Evidence for disrupted copper availability in human spinal cord supports CuII(atsm) as a treatment option for sporadic cases of ALS.

The copper compound CuII(atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). CuII(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 CuII(atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for CuII(atsm) involving copper availability may also be pertinent to sporadic cases of ALS.

Microglial ferroptotic stress causes non-cell autonomous neuronal death.

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 SOD1G37R 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 SOD1G37R mouse model where treatment with a CNS-permeant ferroptosis inhibitor, CuII(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.

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ.

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 CH2, 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 GSK3ß 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.

Determining Copper and Zinc Content in Superoxide Dismutase Using Electron Capture Dissociation Under Native Spray Conditions.

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.

Enhanced Top-Down Protein Characterization with Electron Capture Dissociation and Cyclic Ion Mobility Spectrometry.

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.

Strategies to protect against age-related mitochondrial decay: Do natural products and their derivatives help?

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.

PermaPhos Ser : autonomous synthesis of functional, permanently phosphorylated proteins.

Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this "PermaPhos Ser " system - an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins - we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3ß phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli .

Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments.

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.

Capillary Zone Electrophoresis-Electron-Capture Collision-Induced Dissociation on a Quadrupole Time-of-Flight Mass Spectrometer for Top-Down Characterization of Intact Proteins.

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.

Comparative Structural Analysis of 20S Proteasome Ortholog Protein Complexes by Native Mass Spectrometry.

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.

Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.

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.

Schwann cells orchestrate peripheral nerve inflammation through the expression of CSF1, IL-34, and SCF in amyotrophic lateral sclerosis.

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 SOD1G93A rats, and identify remarkably similar and specific reactive SC phenotypes based on the pattern of S100ß, GFAP, isolectin and/or p75NTR 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 SOD1G93A 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 SOD1G93A 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.

Direct Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.

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.

CD34 Identifies a Subset of Proliferating Microglial Cells Associated with Degenerating Motor Neurons in ALS.

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 SOD1G93A 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 SOD1G93A 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 SOD1G93A 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.

Neuroprotective effect of CuATSM on neurotoxin-induced motor neuron loss in an ALS mouse model.

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 SOD1G93A 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 ß-sitosterol ß-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.

Emergence of Microglia Bearing Senescence Markers During Paralysis Progression in a Rat Model of Inherited ALS.

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 SOD1G93A 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 SOD1G93A rats, numerous cells displayed nuclear p16INK4a as well as loss of nuclear Lamin B1 expression, two recognized senescence-associated markers. The number of p16INK4a-positive nuclei increased by four-fold while Lamin B1-negative nuclei increased by 1,2-fold, respect to non-transgenic or asymptomatic transgenic rats. p16INK4a-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 SOD1G93A rats. Although microglia actively proliferated in cultures, a subset of them developed senescence markers after few days in vitro and subsequent passages. Senescent SOD1G93A microglia in culture conditions were characterized by large and flat morphology, senescence-associated beta-Galactosidase (SA-ß-Gal) activity as well as positive labeling for p16INK4a, 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 p16INK4a staining. These results suggest that cellular senescence is closely associated with inflammation and motor neuron loss occurring after paralysis onset in SOD1G93A rats. The emergence of senescent cells could mediate key pathogenic mechanisms in ALS.

Mast cells and neutrophils mediate peripheral motor pathway degeneration 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.

Sequencing Grade Tandem Mass Spectrometry for Top-Down Proteomics Using Hybrid Electron Capture Dissociation Methods in a Benchtop Orbitrap Mass Spectrometer.

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.

Oxidative Release of Copper from Pharmacologic Copper Bis(thiosemicarbazonato) Compounds.

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 CuII(btsc) and ZnIIATSM (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 CuII(btsc) by H2O2 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, ONOOCO2-) 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 (NO2•, •OH, and CO3•-). Because the potentials for CuII(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.