Autoantibodies can contribute to peripheral and central sensitization through activation of complement pathway, activation of neuronal Fc gamma receptors, and disrupted function of neuronal ion channels. Chronic pain is an unpleasant and debilitating condition that is often poorly managed by existing therapeutics. Reciprocal interactions between the nervous system and the immune system have been recognized as playing an essential role in the initiation and maintenance of pain. In this review, we discuss how neuroimmune signaling can contribute to peripheral and central sensitization and promote chronic pain through various autoimmune mechanisms. These pathogenic autoimmune mechanisms involve the production and release of autoreactive antibodies from B cells. Autoantibodies—ie, antibodies that recognize self-antigens—have been identified as potential molecules that can modulate the function of nociceptive neurons and thereby induce persistent pain. Autoantibodies can influence neuronal excitability by activating the complement pathway; by directly signaling at sensory neurons expressing Fc gamma receptors, the receptors for the Fc fragment of immunoglobulin G immune complexes; or by binding and disrupting ion channels expressed by nociceptors. Using examples primarily from rheumatoid arthritis, complex regional pain syndrome, and channelopathies from potassium channel complex autoimmunity, we suggest that autoantibody signaling at the central nervous system has therapeutic implications for designing novel disease-modifying treatments for chronic pain. Managing the symptoms of chronic pain has become a pervasive problem. From 2015 to 2018, 10.7% of all adults in the United States used prescription pain medication to manage their symptoms.57 The underlying mechanisms of neuromodulation and dysfunctional synaptic plasticity from neuroimmune signaling—principally mediated through cytokines, reactive oxygen and nitrogen species, growth factors, and bioactive lipids—have been described elsewhere in detail.152The immune system works in tandem with the somatosensory nervous system to coordinate host defense. Dysregulation of homeostatic reciprocal signaling between nociceptors and immune cells can lead to heightened excitation of sensory neurons and induce pain.40 Although autoimmune diseases are differentiated by disease-specific immune responses, pathological pain is a shared symptom for most autoimmune diseases.116 Although autoimmune signaling has received less attention than other forms of neuroinflammatory signaling, there is now a growing appreciation that autoimmune mechanisms may be a conduit for the development of chronic pain.27Excessive inflammation and/or tissue damage can lead to autoimmune responses, characterized by activation of T-cell clones that recognize self-antigens.143 The evidence supporting the role of T cells as contributing to both the onset and resolution of chronic pain has been recently reviewed.89 Here, we will instead focus on the emerging evidence for the role of autoantibodies  as actively participating in states of persistent pain through their interactions with the complement system, immune receptors, and neuronal self-antigens.186In the following review, we will discuss how the immune system can contribute to chronic pain through a variety of specific autoimmune mechanisms. Loss of immunological tolerance  can occur when autoreactive T-cell clones expand and induce antibody production by autoantigen-specific B cells.The framework of this review follows the sequence of events for an autoimmune reaction and examines evidence at each step for its contribution to pain. These events include 1) the recognition of self-antigen by B cells; (2) the production of autoantibodies by B cells, focusing on immunoglobulin G (IgG) and immunoglobulin M (IgM); (3) activation of complement; (4) autoantibody signaling at antibody receptors (Fc receptors); and (5) direct interactions of autoantibodies at neuronal antigens  class II molecules from antigen-presenting cells through their surface-bound T-cell receptors.89 On encountering foreign or host antigen presented by helper T cells and recognized by cognate B-cell receptors, B cells become activated and undergo further proliferation and maturation either into memory B cells or antibody secreting cells, both short-lived plasmablasts and long-lived plasma cells.123 B-cell activation and differentiation can also occur through T-cell–independent pathways and occur outside of germinal centers.85 During B-cell maturation, there are several checkpoints to eliminate B cells that recognize self-antigen through receptor editing, negative selection, or anergy, which occur throughout the developmental lifespan of B cells.120 However, if these checkpoints are disrupted, self-reactive B cells can escape removal and differentiate into plasmablasts or plasma cells that produce autoantibodies . During disease progression, the synovium is typically infiltrated by innate immune cells  that release proinflammatory cytokines and chemokines, which recruit additional cells of adaptive immunity (including T cells and B cells).66 Release of cytokines, such as tumor necrosis factor, interleukin-1β, interleukin-6, interferon gamma, and other proinflammatory mediators, from cells near the inflamed joint may act to directly activate peripheral nociceptors, and there is evidence that plasticity in both peripheral and central pain processing may further contribute to aberrant pain.106 Patients can experience pain localized to the joints, as well as neuropathic symptoms, such as burning, electrifying sensations, or pain to light pressure and dynamic brushing. These symptoms further suggest that alterations in peripheral and central processing occur in rheumatoid arthritis, and these mechanisms may share some similarities with neuropathic pain conditions.177Most patients with rheumatoid arthritis indicate pain is the symptom they would most like to improve.91 Even when edema and inflammation of the joints are relieved by disease-modifying antirheumatic drugs, pain can persist in a substantial portion of patients.162 Beyond joint pain, patients with rheumatic diseases also report higher incidences of both migraine and neuropathic pain despite clinical control of inflammation-related symptoms.111 Identifying how pain arises in rheumatoid arthritis separate from ongoing inflammation could provide new avenues for clinically relevant pain relief.There is accumulating evidence that pain in rheumatoid arthritis may be maintained independently from ongoing inflammation. Joint pain frequently occurs before diagnosis and before signs of inflammation are present.98 Delayed treatment of anti-CD20 was also successful at attenuating the severity of mechanical allodynia and weight bearing on the injured paw, indicating both development and maintenance of CRPS-like nociceptive symptoms involve B cells.98 Similar attenuation of fracture-induced allodynia and edema was observed in transgenic B-cell-deficient μMT mice.98 These preclinical results demonstrate a likely role for B cells in CRPS-associated pain, although this hypothesis has yet to be clinically tested.A role for B cells has also been demonstrated in the tibial fracture and casting model, a rodent model of complex regional pain syndrome (CRPS) . DepletiText box 2.186 In addition to the inciting trauma, immobilization of the injured limb (such as wearing a cast) is a risk factor for developing CRPS.165Complex regional pain syndrome (CRPS) is a debilitating form of posttraumatic pain. The etiology of CRPS is unclear, but typically emerges after injury or surgery of a limb. Symptoms between patients are highly heterogenous but can include sensory abnormalities , motor dysfunctions (loss of motor coordination and dystonia), autonomic dysfunctions , and trophic changes (increased or decreased hair and nail growth and tissue or bone dystrophy).44 Evidence from patients with CRPS and animal models of the disease points to immune cell activation, especially in the acute phase of the disorder.181 Elevated concentration of tumor necrosis factor, interleukin-1β (IL-1β), IL-6, IL-8, soluble tumor necrosis factor receptors, monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, soluble receptor for advanced glycation end products, and other neuroimmune signaling factors were detectable in patients skin, blood, or serum.131 Biopsies of the affected skin reveal keratinocyte activation, mast cell accumulation and degranulation, and increased number of tissue-resident Langerhans cells, although both mast cell and Langerhans cell numbers may resolve in advanced stages of the disease.130 Mass cytometry performed on blood from patients with CRPS reveals an expanded population of T-lymphocyte subpopulations, including memory CD4+ and CD8+ T cells, suggesting an ongoing inflammatory lymphocyte response in CRPS.149 Activation of spinal cord glial cells may also play a role in altering central processing of nociceptive signals.2 Noninvasive positron emission tomography imaging of translocator protein-18 kDa (TSPO) to monitor microglia and myeloid cells reveals early and persistent myeloid cell activation in the tibial fracture/casting mouse model, along with transient activation of spinal cord and brain microglia.24 Understanding the temporal dynamics of immune cell activation along the pain neuraxis in CRPS may offer new insights into disease progression.The complexity of the clinical manifestation has made it difficult for researchers to understand the mechanistic underpinnings of this syndrome. Heterogeneity of the clinical presentation signifies a likely multisystem pathology involving inflammatory, neuronal, musculoskeletal, and microvascular contributions that may vary over disease progression.71 A case study with intractable Morvan syndrome and anti-VGKC complex antibodies also described that pain relief for the patient was achieved with rituximab treatment.128Depletion of B cells may also be a disease-modifying treatment for pain in voltage-gated potassium channel (VGKC) complex autoimmunity , or in some circumstances, pain may be the only symptom of a diagnosed autoantibody disorder (such as in VGKC complex autoimmunity).153 There are 5 classes of Ig in mammals: IgA, IgD, IgE, IgG, and IgM, each with different structures and functions for host immunity. To date, IgG and IgM autoantibodies in particular have been implicated in persistent pain states.186Antibodies (Ig for immunoglobulin) are Y-shaped proteins segregated into 2 domains: the variable antigen-binding domain (Fab region) at the top that can recognize unique epitopes on antigens and the invariant fragment crystallizable domain (Fc region) at the bottom, which enables the antibody to interact with Fc receptors and elements of the complement system.153 Antibodies recognize epitopes from pathogens but are also important for wound healing and removal of cellular debris after injury because both IgM and IgG accumulate at the site of sciatic nerve crush injury and contribute to clearance of myelin debris.173 Autoantigenic antibodies in autoimmune diseases are most frequently of the IgG isotype, although other Ig classes can also promote autoimmunity.46On binding antigen, IgM or IgG function to neutralize targets by agglutination, targeting cells for opsonization or antibody-dependent cell cytotoxicity, and recruiting elements of the complement cascade. Antibodies can also form immune complexes—clusters of multiple antibodies and antigens bound together—and these complexes can signal at their respective Fc receptors, either Fc mu receptor (FcµR) for IgM or Fc gamma receptor (FcγR) for IgG.151 One possibility identified in rheumatoid arthritis for generating self-reactive antibodies is through posttranslational modifications of proteins. Posttranslational modifications—such as citrullination, glycosylation, carbamylation (or homocitrullination), acetylation, phosphorylation, nitrosylation, and sulfation—alter the chemical, functional, and antigenic properties of naturally occurring proteins or peptides in various ways, including the addition or removal of functional groups, sugars, or other chemical groups to particular sites on proteins.175 These modifications to protein structures or amino acid sequences can generate new antigenic epitopes capable of producing a CD4+ T-cell- and MHC class II-dependent autoantibody response against native protein that breaks immunological tolerance.42Although considerable advancements have been made toward understanding the role of autoantibodies across diseases, for the most part it is still unclear how initial tolerance is broken leading to B-cell expansion, autoantibody production, and epitope spreading .17 Although the etiology of the disease is unknown, autoantibodies against cyclic citrullinated peptide and rheumatoid factor can be detected in blood several years before diagnosis.140 Anti-citrullinated protein antibodies (ACPAs) can interact with proteins or peptides expressing the amino acid citrulline.43 The generation of autoantigenic proteins expressing citrulline occurs through the process of citrullination, a posttranslational modification in which the amino acid arginine is replaced by citrulline through the enzymatic activity of peptidylarginine deiminases (PADs).26 Peptidylarginine deiminase enzymes have been implicated in generating modified citrullinated proteins  that are targeted by ACPAs, and PADs may themselves become targets of autoantibodies during disease progression.26 Anti-citrullinated protein antibody–IgG is reduced but not completely eliminated by rituximab, indicating that ACPAs are produced by CD20+ B cells, with a persisting contribution by stable, terminally differentiated plasma cells.151 The observation that both joint pain and the presence of autoantibodies occur before clinical onset of arthritis suggests that autoimmune antibody signaling could be implicated as a distinct mechanism for arthralgia.Autoantibodies have been suggested as a potential mechanism driving pain in rheumatoid arthritis.183 These results indicate that anti-citrullinated antibodies from patients with rheumatoid arthritis are sufficient to promote pain. Anti-citrullinated protein antibodies accumulate in disease-relevant tissues including the joints, skin, bone marrow, DRG, adipose tissue, and spleen. Patient-derived ACPAs localize with CD68+ macrophages or osteoclasts, alongside CGRP+ neurons in the bone marrow, and its effects on pain was related to increased expression of the chemokine interleukin-8 (IL-8).183 Anti-citrullinated protein antibody stimulation of IL-8 could contribute to arthralgia through direct action of IL-8 at its receptor cysteine-x-cysteine receptor1/2 on small fiber nociceptors, thereby increasing voltage-gated sodium currents, or indirectly through recruitment of neutrophils.178 Likewise, when monoclonal antibodies are cloned from the sequences of citrulline-autoreactive B-cell receptors captured from ACPA+ patients, some  patient-derived antibodies can produce mechanical hypersensitivity in mice after injection of lipopolysaccharide (LPS) to model an inflammatory stimulus.166 Thus, the mechanism for how ACPAs produce pain may require an additional inflammatory event and result from the formation of local immune complexes within the joints.43Anti-citrullinated protein antibodies are a biomarker of rheumatoid arthritis and may independently promote pain-related behavior. Intravenously administered IgG isolated from ACPA seropositive patients is sufficient to produce mechanical and thermal hypersensitivity in mice, along with nonevoked reductions in spontaneous activity without producing joint inflammation.12 Fab fragments of these anti-CII antibodies are ineffective at producing pain-related behavior; these data indicate that the Fc fragment of these anti-CII autoantibodies may be necessary for their effects on pain, and further research is needed to determine if the Fc fragment is sufficient to account for pain in this model.12 When applied to primary DRG cell cultures, immune complexes of anti-CII IgG bound to collagen type II stimulate intracellular calcium release and evoke inward currents in around 20% of cells , whereas monomeric antibodies that are not in immune complexes fail to evoke any DRG activity.12 This may explain why treatment of cultured DRGs with patients' ACPA does not on its own stimulate depolarizing currents becasue formation of immune complexes (self-antigen bound to autoantibodies) is likely necessary for autoantibodies to cause neuronal hyperexcitability.183Beyond ACPAs, autoantibodies in the collagen antibody-induced arthritis (CAIA) preclinical model are also pronociceptive. Using the CAIA model, antibodies targeting collagen type II (anti-CII) are transferred to animals, followed by LPS injection to trigger an inflammatory response. Mice treated with anti-CII antibodies develop mechanical hypersensitivity and show reduced spontaneous activity several days before any signs of inflammation or extracellular remodeling are observed in the joints.52 Early evidence for an autoantibody hypothesis of CRPS was derived from experiments in which intravenous immunoglobulin (IVIg) treatment (pooled human serum consisting mainly of IgG) resulted in pain relief for small samples of patients with CRPS.53 In a larger multicenter trial, however, IVIg was not more effective than placebo for relieving pain, suggesting that this method may only be efficacious for a certain subpopulation of patients.51 Although this evidence is partially supportive of circulating autoantibodies as contributing to pain in a subset of CRPS, it should be noted that the therapeutic mechanism of action for pooled IVIg is still debated; the salutary effects of IVIg could involve inhibition of antibodies binding to activating FcγRs, upregulation of inhibitory FcγRIIB, neutralization of circulating IgG, or through alternative methods of immunomodulation.94 Plasma exchange therapy has also been retrospectively associated with pain relief in a high proportion of patients with long-standing CRPS.7 Similar to the caveats for IVIg, plasma exchange might remove alternative disease-relevant molecules or proteins other than pathological autoantibodies, such as miRNA-containing exosomes.13The development of CRPS comprises elements of sterile inflammation and autoantibody-mediated pathology, leading some to argue that CRPS should be considered as an autoimmune syndrome.27 Serum IgG from patients with chronic CRPS transferred intraperitoneally to female mice, followed by unilateral plantar skin and muscle incision to model limb injury, enhances unilateral mechanical hyperalgesia and paw edema compared with IgG from healthy matched controls.163 With daily transfer of IgG from patient with CRPS, mechanical hypersensitivity of the paw quickly develops and remains heightened over time. By contrast, paw edema and concentration of the inflammatory cytokines tumor necrosis factor, IL-1β, and IL-6 in the paw increases at the time of injury but resolves by day 13, when mechanical allodynia is still elevated.67 Reminiscent of the passive IgG transfer effects from patients with CRPS, serum or purified IgM from wild-type mice 3 weeks after tibial fracture, as well as IgM purified from patients with CRPS, was pronociceptive when transferred to B-cell–deficient mice.62 These observations support the notion that injury-induced self-antigens trigger production of autoantibodies that could produce pain through activation of nociceptive neurons.61 In agreement with this hypothesis, IgG purified from patients with CRPS and treated on mouse skin–saphenous nerve preparations evokes activity of Aδ and C mechanonociceptors.25 The recapitulation of these basic CRPS-like pain symptoms in the rodent model after passive antibody transfer suggests that pathogenic autoantibodies are present in CRPS and directly contribute to pain-related behavior after injury.Passive IgG transfer, in which IgG fractions are purified from serum of patients with CRPS or rodent models and delivered to naive subjects, can address if autoantibodies are necessary for pain in CRPS.80 Epidermal adrenergic signaling has been associated with pain and inflammation in CRPS; keratinocytes in the epidermis also express β2 adrenergic receptors, and stimulation of these receptors by sympathetic neurons contributes to local release of IL-6 in a rat model of CRPS.96 This pathway could indirectly increase cutaneous sensory neuron excitability and promote mechanical hypersensitivity seen in CRPS.96 Alternatively, skin-nerve preparations demonstrate that IgG from patients with CRPS may directly stimulate activity from peripheral nociceptors, although additional in vivo evidence of electrophysiological properties is needed to confirm these observations.25 Injury-induced changes in neuronal neuropeptide activity may also influence the localized immune response. Transgenic mice with deficient expression of the neuropeptides substance P (Tac1−/−) or CGRP (Ramp1−/−) show attenuated mechanical allodynia in the tibial fracture and casting model of CRPS.97 In addition, Ramp1−/− mice have reduced deposition of IgM in the skin, sciatic nerve, and spinal cord compared with wild-type animals 3 weeks after fracture.97 Thus, neuropeptide signaling may influence IgM autoantibody deposition after injury, and the antigenic targets of IgM may be localized to the skin and nerves. Indeed, IgM from the mouse model of CRPS and IgM from patients with CRPS have been shown to be autoreactive to keratin 16 (KRT16) from the epithelial tissues and cross-reactive with histone 3.2, γ-actin, and α-enolase.161 Immunohistochemical staining indicates microglial and astrocyte activation also occurs in the dorsal horn of the spinal cord after intraperitoneal transfer of IgG from patients with CRPS.67 Systemic IL-1 inhibition or microglia-specific elimination of IL-1β alleviates the pain-related behavior from IgG transferred from patients with CRPS, supporting a role for central glial activation in the development of mechanical allodynia after CRPS IgG transfer. Collectively, these data indicate that IgG and IgM autoantibodies in CRPS may function by activating nociceptors, leading to pain through changes in neuronal and glial functions.67The characteristics of the cellular and molecular targets for autoantibodies in CRPS are not fully understood. One possibility is the generation of antibodies that recognize G protein-coupled receptors on neurons or vascular tissue after traumatic injury. Antibodies isolated from patients with CRPS have demonstrated to be agonists of autonomic neuronal receptors, including β2 adrenergic, muscarinic 2, and α-1a adrenergic receptors.20 One possibility is that autoantibody accumulation at self-antigenic targets in discrete tissues could recruit complement or produce localized inflammation in tissue niches, resulting in continued local deposition and restricted effects on nociceptors. The generation of autoreactive antibodies may also be regionally restricted. A recent study found that the mouse tibial fracture and cast model of CRPS results in lymph node enlargement and the emergence of germinal center B cells only in the popliteal lymph node ipsilateral to injury.99 This localized development of lymphocytes could account for the regionally restricted deposition of autoantibodies seen in this model, although autoreactive antibodies would likely be released into lymphatic circulation. In addition, the localized expression of self-antigens could support the induction and secretion of autoantibodies in discrete tissues. The subsequent formation of antigen-bound IgG-immune complexes may be sufficient to trigger complement activation or FcγR-mediated activity on nociceptors near the site of injury.An outstanding question is how autoantibody production contributes to chronic pain that is regionally restricted. For instance, although some patients with CRPS develop pain that spreads widely from the site of injury, most cases experience pain localized to the injured limb. If autoantibodies are detectable in the serum, why are symptoms typically limited to one limb?98 Moreover, the authors found transfer of IgM, but not IgG, from either wild-type CRPS mice or patients with CRPS was pronociceptive to B-cell–lacking μMT mice,62 which differs from other reports on the pronociceptive role of CRPS IgG transfer.67 This contradiction may be due to the use of wild type vs B-cell–deficient μMT mice, as well as large differences in doses and duration of IgG transfer. Future research should investigate what brings about self-reactive IgG and/or IgM under conditions of pain.Further research is needed to explore the similarities and differences between IgG and IgM and their contributions to nociceptive hypersensitivity. Although there is evidence (presented below) that IgG autoantibodies exert some of their contributions to pain through interactions at FcγRs, there is considerably less known about how IgM can influence pain, although its effector functions are presumably due to complement recruitment or signaling at FcµRs. In 2 reports using the CRPS tibia fracture and casting model, IgM but not IgG protein was elevated in injured hind paw skin and sciatic nerve.168 Complement is composed of more than 30 proteins found circulating in plasma and attached to cell surfaces. These complement elements are produced mainly by hepatocytes in the liver and by innate immune cells, including macrophages, monocytes, mast cells, and dendritic cells.107 There are several pathways to activation, but all result in a series of proteolytic cleavages of complement components, activating subsequent complement proteins in a well-characterized cascade. IgG and IgM antibodies are known to activate complement through the classical pathway, in which the C1 complex binds to the Fc region of antigen-bound antibodies.The complement system supports host immunity by engaging a set of effector molecules to remove pathogens or damaged cells, amplify inflammation, and augment antibody responses.137 Early descriptive studies noted that elements of the complement system protein are elevated at the site of nerve injury, in the spinal cord dorsal horn and brainstem after peripheral nerve injury.105 Transcriptional analyses of spinal dorsal horn confirm that complement expression is induced after different models of nerve injury.93 Complement induction is functionally significant because either intrathecal or perisciatic delivery of soluble complement receptor 1 as a method to block C3a, C5a, and C5b-9 is sufficient to reverse mechanical allodynia from sciatic inflammatory neuropathy and chronic constriction injury.171 More specific pharmacological or genetic disruption of C5a or its receptor C5aR  reduces cold allodynia and mechanical allodynia after partial nerve injury, reduces paw inflammation and pain after postsurgical paw incision, and attenuates inflammatory pain.156Elements of the complement system have been implicated in various pain conditions, including neuropathic pain, postoperative pain, and inflammatory pain.168 However, it is not yet clear if the complement system contributes directly to joint pain. In the CAIA mouse model of rheumatoid arthritis, pharmacological antagonism of the C5a receptor did not block the development of pain-related behavior in response to CII autoantibodies, and complement 5 knockout (C5−/−) mice likewise develop pain-related behavior like wild-type mice.12 These results indicate, at least for the CAIA model, that pain hypersensitivity develops independently from C5.Complement has been implicated in disease progression in arthritis; biomarkers of complement are elevated circulating in blood and in synovial tissues of patients with rheumatoid arthritis, and genetic deficiencies in receptors for C3a, C5a, or C6 protein all reduce disease severity in mice.98 Depletion of B cells with anti-CD20 attenuates C5b-9 expression in the skin and nerve, implying that autoantibody release is a prerequisite for complement accumulation.98 Thus, binding of IgM bound to self-reactive targets at the site of traumatic injury and along primary afferents could lead to excessive complement recruitment, which could promote pain by damaging cells and peripheral nerves through membrane attack complexes or by elevating proinflammatory factors.138 CRPS is not typically associated with excessive tissue destruction, but localized damage to small-fiber peripheral nerves is consistent with the clinical presentation of CRPS. In patients with CRPS, a loss of intraepidermal nerve fibers has been observed in skin biopsies from the CRPS-affected limb compared with contralateral tissue,126 whereas other reports have found bilateral reductions in intraepidermal nerve fiber density in patients with CRPS compared with healthy controls.142 We speculate that the degeneration of nociceptive nerve fibers in affected tissue might be driven by complement. Cell lysis by membrane attack complexes could lead to focal damage at mixed peripheral sensory, motor, and autonomic nerves directly, or indirectly injure nerves by damaging vascular endothelial cells, keratinocytes, or other support cells.124 Although speculative, complement activation in CRPS may be restricted to autoantibody-bound targets in the periphery, accounting for localized tissue damage and inflammation.In CRPS, it is also still unclear if complement contributes directly to pain, but there is circumstantial evidence linking autoantibody accumulation to pain. After tibia fracture and casting in the mouse model of CRPS, IgM accumulates in the injured hind paw skin and sciatic nerve, and there is also increased expression of complement C5b-9 membrane attack complexes at these sites.156 However, more clarity is needed to determine the downstream effects of complement activation that lead to pain. Additional research is also needed to determine if specific IgG or IgM autoantibodies in different disease models engage similar or separate elements of complement to produce nociceptor activation. Furthermore, during development in autoimmune demyelinating diseases, there is evidence that complement can tag neuronal synapses for microglial engulfment and elimination.182 Considering the importance of synaptic remodeling in the generation of chronic pain, there is a possibility that complement signaling to microglia could be an unexplored pathway for synaptic circuit reorganization contributing to pathological pain processing.Complement activation is important for coordinating host defense, but its recruitment may lead to detrimental amplification of pain under certain disease conditions. Recruitment of complement by autoantibodies could be pronociceptive through stimulation of proinflammatory cytokines locally in tissues, resulting in nociceptor activation.103 FcµR, the receptor for IgM, may be expressed by oligodendrocytes, but it is not yet clear if it plays a role in nociception.119Considering that the preponderance of research to date has explored the role of autoimmune IgG, this section will focus primarily on the role of FcγRs in chronic pain. However, there may be unexplored roles for other classes of Fc receptors that could be involved in pain or other sensory modalities. For example, expression of Fc epsilon receptor, the receptor for IgE, has been shown to co-localize to peripheral neurons and its activity may be important for allergic itch.69 The FcγR classes can be functionally categorized as activating or inhibiting. Activating receptors promote phagocytosis, proinflammatory cytokine production, antibody-dependent cellular toxicity, oxidative burst, and cellular proliferation, whereas the downstream signaling of inhibiting receptors opposes these outcomes.121 There are several classes of activating FcγRs that differ between species, from humans  to mice , to rats .158 In humans, mice, and rats, FcγRIIB is the sole inhibiting FcγR. For nearly all of these receptors, recognition of IgG occurs only when it has aggregated into IgG-immune complexes. Among the activating receptors, FcγRI is distinct in its high affinity for certain IgG isotypes, as well as its capability of binding both monomeric IgG along with IgG-immune complexes.The classic surface-expressing FcγRs are transmembrane glycoproteins that recognize the Fc region of IgG. There are several classes of FcγRs that differ in their affinity for IgG isotypes, in their cellular localization, and in the downstream signal transduction pathways they engage.121 For instance, the interactions between the complement system and FcγR signaling have long been studied for their role in rheumatoid arthritis pathogenesis.157 Our understanding of FcγRs has mostly derived from myeloid cells, although the functional outcomes they engage will naturally differ depending on the identity of the cell expressing them.60The role of FcγRs in regulating innate and adaptive immune responses in infection and autoimmunity has been extensively studied.172 Microglia isolated from both humans and rats express FcγRs,174 and astrocytes from developing mouse and rat brains also have been characterized as expressing functional FcγRs.158 Importantly, the expression patterns of activating and inhibitory FcγRs on cells can change in states of injury, inflammation, and immune cell differentiation.60 Thus, in conditions of pathological pain, there may be dynamic alterations in FcγR expression among cell populations.Fc gamma receptors are expressed most prominently on immune cells, including macrophages, dendritic cells, natural killer cells, neutrophils, eosinophils, mast cells, and B cells. Beyond immune cells, there is evidence that cells within the central nervous system (CNS) also express FcγRs. Expression of FcγRI, FcγRII, and FcγRIII is observed in resected human brain tissue, although it is still unclear which cell types express these proteins.5 Several different approaches have confirmed this observation. Using immunofluorescence, it was verified that rat FcγRI is expressed at varying proportions on DRG cell bodies that co-express markers for peptidergic (CGRP or substance P) and nonpeptidergic (IB4) nociceptive neurons, and most small-diameter FcγRI+ DRG neurons express transient receptor potential V1.136 Transcriptional profiling of bulk samples indicates that mRNA of all 4 mouse FcγRs  are expressed in DRG.144 Labeling nociceptors with retrograde tracers reveals Fcgr1 expression in ∼28% of joint-innervating neurons.179 Single-molecule fluorescent in situ hybridization further confirms expression of Fcgr1 and Fcgr2b in mouse sensory neurons, and localization experiments suggest these transcripts are potentially transported along nerve fibers and accumulate at sites of nerve injury.12 Furthermore, immunoreactivity of mouse DRG cultures demonstrates co-localization of FcγRI and FcγRIIB with the neuronal marker βIII-tubulin, although their distribution differed: FcγRI was primarily localized to axons and neurites, whereas FcγRIIB immunoreactivity localized primarily on the cell bodies.12 Human DRG samples also express mRNA for all classes of FcγRs,144 although more evidence is needed to clarify the cellular and spatial distribution of FcγR protein, ie, whether it resides at DRG cell bodies, satellite glial cells, or primarily at neuronal projections in the periphery and axonal terminals in the spinal cord.In addition to their effect on immune cells, IgG may directly interact with FcγRs on sensory neurons to modulate nociceptive activity. This notion was derived from initial observations that primary sensory neurons from the mouse DRG express FcγRI as determined by immunostaining and Western blot.136 Elevated neuronal activity in response to FcγR activation may result in signaling through spleen tyrosine kinase to trigger opening of transient receptor potential canonical 3 ion channels , knockout mice lacking all 4 murine FcγRs show diminished bone erosion, and in wild-type mice, the extent of bone erosion positively correlates with the number of joint-infiltrating neutrophils.31 Bone erosion can be accelerated by neutrophil-derived tumor necrosis factor and IL-6 signaling to osteoclasts, but autoantibodies can also signal directly to FcγRs expressed by osteoclasts to further promote cell differentiation and joint destruction.154 Inhibiting FcγR signaling has shown promise for halting disease progression.94 Using the collagen-induced arthritis model in mice, injections of soluble recombinant human FcγRI to scavenge IgG-immune complexes reduced joint swelling and synovial tissue destruction.35 Thus, there remains a prominent role for FcγRs in mediating arthritis disease activity.157Autoantibodies can contribute to rheumatoid arthritis development through FcγR-mediated effector functions. Autoreactive IgG bound to self-antigens form immune complexes that can signal at FcγRs and disrupt the homeostatic balance between activating and inhibitory FcγRs, leading to improperly contained inflammation.179 As previously mentioned, in the CAIA model of rheumatoid arthritis, injections of collagen-targeting anti-CII antibodies and LPS are sufficient to produce pain-related behavior before any signs of joint inflammation.12 Mechanical hypersensitivity to CAIA was also observed in mice lacking FcγRs on myeloid cells, but no pain-related behavior was seen if only hematopoietic cells expressed FcγRs, supporting the notion that pain-related behavior in this model arises from FcγR signaling on nonimmune cells.12 Cultured mouse DRG neurons show increased intracellular calcium signaling and release of CGRP neuropeptide in response to anti-CII-immune complexes, but not to monomeric CII antigen or antibody.12 These results align with in situ hybridization results that found around half of Fcgr1+ neurons co-expressed CGRP.179 Autoantibody immune complex–stimulated release of CGRP could signal at CGRP receptors on other neurons to cause nociceptor hyperexcitability. Ligand-receptor interaction analysis also suggests neuronal CGRP could influence the function of DRG glial cells (satellite glia and/or Schwann cells) by signaling at glial-expressed RAMP2.180 The outcome of CGRP treatment to satellite glial cells from trigeminal ganglia is the release of multiple proinflammatory cytokines, which can further exacerbate nociceptor activity.1 Based on these results, it is conceivable that autoantibodies, including ACPAs, accumulate at specific targets in the joint, forming immune complexes that can activate FcγRI on nociceptors at concentrations that do not evoke overt inflammation, producing arthralgia before edema and joint destruction.12Converging evidence from various animal models of rheumatoid arthritis has identified a role for FcγR-coupled signaling on sensory neurons as a mechanism for how autoantibodies could contribute to pathological pain.+ neurons and small-diameter putative nociceptive neurons, and a greater proportion of joint-innervating sensory neurons were sensitive to IgG-immune complex stimulation after AIA induction.179 To investigate the functional role of neuronal FcγRI, conditional knockout mice and rats were generated in which Fcgr1 is deleted from sensory neurons expressing the membrane protein phosphoinositide-interacting regulator of transient receptor potential (Pirt), offering a more selective cell-type–specific genetic perturbation.179 Accordingly, in both mice and rats, AIA-induced pain-related behaviors are alleviated when Fcgr1 is deleted from Pirt+ sensory neurons.179 Likewise, pharmacological inhibition of FcγRI by delivering anti-FcγRI antibody  into the joint after AIA attenuates mechanical and thermal sensitivity with no effect on joint swelling.179 As previously mentioned, these results corroborate the notion that disease mechanisms driving joint pain may be partly uncoupled from joint destruction and inflammation.159The role of neuronal FcγRI contributing to pain was confirmed using a separate model of arthritis (the AIA model). Both mice and rats show increased FcγRI expression in the DRG after AIA, particularly in CGPRFcgr1 and Fcgr2b in mouse sensory neurons.12 It is unclear if this trafficking of Fcgr transcript in neurons is functionally relevant for pain. An intriguing report demonstrated expression of voltage-gated sodium channel Nav1.7 on dendrites of dorsal horn spinal neurons despite the lack of Nav1.7 mRNA and argues that mRNA or protein may be transsynaptically transferred from primary afferent sensory neurons to dorsal horn neurons.4 Although this may not be the case for FcγRs, these observations make it clear that it will be important to use multiple methods that can resolve cell-type expression  to determine where FcγRs are expressed in DRG neurons, spinal cord, and brain. Fc gamma receptor expression has been identified on most immune cells as well as on somatosensory neurons, microglia, and astrocytes, but further characterization is needed to determine their cellular expression pattern in pain-relevant regions and how their expression may change in the maintenance of chronic pain.It remains an open question if FcγRs other than FcγRI are involved in pain. The repeated observation that somatosensory neurons express several classes of FcγRs raises the possibility that other FcγR classes promote nociceptive hypersensitivity similar to FcγRI. Given similarities in their structure and intracellular signaling pathways, there might be overlapping roles for activating FcγRs  to influence nociceptor hyperexcitability, or there may be loss of some unknown inhibitory function of FcγRIIB. Alternatively, there may be spatial restriction of FcγR expression within cellular compartments of neurons and glia, and their expression could involve dynamic trafficking in response to stimuli, as seen in response to injury with 10 VGKCs are expressed by neurons, including sensory neurons, and they function to hyperpolarize neuronal resting membrane potentials, thereby influencing action potential frequency and resultant neurotransmitter release.169 Proper coordination of VGKC activity with other ligand- and voltage-gated ion channels is therefore critical for normal sensory and motor function.64Activation of voltage-gated cation channels expressed by sensory neurons is a primary mechanism that determines nociceptor excitability to encode the detection of stimuli causing—or threatening to cause—tissue injury.41 Autoantibodies found in these disorders do not target the VGKC Kv1 subunits themselves, but rather most commonly bind to 3 proteins that complex with VGKCs within the cellular membrane: contactin-associated protein-like 2 (CASPR2), leucine-rich glioma inactivated 1, and contactin-2.70 Neuropathic pain as a symptom is particularly prevalent in patients with confirmed CASPR2-IgG, with the extremities being the most common location of reported pain.79 Contactin-associated protein-like 2 is a cell adhesion molecule in the neurexin family and is primarily localized at juxtaparanodal regions—sites of contact between myelinating glial cells and axons both in the CNS and at nodes of Ranvier in the peripheral nervous system—and in association with contactin-2 form complexes to cluster VGKCs at these regions.167 As such, autoantibody-mediated inhibition of VGKC complex expression or function can disrupt normal hyperpolarizing currents mediated by properly clustered potassium channels, thus leading to pain from increased neuronal excitability.Several neurological disorders have been described that are identified by the presence of VGKC complex autoantibodies . These d155 In this in vitro preparation, CASPR2-IgG treatment causes a reduction in membrane expression of Kv1.2 VGKCs, which mirrors the reduction in DRG surface VGKC expression seen in Cntnap2−/− mice lacking CASPR2.29 The mechanism of how CASPR2-IgG reduces neuronal VGKC expression is likely not through internalization of CASPR2 but through binding CASPR2 and blocking its normal interactions with contactin-2, disrupting proper clustering of VGKCs on the axon at juxtaparanodal regions.132 Indeed, loss of Cntnap2 is associated with exaggerated behavioral responses to mechanical or noxious chemical stimuli, which was accompanied by in vivo hyperexcitability of primary afferents and dorsal horn neurons in response to mechanical stimulation.29 Furthermore, similar to rheumatoid arthritis and CRPS, passive transfer of IgG from CASPR2-IgG-positive patients produces pain-related behavior in mice.29There is an increasing understanding of how VGKC complex antibodies are causative agents to pain hypersensitivity in these autoimmune neurological diseases. Neurons treated with VGKC complex antibodies show properties of hyperexcitability; IgG purified from patients with neuromyotonia causes repetitive firing of cultured mouse DRG neurons, and similar results are obtained when cells are treated with serum from CASPR2-IgG-positive patients, demonstrating the possibility of antibody interactions with DRG neurons.79 In addition to rituximab, IVIg and plasma exchange were effective at improving symptoms for some patients with leucine-rich glioma inactivated 1–targeting antibodies.71Therapies of immunomodulation have shown some disease-modifying effects for pain. Treatment with different immunotherapies  is frequently associated with pain improvement.28 Future research should determine if different classes of somatosensory neurons are differentially affected by VGKC complex autoantibodies. Autoantibodies that are reactive with other neuronal antigens, such as antibodies targeting the NMDA glutamate receptor, are not necessarily associated with pain as a major symptom. However, autoantibodies for CNS targets have been implicated in psychiatric and neurological disorders. Antibodies targeting NMDA glutamate receptor can result in neurological deficits and psychosis; myasthenia gravis is a neuromuscular disorder caused primarily by autoantibodies against the acetylcholine receptor, and autoantibodies targeting receptors for the inhibitory neurotransmitter glycine have been implicated in encephalitis and stiff-person syndrome.68 This leaves open the possibility that autoantibodies reactive to neuronal or glial targets could influence pain sensation and perception.These results collectively indicate that autoantibodies can target potassium channel complexes on neurons producing heightened neuronal activity, thereby providing an explanation for the development of chronic pain in these neurological disorders. These autoantibodies presumably affect the firing rates of myelinated fibers; this would implicate activity from Aδ nociceptors, as well as the Aβ mechanoreceptors for innocuous touch whose dysfunction is implicated in mechanical allodynia.The cumulative evidence reviewed here represents the remarkable progress made toward illuminating the ways in which autoimmune mechanisms contribute to pain in various disorders. Nevertheless, there are many questions related to autoimmunity and pain that remain to be answered. We highlight several of these questions here related to the development of autoimmunity, the influence of biological sex, the role of the brain in shaping immune function, and the unexplored role of autoantibody signaling at neurons and glia in the spinal cord and brain.151 One could speculate that glycosylation of autoantibodies in specific tissue niches could shift the binding affinity to certain FcγRs expressed by neurons or immune cells.121For conditions in which autoantibodies contribute to chronic pain, it is not clear what causes the initial shift from immune tolerance to autoimmune activation. This is a significant unanswered question with clinical implications for treating and preventing these conditions. There is still more research needed to identify what molecular triggers lead autoantigen-specific T cells and B cells to the production of autoantibodies during conditions of chronic pain. There may also be modifications to antibodies produced by B cells, such as glycosylation, that could alter the affinity of the Fab region to bind self-antigenic epitopes, or alter the Fc region to affect its ability to bind certain activating or inhibiting FcγRs, and/or change complement system recruitment. These modifications to self-IgG may shift “nonpathogenic” IgG into more disease-promoting autoantibodies.110 Given the vast differences in the probable risk factors and etiologies of different autoimmune diseases, there is unlikely to be a single biological pathway explaining these sex differences. However, there are potentially shared mechanisms associated with adaptive immune function. For instance, the autoimmune diseases systemic lupus erythematosus and Sjögren syndrome, which affect women at a rate 9 times higher than men, have the strongest genetic association with class II MHC human leukocyte antigen genes at the MHC locus. Variations in alleles for human leukocyte antigen genes have been associated with an increased risk for various autoimmune diseases.145 More recently, variations in complement genes C4A and C4B, found at the same MHC locus, strongly predict vulnerability for developing systemic lupus erythematosus and Sjögren syndrome in women, with women on average expressing lower protein expression of C4.76 Although further research is needed to elucidate the functional association between complement gene expression and autoimmune disease risk, reduced expression of complement could prolong adaptive immune cell interactions with self-antigens at sites of cellular injury or increase immune complex–mediated signaling at nociceptors.76Biological sex is an important factor that should be considered in determining cellular and molecular explanations for autoimmune mechanisms of pain. Women have higher incidence of both chronic pain and likelihood of developing most autoimmune diseases.20 Sympathetic innervation of primary and secondary lymphoid organs has been well characterized, and sympathectomy has been shown to influence the production of antibodies in the spleen.108 Immune cells across tissues have been shown to express neurotransmitter and neuroendocrine receptors, allowing for this neuronal control of immunity.48 To this end, a recent report identified a brain-to-spleen pathway controlling adaptive immunity, in which top-down activity from this neural circuit from central amygdala and paraventricular nucleus could directly influence the T-cell–dependent production of antibodies in the spleen.189 These observations hint at the exciting possibility that specific neural pathways could be targeted to more precisely influence antibody production. It has been thoroughly demonstrated that neural activity can regulate peripheral cytokine release and innate immune responses through vagal reflex circuits, and optogenetic activation of dopamine neurons in the brain has been shown to influence innate and adaptive immunity.146 Central mechanisms could conceivably influence autoimmune cellular function as well across tissues because neuronal release of CGRP can constrain cytokine production and proliferation of type 2 innate lymphoid cells in both the lung and the small intestine.185 Similarly, a newly described circuit of gut-innervating nociceptors can control localized immune responses to mediate host defense against pathogenic gut bacteria.87 These investigations highlight the sophisticated ways in which the CNS can influence immune function, and future challenges for treating pain arising from autoimmune mechanisms will involve advancing our knowledge of how the CNS can influence immune responses in health and disease.Considering the bidirectional signaling between the immune system and the nervous system, it is possible that neural circuits from the brain to immune targets could contribute to autoimmune function and thereby influence pain.83 It will be important for future research concerning this topic to determine whether or not autoimmune mechanisms in the spinal cord and brain are initiated by changes in activity from peripheral input or if they are sustained independently from DRG activity.Finally, most of the studies reviewed here focus on the role of autoimmune signaling at peripheral sensory input (in particular the DRG neurons) as contributing to pain. There is an established role of spinal cord neuroimmune signaling and plasticity of brain circuits as contributing to chronic pain, but the exact involvement of neural, glial, and immune cell signaling in spinal and brain circuits as contributing to autoimmune regulation of pain remains to be discovered.186 In this review, we have focused on the sequence of events for how autoimmunity is established—from activation of B cells to autoantibody production, to the actions of autoantibodies recruiting complement, signaling at Fcγ receptors, and binding to self-reactive targets—and we have demonstrated how these autoimmune mechanisms can promote chronic pain across diverse conditions, including rheumatoid arthritis, CRPS, and autoimmune channelopathies. Beyond these chronic pain conditions, there is evidence in neuropathic pain that nerve injury upregulates FcγR expression in rodent spinal cord and DRG.101 This raises the possibility that autoantibodies and autoimmune signaling represent a generalized phenomenon across painful conditions, with different diseases involving distinct autoimmune mechanisms that contribute to the manifestation of chronic pain. Further elaboration of these autoimmune signaling properties and their contributions to pain could be critical for developing novel analgesic therapies.Autoantibodies have been proposed as an important neuroimmune mechanism causing enhanced pain.The authors have no conflicts of interest to declare.


By reporting themolar abundance of proteins, absolute quantificationdetermines their stoichiometry in complexes, pathways, or networks.Typically, absolute quantification relies either on protein-specificisotopically labeled peptide standards or on a semiempirical calibrationagainst the average abundance of peptides chosen from arbitrarilyselected proteins. In contrast, a generic protein standard FUGIS  requires no isotopic labeling,chemical synthesis, or external calibration and is applicable to quantifyingproteins of any organismal origin. The median intensity of the peptidepeaks produced by the tryptic digestion of FUGIS is used as a single-pointcalibrant to determine the molar abundance of any codigested protein.Powered by FUGIS, median-based absolute quantification (MBAQ) outperformedother methods of untargeted proteome-wide absolute quantification. Relative quantification determineshow the abundance of the same protein changes across multiple conditionson a proteome-wide scale. In contrast, absolute quantification determinesthe exact molar quantity of each protein in each condition. In thisway, it is possible to relate the molar abundance of different proteins,estimate their expression level, or determine their stoichiometrywithin a variety of molecular constellations from stable complexesto organelles or metabolic pathways and interaction networks.13 It is usually presumed that the average abundanceof a few selected peptides faithfully represents the abundance ofthe corresponding source protein. In turn, peptides quantificationrelies either on isotopically labeled standards having exactly thesame sequence or on a semiempirical calibration against the abundanceof selected  peptides originatingfrom arbitrarily chosen standard proteins.14 Targeted approaches are more accurate, yet they only cover a smallselection of proteins that cannot be changed during the experiment.The latter methods work proteome-wide; however, they rely on arbitraryassumptions, and their accuracy is biased by experimental conditionsand the properties of individual proteins.A broadrepertoire of absolute quantification techniques tailoredtoward common analytical platforms, biological contexts, and researchaims was developed.15 uses a set of isotopically labeledsynthetic peptide standards identical with proteotypic peptides fromendogenous proteins. Alternatively, QconCAT,16 PSAQ,17 PrEST,18 PCS,19 MEERCAT,20 DOSCAT,21 and GeLC-based MS Western22 employ metabolically labeled protein chimerasthat, upon proteolytic cleavage, produce the desired peptide standards.MS Western relies on quantifying multiple proteotypic peptides perprotein and validates the concordance of protein determinations bymonitoring the intensity ratios between the XIC peaks of the standardsand the corresponding endogenous peptides. Common discrepancies inthese ratios point to an unreliable quantification and are typicallydue to miscleaved peptides or unexpected post-translational modifications.AQUA23 and SCAR24 standards use minimal sequencepermutation or scrambling. It is assumed that scrambled and endogenouspeptides share key physicochemical properties that result in equalinstrument response,26 which depends on the analyticalconditions and requires extensive validation.To circumvent isotopic labeling, MIPA27 APEX,28 SCAMPI.30 Methods like Top3/Hi-3,6 iBAQ,31 ProteomicRuler,32 xTop33 and Pseudo-IS34 use averaged XIC intensitiesof selected or of all peptides matching the protein of interest. Becauseof limited interlaboratory consistency, they are mostly used for supportingconventional proteomics workflows.Advances in robustand reproducible LC-MS/MS have led to the notionthat generic measures of a protein’s molar abundance couldbe deduced either from raw intensities or spectral counts of peptidepeaks, e.g., emPAI,Hence, there is a need todevelop a technology combining the accuracyand precision of the internal standards-based targeted quantificationwith broad  coverage and ease of use ofuntargeted methods. To this end, we developed an untargeted proteome-widequantification workflow termed median-based absolute quantification(MBAQ) that rely upon a fully unlabeled generic internal standard(FUGIS) based on the common physicochemical properties of proteotypicpeptides.6 cells werelysed for 30 min on ice in either 1 or 0.5 mL of RIPA buffer containingCLAAP protease inhibitors cocktail ). Subsequently, the cellswere further lysed by passing them 10 times through a 25 Gauge syringe.A postnuclear supernatant was obtained by 15 min centrifugation at4°C and 14 000g. The supernatant wasused for further analysis by GeLC-MS/MS  with MS-Western and FUGIS standardsin separate experiments.HeLa Kyoto cellswere cultured in Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal calf serum and 1% penicillin–streptomycin(Gibco Life Technologies). HeLa cells were trypsinized, counted, andwashed 2 times with PBS before 1 × 1022 The total protein content fromHeLa cells from both dilutions was loaded onto precast 4–20%gradient 1 mm thick polyacrylamide minigels purchased from AnamedElektrophorese  for 1D SDS PAGE. Separate gels wererun for 1 pmol of BSA and isotopically labeled lysine (K) and arginine(R) incorporated chimeric standard containing 3–5 unique quantitypicpeptides from the target proteins The sample was cut into 3 gel fractions,and each fraction was codigested with a known amount of BSA and thechimeric standard using Trypsin Gold, mass spectrometry grade . The digest was analyzed using the GeLC-MS/MS workflow . Peptidesmatching and chromatographic peaks alignment was carried out as describedin the Supporting Information (Databasesearch and data processing). The quantification was performed usingthe software developed in house.9Absolute protein quantification was performedusing the MS Westernprotocol.Supporting Information, GeLC-MS/MS).The on-column amount of FUGIS was 200–400 fmol; the loadedamount of chimeric proteins CP01 and CP02 was 300 fmol. Peptides matching and chromatographic peaks alignmentwere carried out as described in the Supporting Information (Database search and data processing). The output.csv files with sequences of matched peptides and areas of their XICpeaks were further processed by GlobeQuant software.Similarto the MS Western experiments, the total HeLa cell lysatefrom both dilutions was separated by1D SDS PAGE. Separate gels wererun for 1 pmol of BSA and the fully unlabeled generic internal standard(FUGIS). The gel lane was cut into three gel slices, and each slicewas codigested with a known amount of BSA and FUGIS and analyzed byLC-MS/MS  for fast access and search in the CSV file. GlobeQuant runs ona Windows 7 workstation with 16 GB of RAM and a 4-core processor.The .csv output from the Progenesis LC-MS v.4.1  with peptide ID’s and their respective raw XIC peak areaswas used by GlobeQuant software. A list of FUGIS peptides was providedas an input. The software calculated the molar amount of the FUGISstandard using the scrambled and native peptide pairs of BSA , relatedit to the median area of XIC peaks of FUGIS peptides. The calculatedmolar amount of the FUGIS standard was related to the median and further used it as a single-point calibrant.GlobeQuantsoftware was developed as a stand-alone Java script-based applicationusing an in-memory SQL database  was taken and averagedto provide the molar amounts of the protein. The software packageis available at 36 as evidenced by the time course and relativeabundance of the rendered peptides.22 Therefore,the peptide concentration could be inferred from the known molar abundanceof the chimeric protein.The MBAQ(median-based absolute quantification) workflow relies on a recombinantprotein standard consisting of concatenated peptides whose sequencesemulate the physicochemical properties shared by typical proteotypicpeptides. Its tryptic cleavage produces peptides in exactly equimolarconcentrations,22 We therefore hypothesized that the peak areascorresponding to an equimolar amount of proteotypic peptides releasedfrom the chimeric protein standard could cluster around some medianvalue irrespective of their sequence. As compared to the targetedquantification by comparing the intensities of the standard and analytepeaks, MBAQ can be less affected by a biased yield of some peptide(s)because the abundance of all clustering peptides is used for calculatingthe median.We therefore propose to determine themedian value of the areas of the XIC peaks of the peptides producedfrom chimeric protein and then use it as a single-point calibrantto calculate the molar abundance of other peptides from any codigestedprotein. We note that proteotypic peptides included into the chimeraprotein standard are selected according to a few common rules, suchas a higher abundance of XIC peaks, no evidence of internal and externalmiscleavages, no internal cysteine and methionine residues, and noaspartic or glutamic acid residue at the peptides N-terminus.9 and CP024 chimeras from our collection22 .If so, we only have to (i) provide a sufficientnumber of suchpeptides to compute the robust median value under the given experimentalconditions, (ii) select suitable peptides from those matched to theprotein of interest, and (iii) check if its quantification by individualpeptides is concordant. In our institute, we systematically producelarge (40–270 kDa) protein chimeras comprised of 40–250proteotypic peptides from various proteins. To test the feasibilityof MBAQ, we further used CP01Caenorhabditis elegans proteins.4 Despite an equimolar concentrationof produced peptides, their peak areas differed by almost 10-fold. However, the abundance of 48% ofall peptides clustered near the median value .In order to ascertain that clustering does not depend on some particularpeptide sequences, we digested another 265 kDa chimera (CP02) harboringproteotypic peptides from 48 proteins from Drosophilamelanogaster.9 We foundthat the peak areas of 42% of the peptides were close to the medianvalue . We concluded that independent ofthe peptide sequences, approximately one-half of the proteotypic peptidesclustered around the same median while others scattered around it.However, the commonality between the peptide sequences within theclustering and nonclustering groups was not immediately obvious.We first askedhow the areas of the XIC peaks of the proteotypicpeptides chosen from different proteins and concatenated into a chimeraare distributed around the median value and how many peptides wouldbe required to estimate it with acceptable accuracy. To this end,we digested 267 kDa chimeric protein (CP01) comprised of 250 proteotypicpeptides selected from 53  10-foldS1. Howev 10-foldS1. HowevFigure S2), we checked whether the median value could faithfully representthe molar abundance of the chimera. We expect that, in this case,possible suppression of peptide ionization by a sample matrix wouldbe likely randomized compared to a hypothetical scenario if all peptideswould be eluting together. For this purpose, we used the CP01 to quantify48 metabolic enzymes from C. elegans by the MS Western protocol and, independently, using a median valuecomputed from the abundance of all CP01 peptides. We underscore thatin the MS Western workflow each enzyme was quantified using severalisotopically labeled peptide standards that exactly matched sequencesof the corresponding native peptides4 withno recourse to other peptides. In contrast, in the MBAQ workflow allpeptides from the digested chimera were taken for calculating a singlemedian value that was subsequently used for quantifying all proteins.MBAQ was concordant with MS Western, showing a Pearson’s correlationof 95% (Since the “near-median” (NM) peptides wereevenlydistributed across the retention time range S2, we chTable S1).In a separate experiment, we quantified30 proteins from the commerciallyavailable UPS2 protein standard  using MBAQ andthe median calculated from CP01 peptides. The Pearson’s correlationwas 96%, and the median quantification error was less than 20%  and fullyunlabeled internal standard (FUGIS).FUGIS was conceived as a relativelysmall protein chimera composed of concatenated proteotypic-like trypticpeptides that, however, share no sequence identity to any known protein.It also comprises a few reference peptides with close similarity tosome common protein standard, e.g., BSA. Upon codigestion with quantifiedproteins, FUGIS should produce an equimolar mix of peptide standardswhose median abundance would support one-point MBAQ of all of thecodetected peptides from all of the proteins of interest. The exactamount of FUGIS is determined by comparison with the known amountof codigested reference protein  in the same LC-MS/MS experiment.We first asked what is the minimum number of peptides requiredto reach a consistent median value? For this purpose, we performeda bootstrapping experiment over the abundance of tryptic peptidesderived from CP01 and CP02. Median values were calculated by repetitiveselection of a defined (3–120) number of peptides 2. The daNext, we altered sequencesof these near-median peptides in severalways such that they become different from any known sequence. Yet,we aimed to preserve the similarity of their physicochemical properties,such as net charge, hydrophobicity index, and location of polar  amino acid residues as compared tocorresponding “source” peptides.Table S2) that was expressed and metabolically labeled with 13C15N-Arg and 13C-Lys in Escherichia coli.22 Itsband was excised from 1D SDS PAGE, codigested with the band of 1 pmolof BSA, and analyzed by LC-MS/MS.22 Similarto a previously published strategy,37 thepeptide abundance was normalized to the abundance of the BSA peptidesin the chimeric protein to check if the normalized median abundance(NMA) is close to unity (∼1.0). A unit NMA means that the medianabundance truly represents the amount of the FUGIS standard, whileany deviation contributes to the error in quantification.We first selecteda set of 40 out of a total of 70 NM peptidesand reversed their amino acid sequences 3A exceptThe NMA for the reversed sequences was 0.45 3B, whichTable S3). Pairwisecomparison of the peak areas of the native and scrambled sequencessuggested that they differed by less than 5%. Similar to GCP01, wecalculated the NMA for peptides in GCP02. Scrambled peptides behavedsimilar to the native sequences with a NMA of 1.02  min. Also, the relative abundance (peptide ratios) similar 3C. ThereFigure S3; Table S4), which harbored 43 scrambled near-medianpeptides and 5 sequences of scrambled reference peptides from BSA.Its peptides were not identical t to any known protein sequence acrossall organisms (Table S4).Taken together, we designed and producedthe FUGIS chimeric proteinhaving the molecular weight of 79.01 kDa . We observedthat the molar abundance determined by MBAQ was much closer to MSWestern than that of Top-3/Hi-3.We then proceeded to quantify the molar amountsof 4 proteins  from HeLa cells using MBAQ,MS Western,Table S5; Table S6). We estimated the concordance of its molar amount independentlycalculated from multiple peptides by the coefficient of variation(% CV).9 If calculated from the BestN peptidesit was 7%, which is significantly better than Hi-3 quantification(18%) .For MBAQ, in each target proteinwe selected peptides whose meanand median values differed by less than 15%. We termed them as “BestN”peptides–in contrast to TopN peptides that corresponded tothe N most abundant peptides. To assess if BestN peptides deliveredbetter accuracy, we looked into the quantification of one of the fourproteins (Table S7). For humancatalase (P04040) and serine/threonine protein kinase (P53350), theTop2 and Best2 peptides were the same (Table S7). Since the BestN peptides is a subset of the TopN peptides a minimumof two peptides was required to provide concrdant molar amounts.To understand why BestN peptides improved the quantification accuracy,we considered the difference between the BestN and the TopN peptidesets. For human G3P (P04406) and tubulin-1 alpha (Q71U36), the mostabundant peptides were excluded from the BestN set that reduced CVdown to less than 10%  are plotted as a ranked cumulativeabundance in Table S8) provide a useful resource for benchmarking of newlydeveloped absolute quantification methods.The GlobeQuant software supports the MBAQ workflow 6A by selFigure S5). This is not surprisingsince both determinations by Proteomic Ruler do not correlate andare also discordant with iBAQ. Since MBAQ corroborated MS Western peptides,MBAQ improved the quantification consistency by disregarding peptideswhose ionization capacity is based on a uniquely favorable sequence.It is also important that in MBAQ the molar abundance of peptidesis referred to a recognized commercial quantitative standard.It has long been noticed thatthe abundance of proteins could beinferred from the abundance of the best detected (TopN) peptides,as in Hi-3 quantification.We speculate that employing MBAQ or similar quantification mightbe an important step toward establishing diagnostically relevant proteinvalues in liquid and solid biopsies. MBAQ could quantify any proteindetectable with multiple (three or more) peptides in any LC-MS/MSexperiment, including data-independent acquisition (DIA). MBAQ doesnot rely on preconceived knowledge of the protein composition or availabilityof MS/MS spectra libraries.Finally, charting the proteome andmetabolome composition in molarquantities will facilitate our understanding of metabolic and signalingpathways that are controlled by molar ratios between multiple enzymesand substrates and help to uncover the molecular rationale of proteotype–phenotyperelationships.


While the identificationof paramagnetic O– radicals and their adsorptioncomplexes with O2 point to the emergence of hole centers,triboemitted electrons become scavenged by molecular oxygen to convertinto adsorbed superoxide anions O2– asmeasured by electron paramagnetic resonance (EPR). By means of complementaryUV-photoexcitation experiments, we found that photon energies in therange between 3 and 6 eV produce essentially the same EPR spectroscopicfingerprints and optical absorption features. To provide insightsinto this effect, we performed density functional theory calculationsto explore the energetics of charge separation involving the ionizationof low-coordinated anions and surface-adsorbed O2– radicals at points of contact. For all selected configurations,charge transfer is not spontaneous but requires an additional drivingforce. We propose that a plausible mechanism for oxygen radical formationis the generation of significant surface potential differences atpoints of contact under loading as a result of the highly inhomogeneouselastic deformations coupled with the flexoelectric effect.Powder compaction-inducedsurface chemistry in metal oxide nanocrystalensembles is important for very diverse fields such as triboelectrics,tribocatalysts, surface abrasion, and cold sintering of ceramics.Using a range of spectroscopic techniques, we show that MgO nanocubepowder compaction with uniaxial pressures that can be achieved bygentle manual rubbing or pressing ( These range from elastic and plastic deformation9 to shearing and fracture of surface elements11 such as edges and corners to coordinatively generate unsaturatedsurface ions of high chemical reactivity.14 Our mechanisticunderstanding of mechanically initiated chemical surface processesat the atomistic scale is scarce. Hence, a rational connection betweentriboinduced chemical activation steps including their energeticsand the application of processing parameters, which induce the triboelectricphenomena, is very much needed.Frictionbetween metal oxide interfaces can have important implicationsfor surface chemistry and occurs naturally in the course of handlingand compaction of nanoparticle powders. While there are a large numberof triboinduced phenomena reported for metal oxides, such as tribocharging,triboelectricity, or triboluminescence,16 adsorption and surface chemistry of complex organic molecules suchas porphyrins,18 and the photoexcitation of specificsurface structures such as nanocube corners and edges with UV lightof energies hν > 4 eV in great detail. Theoreticallypredicted energies of optical absorptions due to corners, edges, and(100) faces20 were found to be in excellentagreement with the experimental values and have even enabled us toisolate spectral contributions from buried interfaces.22 Moreover, photoexcitation studies revealed key insights into theenergetics of charge separation and subsequent electron and hole trappingat surface defects.25 Structural deformation of MgO under loading is alsorelatively well-understood. For example, compression-induced plasticityin bulk MgO materials is related to the nucleation of dislocations,their movement, and intersections.9 Enhanced deformationrates and dislocation densities in compressed bulk MgO samples seemto also promote ion vacancy formation26 and triboemission of charge carriers.27 Meanwhile, progress in experimental techniques has allowed for addressingplastic deformation and dislocation behavior of MgO at the nanoscale.8 Despite these advances, important qualitativeand quantitative information about the chemical species that formduring these early tribochemical activation steps and the energeticsof their formation is lacking.Due to their simple structureand grain morphology, cubic metaloxide particles such as MgO, CaO, FeO, or NiO are extremely well-suitedmodel systems to study tribo- and compaction-induced chemical reactionsinside powders. MgO particles produced using gas-phase synthesis techniquessuch as chemical vapor synthesis (CVS) or flame spray pyrolysis (FSP)yield narrow particle size distributions below 10 nm in combinationwith a characteristic cubic particle morphology that is determinedby the thermodynamically most stable (100) surfaces. The uniformityin size and shape have enabled diverse nanoparticle processes to berationalized, including oriented attachment,6 materials for the harvesting of low-level mechanical energy.There is a so far unexplored opportunity in the field of intergranularsurface chemistry, where we anticipate that our findings would beof interest for new approaches in chemical and cold sintering as wellas for the design of novel composites that can be polymerized at roomtemperature to achieve homogeneously distributed nanoparticles.Here, in this joint experimentaland theoretical study, we findthat MgO nanocube powder compaction in the dark at external pressuresas low as 5 MPa (corresponding to gentle pressing or rubbing by hand)excites electron–hole pairs. Following charge separation, surfaceradicals are produced that are accessible to independent spectroscopictechniques such as electron paramagnetic resonance (EPR) and UV–Visdiffuse reflectance spectroscopy. We show that the resulting charge-separatedconfigurations are equivalent to those that can be generated by UVexcitation with 3–6 eV photons. Therefore, gentle rubbing ofa powder is sufficient to surprisingly access energetically excitedstates and produce reactive surface species. These phenomena couldfind applications in the polishing and chemical treatment of surfacesas well as novel tribovoltaic and tribocatalyticThis study is structured as follows: in the first part, we describethe experimental findings (structure characterization and spectroscopy)we obtained in the course of MgO nanocube powder compaction-inducedcharge separation. We compare the outcome of this process in the darkwith paramagnetic states that originate from UV- excitation to concludethe nature of local defects and the energies required to form them.Then, in the second part, we analyzed different local particle–particleconfigurations and present density functional theory (DFT) resultsrelated to optimized structures of ionized donor and negatively chargedacceptor species. Finally, we discuss potential processes of energydissipation in the regimes of elastic and plastic deformation to explainthe compaction-induced charge separation effects observed.Q(Ar) = 1250 sccm] guides the Mg vapor to the end of theinner glass tube, where the argon/metal vapor mixture gets into contactwith molecular oxygen  streaming through the outer tube. At this position,a highly exothermic reaction in the presence of oxygen is initiated,which leads to the formation of MgO nanoparticles from homogeneousnucleation in the gas phase. Short residence times of resulting nucleiin the hot reaction zone (<2 ms),29 guaranteedby the Ar flow and continuous pumping down to p =50 ± 2 mbar, prevent undesired particle coarsening and coalescence.The nanoparticle agglomerates are collected in a stainless-steel netat room temperature downstream the reactor. Total pressure, gas flows,and furnace temperature are kept constant during the entire periodof particle collection.MgO nanocrystalsobtained from CVS correspond to the controlled combustion of Mg metalvapor in the presence of oxygen under reduced pressure. Further informationis provided in refs , 22), a, a22), ap(O2) < 10–5 mbar and defined gas atmospheres. Sample heatingup to 1123 K is described by a stepwise temperature increase of 100K with heating rates of 5 K/min (room temperature up to 373 K) and10 K/min (rest of the protocol), respectively. The next heating stepis initiated as the pressure falls below p(O2) < 9 · 10–6 mbar. Admission ofpure oxygen [p(O2) = 10 mbar] was performedat 1123 K and dwelled for 10 min. After subsequent evacuation to p(O2) < 9 . 10–6 mbar, thesample was heated to the final temperature  and dwelled for 60 min prior to cooling down to roomtemperature.30Powder annealing was performed in dedicatedfused silica cells attached to a high-vacuum rack, which allows forpressures as low as m = 150 ± 10 mg) wastransferred into the cavity (d = 13 mm) of a compactiontool  and uniaxially compressed with a hydraulicpress  under an appliedpressure between p = 1 MPa and 74 MPa that was dwelled for 1 min toobtain green compacts in a controlled and reproducible way. To minimizethe amount of water adsorption, powder transfer and compaction wasperformed inside glovebags filled with Ar at room temperature.Powder compaction was performed viacold uniaxial pressing resulting in a regular disk-shaped specimen.A defined mass of powder  between 5 and 80.5° 2Θ with a step sizeof 0.02° and opened divergence and antiscatter slits at 0.3 and4°, respectively. A primary and secondary side 2.5° Sollerslit was used to minimize axial divergence and a detector window openingangle of 2.93° was chosen. Data handling and qualitative phaseanalysis was performed with the Bruker software DIFFRAC.EVA V2.1.Crystallite domain sizes were derived from the integral reflectionwidth by applying the Scherrer equation to the (200) main diffractionfeature.Continuous scan powder X-ray diffraction(XRD) data were collected at room temperature in the coupled Θ–Θmode on a Bruker D8 Advance with a DaVinci design diffractometer,having a goniometer radius of 280 mm and equipped with a fast-solid-stateLynxeye detector and an automatic sample changer. Powder samples wereprepared on a single-crystal silicon zero-background sample holder.Data acquisition was performed using Cu KαTransmission electronmicroscopy (TEM) data were acquired using a JEOL JEM-F200 transmissionelectron microscope operating at 200 kV equipped with a cold fieldemission electron source. TEM images were recorded using a TVIPS F2162k by 2k CMOS camera to access morphological and structural information.Evaluation of images acquired during TEM analysis was performed witheither ImageJ (V1.52a) or the EM Measure software from TVIPS.2-sorption analysis wasperformed at 77 K and the specific surface area was calculated byapplying the model of Brunauer–Emmett–Teller (BET).Prior to sorption measurements with an ASAP 2020 adsorption porosimeterfrom Micromeritics GmbH, each sample was degassed under vacuum at573 K for 3 h. The BET surface area (SBET) was evaluated using adsorption data in a relative pressure range p/p0 of 0.06–0.21. Underthe assumption of equally sized and nonporous cubic particles, dBET), derivedfrom the integral sample volume, where ρ depicts the theoreticaldensity of MgO.31 Additionally, pore sizeanalysis was performed by applying the BJH model to the recorded sorptionisotherms.NPlus standard cavity and using an NMR teslameter that allowsfor accurate determination of resonant field values. MgO green compactfragments were placed inside a Suprasil quartz glass tube (d = 5 mm) and connected to the EPR high-vacuum line withbase pressures as low as p(O2) < 10–5 mbar, which also allows for in situ thermal treatmentup to 1173 K as well as addition of pure gas atmospheres [p(O2)] combined with UV-excitation. Polychromaticillumination of reannealed powder compacts [p(O2) < 10–5 mbar and T =1173 K] at the spectrum acquisition temperature was carried out usinga 300 W Xe-arc lamp equipped with a water filter to exclude IR contributionsfrom the excitation spectrum. EPR spectra were recorded at 10 K usinga waveguide cryogen-free system (Oxford Instruments). For spectrumacquisition, a microwave power of 2 mW, at a field modulation frequencyof 100 kHz with an amplitude of 1 G, was chosen.X-band EPR measurements were performedon a Bruker EMXplus-10/12/P/L spectrometer equipped with an EMX32 which is freely available.Spin systems of axial and rhombic g-tensor symmetrieswere modeled using Gaussian and/or Lorentzian line shapes. The bestfit was obtained after performing a least-squares fitting routineof multiple component EPR spectra. The aim of this approach was toperform a qualitative assignment of species, based on a profound knowledgeabout oxygen-related paramagnetic species that are well-known to formon the surface of MgO nanoparticles.35 Further details of thesimulation are provided in Supporting Information.The experimentalspectra were simulated using the Matlab-based computational packageEasySpin,37 A cluster of atoms centered near a region of interest . To prevent spurious spillingof the wave function from the quantum cluster into the classicallymodeled regions, the first and second nearest-neighbor Mg are modeledusing a semilocal effective core pseudopotential possessing no associatedbasis functions  and an electron affinity (EA) of 0.29eV (exp: 0.44 eV), for O2–, dO–O = 1.35 Å (exp: 1.34 Å), and for OH–, dO–H = 0.98 Å(exp: 0.97 Å).To calculate the total energiesof the various donor and acceptor configurations, we employ a quantummechanical embedded cluster approach, which combines quantum mechanicaland pair-potential levels of theory to describe large aperiodic systemssuch as MgO nanocrystals.2-sorption analysis (p = 74 MPa) reduces the surface area of the nanocube powderby roughly 30%, from 300 to 200 m2/g  of MgO nanocube powders in an oxygen atmosphere.35 Transformation of the optical absorption propertiesof the wide band gap material (Eg = 7.8eV)41 from a white scattering powder   1. A deta35 Although the individual EPR-signal components arebroadened in comparison with the UV-excitation results,35 the g-parameters are in very good agreement withthe earlier reported EPR signatures of trapped hole centers  and superoxide and ozonideanions  (Supporting Information). The signal broadening observed is attributedto two major effects:(i)powder compaction leads to higherconcentrations of paramagnetic defects which can emerge in close vicinityto each other and undergo spin-exchange interactions;(ii)a fraction of paramagnetic oxygenmolecules remains trapped inside the internal voids and closed poresof the compacts 2d. At crThe oxygen radical species that form upon compaction 2 have alp(O2) < 10–5 mbar at room temperature leadsto the decomposition of the paramagnetic O3– adduct into its components O– and O2. In the present study, however, we find that the ozonide ions remainafter pumping at room temperature, which serves as clear evidencefor their encapsulation in the internal pores .Previous studies on nonconsolidated loosenanoparticle powdershave shown that pumping to pressures as low as g = ±10–3 orless. This can be attributed to the altered microstructure of theMgO nanocube ensembles after powder compaction. New particle contactsand interface configurations emerge and give rise to qualitativelynew adsorption sites for O2– and O3– complexation that can participate in thepressure-induced charge separation process. Moreover, at the EPR acquisitiontemperature of Tacqu. = 10 K, residualoxygen and traces of water, which unavoidably remain trapped as aresult of the compaction step in the nitrogen-filled glovebag, alsoproduce slightly altered local electronic environments around theparamagnetic adsorbates .Deviations inthe EPR signatures of oxygen radicals obtained bycompaction versus UV-excitation of uncompacted powders are in therange Δ14 spins(N > 1011), this concentration corresponds roughly to 10–3 spins per particle. The application of higher pressures and, concomitantly,higher values for the compressive engineering stress (σ ≥37 MPa) by a hydraulic press increases the compacts’ translucencyand -at the same time -the intensity of its yellow/brown color  and 75 MPa:charge separation 2c, color14 spins3. Clearlwn color 3. Througg-parameter values, we also included UV-excitationexperiments in this study . Subsequent UV excitation in an O2 atmosphere generates oxygen radicals with an EPR powder spectrum and superoxide (O2–) anion formation.Asan additional process, photoexcitation with a threshold energyof 4.0 eV was found to initiate an oxygen adsorption-assisted processof ozonide as the electron-trapping species (acceptor). The hole-trapping species(donor) is thought to be a low-coordinated oxygen ion (either at afree corner or a point of contact such as a corner-terrace feature).One question that emerges is whether the formation of new structuralfeatures at particle–particle points of contact by compressioncould be sufficient to explain the observed charge transfer by significantlyreducing the energy cost for electron–hole separation. Here,we attempt to answer that question by calculating total (ground state)energy differences between candidate donor and acceptor features inthe nanopowder using DFT and the embedded cluster method  should be relativelylow including the oxygen-terminated corner-terrace and a surface-adsorbedhydroxyl group (OH–). For the former, we considerthe interface with and without compressive strain  or near a point of contactbetween nanocrystals (CT).1) is 4.9 eV. Application of a 4% compressivestrain, followed by full geometry optimization is found to reducethe length of the O corner–Mg terrace bond by about 3% (D2). However, this leads to only a 0.1 eV reduction in the IEsuggesting by itself that local strain of bonds due to compressionis not likely responsible for inducing charge transfer. The calculatedIE for the hydroxyl adsorbed on an edge is 3.2 eV. Turning now tothe acceptor features, we find the EA of the oxygen molecule adsorbedat the terrace is 2.6 eV (A1), at the oxygen-terminatedcorner-terrace is 1.5 eV (A2), and at the edge is 2.9 eV(A3).3–A3) has the lowestΔ of 0.3 eV. However, this is unlikely to explain the experimentallyobserved compression-induced charge separation as the hole that isleft on the donor remains localized on the hydroxyl oxygen. If thiswere the correct charge-separated state, one would see evidence forit in the form of hyperfine splitting in the EPR spectra, but thisis not seen experimentally. Of the charge-transfer processes directlyinvolving a point of contact, the lowest energy difference is forelectron transfer from a strained oxygen-terminated corner-terraceto an oxygen molecule adsorbed at an adjacent edge (D2-A3) with Δ = 1.9 eV.To compare the feasibility of the various possible charge-transferprocesses, we summarize the energy difference Δ = IP –EA for the donor–acceptor pairs in The analysis performed here at the quantum level pointsto a numberof local contact configurations that would facilitate the separationand consecutive charge transfer. However, while all the energy costscomputed are below 4.6 eV , they still will require energies ≥ 1.9eV. Thus, in the absence of additional driving forces, the local defectgeometries alone cannot explain the spontaneous occurrence of chargeseparation.46 addressedthe question whether flexoelectric potential differences that areinduced by inhomogeneous strain at nanoscale asperities can drivetribocharge separation and transfer. Based on Hertzian and Johnson–Kendall–Robertscontact models for elastic deformation, their analysis revealed thatlarge strain gradients can give rise to surface potential differencesin the range of ±1–10 V. Such inhomogeneous strain effectswith their intrinsic asymmetry can emerge on a variety of solid partsin movement, such as particles during milling and inside powders duringcompaction. In fact, MgO nanocubes and their aggregates exhibit alarge abundance of such asperities in the form of nanocube edges,step edges, or corners. Upon powder compaction, all of these surfacefeatures get in contact with surface elements present on adjacentparticles. Based on the computed energies mentioned above, the typicalpotential differences that can be generated by the flexoelectric effectcould be sufficient to induce charge transfer under compaction.One example of a plausible additional driving forceis the flexoelectriceffect. The flexoelectric effect can occur in essentially all typesof insulating materials (ceramics and polymers) and is characterizedby a linear coupling between a local strain gradient and polarization.In recent work Mizzi et al.47 This low density may arise from the tendency of the cubicMgO particles to stack and form elongated clusters  surfaces,corners, and edges. Evidence for the interaction between dislocationswas primarily obtained for larger cubes. Despite these advances, fornanoparticles present in a powder, we still are limited in the factthat the contact area between the particles, which will determinelocal stresses, is difficult to define and is strongly dependent onthe local microstructure of the powders.Recent advances in in situTEM measurements in combination withthe use of nanocubes of MgO smoke and complementary molecular dynamics(MD) simulations revealed details on elastic and plastic deformationat the level of individual particles.46When a powder of particlesis compacted under pressure, the pointcontacts between the particles flatten to areal contacts. In sucha porous compact, any transmission of force takes place mainly throughthis contact region. From the present study, it is clear that thelow density of the powder compact, as well as the heterogeneity inpore sizes (presence of mesopores, Here, it is also important to note that compaction-inducedchargeseparation was observed on nanocrystalline MgO with dehydroxylatedparticle surfaces and in pristine environments. The next logical stepwill be to explore the impact of the environmental gas atmosphere,such as the presence of water vapor on charge separation yield andto expand this investigation to other metal oxides of comparable dispersion.25 This suggeststhat compression alone is likely to be inducing a very similar charge-transferprocess; however, the mechanism is not currently known.As a summary of the here-discussed results, we can state that ourexperimental investigations demonstrate that MgO powder compactioninduces the formation of oxygen radicals by charge separation essentiallyindistinguishable from those generated by UV-excitation of the samecompacted material. The mechanism for charge separation by UV- excitationis well-understood, involving the photoexcitation of low-coordinatedanion features leading to electron transfer to acceptor species suchas adsorbed oxygen molecules. This process requires photon energiesof around 4.6 eV or higher.To providesome insights into possible mechanisms, we have performeda number of DFT calculations to assess the electronic properties offeatures at points of contact between nanocrystals as well as at dislocationsthat may be generated under loading, facilitating plastic deformation.We have also provided estimates of the energy that may be releasedby compression-induced plastic deformation. The DFT calculations considerthe electronic properties of various features in local equilibriumto assess whether their structure alone is sufficient to induce chargetransfer. While the answer is negative, we discuss how the flexoelectriceffect that has recently been invoked to explain tribocharge separationand transfer at nanoscale asperities can provide an additional drivingforce for such charge-transfer processes. We also show that it islikely that under compression, some degree of plastic deformationmay take place involving the formation and motion of dislocations.We show that the presence of dislocations in local equilibrium alonecannot explain charge transfer. However, under compression, theirnonequilibrium dynamics that may involve direct excitation of phononsor conceivably electrons cannot be ruled out as a mechanism for chargetransfer.Uniaxial pressing ofMgO nanocube powders was found to producecharacteristic spectroscopic property changes in the material thatare indicative of charge separation effects at the surfaces and interfacesof the nanograins. In complementary theoretical calculations, we analyzedselected local contact configurations between MgO nanocubes and calculatedthe associated energetics upon external mechanical load. While referenceexperiments on light-induced charge separation, producing identicalEPR and optical spectroscopic fingerprints, have revealed 4.6 eV asa minimum excitation energy for charge separation, the computationalresults indicate energies below this value.49 Moreover, the triboemission-inducedgeneration of surface radicals offers a novel opportunity region toinitiate radical polymerization reactions that can evolve in the confinedspace in between the grains, which may lead to nanoparticle–polymercomposites of superior homogeneity and density.In the field oftribocharging, this study provides for the firsttime molecular-level information on the pressure-induced separationand interfacial transfer of charge carriers on MgO grains. Since theseprocesses can occur prior to the step of sintering, such insightsserve as an important basis for a knowledge-based design and engineeringof interfaces in compressed metal oxide nanoparticle powders. Relatedprocessing steps that trigger interesting interface chemistry canbe performed at temperatures significantly below those typically requiredfor the sintering of ceramics. Hence, so far unavailable mechanisticdetails will become accessible for the newly developed process ofcold sintering.