provide evidence for a subpopulation of proliferating microglial cells in SOD1 (G93A) rats that undergo a phenotypic transition into AbA cells after onset of paralysis that may promote the fulminant disease progression. These cells could be a therapeutic target for slowing paralysis progression in ALS.

More than 130 different mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been associated with amyotrophic lateral sclerosis but the mechanism of this toxicity remains controversial. To gain insight into the importance of the zinc site in the pathogenesis of SOD1 in vivo, we generated a Drosophila model with transgenic expression of a zinc-deficient human SOD1. Expression of zinc-deficient SOD1 in Drosophila resulted in a progressive movement defect with associated mitochondrial cristae vacuolization and reductions in adenosine triphosphate (ATP) levels. Furthermore, these flies are sensitized to mitochondrial toxins, paraquat, and zinc. Importantly, we show that the zinc-deficient SOD1-induced motor defect can be ameliorated by supplementing the endogenous fly respiratory chain machinery with the single-subunit NADH-ubiquinone oxidoreductase from yeast (NADH is nicotinamide adenine dinucleotide, reduced form.). These results demonstrate that zinc-deficient SOD1 is neurotoxic in vivo and suggest that mitochondrial dysfunction plays a critical role in this toxicity. The robust behavioral, pathological, and biochemical phenotypes conferred by zinc-deficient SOD1 in Drosophila have general implications for the role of the zinc ion in familial and sporadic amyotrophic lateral sclerosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase-dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2'(3')-O-(4-Benzoylbenzoyl)-ATP. The P2X7 inhibitors BBG, oATP, and KN-62 prevented 2'(3')-O-(4-Benzoylbenzoyl)-ATP-induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1-100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7-induced motor neuron death was dependent on neuronal nitric oxide synthase-mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well-established peroxynitrite/FAS death pathway in motor neurons., (© 2013 International Society for Neurochemistry.)