Nurture outpaces nature: fostering with an attentive mother alters social dominance in a mouse model of stress sensitivity.

Maternal care is critical for epigenetic programming during postnatal brain development. Stress is recognized as a critical factor that may affect maternal behavior, yet owing to high heterogeneity in stress response, its impact varies among individuals. We aimed here to understand the connection between inborn stress vulnerability, maternal care, and early epigenetic programming using mouse populations that exhibit opposite poles of the behavioral spectrum (social dominance [Dom] and submissiveness [Sub]) and differential response to stress. In contrast to stress-resilient Dom dams, stress-vulnerable Sub dams exhibit significantly lower maternal attachment, serum oxytocin, and colonic Lactobacillus reuteri populations. Sub offspring showed a reduced hippocampal expression of key methylation genes at postnatal day (PND) 7 and a lack of developmentally-dependent increase in 5-methylcytosine (5-mC) at PND 21. In addition, Sub pups exhibit significant hypermethylation of gene promoters connected with glutamatergic synapses and behavioral responses. We were able to reverse the submissive endophenotype through cross-fostering Sub pups with Dom dams (Sub/D). Thus, Sub/D pups exhibited elevated hippocampal expression of DNMT3A at PND 7 and increased 5-mC levels at PND 21. Furthermore, adult Sub/D offspring exhibited increased sociability, social dominance, and hippocampal glutamate and monoamine levels resembling the neurochemical profile of Dom mice. We postulate that maternal inborn stress vulnerability governs epigenetic patterning sculpted by maternal care and intestinal microbiome diversity during early developmental stages and shapes the array of gene expression patterns that may dictate neuronal architecture with a long-lasting impact on stress sensitivity and the social behavior of offspring.

Functional IgG Autoantibodies against Autonomic Nervous System Receptors in Symptomatic Women with Silicone Breast Implants.

The association between the clinical picture of symptomatic women with silicone breast implants (SBI) and dysregulated immunity was in dispute for decades. In the current study, we describe for the first time the functional activity of purified IgG antibodies derived from symptomatic women with SBIs (suffering from subjective/autonomic-related symptoms), both in vitro and in vivo. We found that IgGs, derived from symptomatic women with SBIs, dysregulate inflammatory cytokines (TNFα, IL-6) in activated human peripheral blood mononuclear cells, compared to healthy-women-derived IgGs. Importantly, behavioral studies conducted following intracerebroventricular injection of IgGs derived from symptomatic women with SBIs (who have dysregulated circulating level of IgG autoantibodies directed against autonomic nervous system receptors) into mice brains demonstrated a specific and transient significant increment (about 60%) in the time spent at the center of the open field arena compared with mice injected with IgG from healthy women (without SBIs). This effect was accompanied with a strong trend of reduction of the locomotor activity of the SBI-IgG treated mice, indicating an overall apathic-like behavior. Our study is the first to show the potential pathogenic activity of IgG autoantibodies in symptomatic women with SBIs, emphasizing the importance of these antibodies in SBI-related illness.

Link between temperament traits, brain neurochemistry and response to SSRI: insights from animal model of social behavior.

BACKGROUND: Dominant-submissive relationships depend upon functionality of the neural circuits involving monoaminergic neurotransmission. Behavioral profiles of selectively bred dominant (Dom) and submissive (Sub) mice have been proposed to mimic hyperthymic- or depressive-like temperaments observed in patients with affective disorders. These mice differentially respond to psychotropic agents and stressful stimuli, however, the mechanisms underlying these differences remain unclear. To address these mechanisms, we analyzed the brain monoamine content and responses to paroxetine (PXT) in Dom and Sub mice. METHODS: The behavioral effects of PXT (3 mg/kg, single injection) were assessed with the Elevated Plus Maze (EPM) and Forced Swim Test (FST). Monoamine tissue content was analyzed by HPLC-ECD. RESULTS: Compared to Dom, Sub mice had decreased levels of serotonin (5-HT) in the brainstem (BS), reduced levels of norepinephrine (NE) in the prefrontal cortex (PFC), hippocampus (HPC), and striatum (STR) and elevated levels of dopamine (DA) in PFC, HPC, STR and BS. In EPM, PXT administration increased locomotion and exploration in Dom mice, with no effect in Sub mice. In FST, PXT disrupted immobility in Dom mice only. The PXT-produced differences in regional monoamine content were strain-dependent and consistent with the behavioral alterations. LIMITATIONS: Chronic PXT treatment, in vivo monoamine assays and sex-dependent analysis were out of the scope of this study and will be performed in the future in order to provide an in-depth evaluation of the neurochemical mechanisms underlying temperament-dependent responses to SSRIs. CONCLUSIONS: Our findings suggest neurochemical mechanisms that underlie temperament-based response to antidepressant treatment.

Link between personality and response to THC exposure.

The effects of cannabis reported by users range from experiences of euphoria and anxiolytic effects to paranoia, anxiety, and increased risk of depression. Attempts to reconcile the apparent contradictions in user response have not been conclusive. Here, we utilized selectively-bred stress-resilient socially dominant (Dom) and stress-sensitive socially submissive (Sub) mice to elucidate this contradiction. Following short-term, repeated treatment with delta-9-tetrahydrocannabinol (THC) at two different doses (1.5 mg/kg and 15 mg/kg), Sub mice presented significant place-aversion in a Conditioned Place Preference paradigm at a high dose, whereas Dom mice displayed no place preference or aversion. Forced Swim test conducted after 6-week of washout period, revealed differential impact of the two THC doses depending upon behavioral pattern. Specifically, the low dose alleviated depressive-like behavior in Sub mice, while the high dose produced the opposite effect in Dom mice. Interestingly, corticosterone concentration in serum was elevated at the high dose regardless of the mice-population tested. We conclude here that differences in dominance behavior and stress vulnerability are involved in the regulation of cannabis response among users and should be considered when prescribing THC-containing medications to patients.

Innate sensitivity to stress facilitates inflammation, alters metabolism and shortens lifespan in a mouse model of social hierarchy.

It is known that stress alters homeostasis and may lead to accelerated aging. However, little is known about the contribution of innate susceptibility to stress to the deterioration of physiological functions, acceleration of aging and developing of age-related diseases. By using socially-submissive stress susceptible (Sub) and socially-dominant stress resilient (Dom) selectively bred mouse model we observed a marked reduction in the lifespan of both male and female Sub mice. We found that innate susceptibility to stress correlates with chronic inflammation, development of splenomegaly and a significant increase in the levels of circulating pro-inflammatory cytokines IL-1ß and IL-6. Furthermore, Sub mice showed a marked hypoglycemia, reduction of insulin levels, increase in GSK3 activity and elevation of IGF-1 serum levels, as well as low skin surface temperature and body weight. Interestingly, lifelong exposure of Sub mice to chronic mild stress did not further reduce their lifespan, indicating a high level of intrinsic stress. Taken together, our data reveal that social submissiveness coupled with innate stress sensitivity coincides with inflammation, leading to the deterioration of physiological functions and early aging independent of whether an individual is exposed to stress or not.

Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury.

Individuals with spinal cord injury (SCI) usually suffer from permanent neurological deficits, while spontaneous recovery and therapeutic efficacy are limited. Here, we demonstrate that when given intranasally, exosomes derived from mesenchymal stem cells (MSC-Exo) could pass the blood brain barrier and migrate to the injured spinal cord area. Furthermore, MSC-Exo loaded with phosphatase and tensin homolog small interfering RNA (ExoPTEN) could attenuate the expression of PTEN in the injured spinal cord region following intranasal administrations. In addition, the loaded MSC-Exo considerably enhanced axonal growth and neovascularization, while reducing microgliosis and astrogliosis. The intranasal ExoPTEN therapy could also partly improve structural and electrophysiological function and, most importantly, significantly elicited functional recovery in rats with complete SCI. The results imply that intranasal ExoPTEN may be used clinically to promote recovery for SCI individuals.

Importin α5 Regulates Anxiety through MeCP2 and Sphingosine Kinase 1.

Importins mediate transport from synapse to soma and from cytoplasm to nucleus, suggesting that perturbation of importin-dependent pathways should have significant neuronal consequences. A behavioral screen on five importin α knockout lines revealed that reduced expression of importin α5 (KPNA1) in hippocampal neurons specifically decreases anxiety in mice. Re-expression of importin α5 in ventral hippocampus of knockout animals increased anxiety behaviors to wild-type levels. Hippocampal neurons lacking importin α5 reveal changes in presynaptic plasticity and modified expression of MeCP2-regulated genes, including sphingosine kinase 1 (Sphk1). Knockout of importin α5, but not importin α3 or α4, reduces MeCP2 nuclear localization in hippocampal neurons. A Sphk1 blocker reverses anxiolysis in the importin α5 knockout mouse, while pharmacological activation of sphingosine signaling has robust anxiolytic effects in wild-type animals. Thus, importin α5 influences sphingosine-sensitive anxiety pathways by regulating MeCP2 nuclear import in hippocampal neurons.

Social rank-associated stress vulnerability predisposes individuals to cocaine attraction.

Studies of personality have suggested that dissimilarities in ability to cope with stressful situations results in differing tendency to develop addictive behaviors. The present study used selectively bred stress-resilient, socially-dominant (Dom) and stress-vulnerable, socially-submissive (Sub) mice to investigate the interaction between environmental stress and inbred predisposition to develop addictive behavior to cocaine. In a Conditioned Place Preference (CPP) paradigm using cocaine, Sub mice displayed an aversion to drug, whereas Dom mice displayed drug attraction. Following a 4-week regimen of Chronic Mild Stress (CMS), Sub mice in CPP displayed a marked increase (>400%) in cocaine attraction, whereas Dom mice did not differ in attraction from their non-stressed state. Examination of hippocampal gene expression revealed in Sub mice, exposure to external stimuli, stress or cocaine, increased CRH expression (>100%), which was evoked in Dom mice only by cocaine exposure. Further, stress-induced decreases in DRD1 (>60%) and DRD2 (>50%) expression in Sub mice differed markedly from a complete lack of change in Dom mice. From our findings, we propose that social stratification dictates vulnerability to stress-induced attraction that may lead to addiction via differential regulation of hippocampal response to dopaminergic input, which in turn may influence differing tendency to develop addictive behaviors.

The Dual Function of the Polybasic Juxtamembrane Region of Syntaxin 1A in Clamping Spontaneous Release and Stimulating Ca2+-Triggered Release in Neuroendocrine Cells.

The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca2+-triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca2+-dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca2+SIGNIFICANCE STATEMENT Syntaxin 1A (Syx) is a central protein component of the SNARE complex, which underlies neurotransmitter release. Although widely studied in relation to its participation in SNARE complex formation and its interaction with phosphoinositides, the function of Syx's polybasic juxtamembrane region (5RK) remains unclear. Previously, we showed that a conformational transition of Syx, related to calcium-triggered release, reported by a Syx-based FRET probe, is abolished upon charge neutralization of 5RK (5RK/A). Here we show that this conformational transition is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and is related to SNARE complex formation. Subsequently, we show that the 5RK/A mutation enhances spontaneous release and inhibits calcium-triggered release in neuroendocrine cells, indicating a previously unrecognized role of 5RK in neurotransmitter release.

Implantation of 3D Constructs Embedded with Oral Mucosa-Derived Cells Induces Functional Recovery in Rats with Complete Spinal Cord Transection.

Spinal cord injury (SCI), involving damaged axons and glial scar tissue, often culminates in irreversible impairments. Achieving substantial recovery following complete spinal cord transection remains an unmet challenge. Here, we report of implantation of an engineered 3D construct embedded with human oral mucosa stem cells (hOMSC) induced to secrete neuroprotective, immunomodulatory, and axonal elongation-associated factors, in a complete spinal cord transection rat model. Rats implanted with induced tissue engineering constructs regained fine motor control, coordination and walking pattern in sharp contrast to the untreated group that remained paralyzed (42 vs. 0%). Immunofluorescence, CLARITY, MRI, and electrophysiological assessments demonstrated a reconnection bridging the injured area, as well as presence of increased number of myelinated axons, neural precursors, and reduced glial scar tissue in recovered animals treated with the induced cell-embedded constructs. Finally, this construct is made of bio-compatible, clinically approved materials and utilizes a safe and easily extractable cell population. The results warrant further research with regards to the effectiveness of this treatment in addressing spinal cord injury.

Effect of social interactions on hippocampal protein expression in animal dominant and submissive model of behavioral disorders.

PURPOSE: Psychiatric conditions, in many cases, arise from social interactions necessary for optimal mental functioning. Dominance and submissiveness are two opposite poles of behavior, stemming from processes of social interactions between members inside one group or species. Extreme dominance and submissiveness expressions in humans is accompanied by mental impairments, including mania and depression. Here, taking advantage of animals bred selectively for traits of dominance and submissiveness, we assess protein expression profiles in dominant and submissive mice in the context of social interaction. EXPERIMENTAL DESIGN: Proteins extracted from hippocampi of naïve and social interaction subjected dominant, submissive and wild type mice (15 mice per each group) are quantified using label-free quantitative LC/MS/MS analysis. Complexity of social interaction-related protein expression is resolved by factor analysis and enriched with GO and protein-protein interaction functional network analyses. RESULTS: In total, 1146 proteins exhibiting expression changes in the wild type mice, as well as dominant and submissive mice are enriched in protein datasets responsible for: 1) socially triggered dominance (90 proteins), 2) inherent submissiveness (75 proteins), 3) socially triggered submissiveness (117 proteins), and 4) social interaction triggered protein expression changes, related to resilience/adaptation to stress (69 proteins). Among the most enriched categories, extensive changes are found in proteins related to presynaptic release, ion channel regulation, circadian rhythm, MAPK, ErbB and NF-kB pathways. CONCLUSION: Data extracted from this first extensive proteomic study of a social interaction paradigm may facilitate decoding of molecular mechanisms responsible for pathogenesis of psychiatric disorders.

A Systems-Level Analysis of the Peripheral Nerve Intrinsic Axonal Growth Program.

The regenerative capacity of the injured CNS in adult mammals is severely limited, yet axons in the peripheral nervous system (PNS) regrow, albeit to a limited extent, after injury. We reasoned that coordinate regulation of gene expression in injured neurons involving multiple pathways was central to PNS regenerative capacity. To provide a framework for revealing pathways involved in PNS axon regrowth after injury, we applied a comprehensive systems biology approach, starting with gene expression profiling of dorsal root ganglia (DRGs) combined with multi-level bioinformatic analyses and experimental validation of network predictions. We used this rubric to identify a drug that accelerates DRG neurite outgrowth in vitro and optic nerve outgrowth in vivo by inducing elements of the identified network. The work provides a functional genomics foundation for understanding neural repair and proof of the power of such approaches in tackling complex problems in nervous system biology.

Dynamics of Hippocampal Protein Expression During Long-term Spatial Memory Formation.

Spatial memory depends on the hippocampus, which is particularly vulnerable to aging. This vulnerability has implications for the impairment of navigation capacities in older people, who may show a marked drop in performance of spatial tasks with advancing age. Contemporary understanding of long-term memory formation relies on molecular mechanisms underlying long-term synaptic plasticity. With memory acquisition, activity-dependent changes occurring in synapses initiate multiple signal transduction pathways enhancing protein turnover. This enhancement facilitates de novo synthesis of plasticity related proteins, crucial factors for establishing persistent long-term synaptic plasticity and forming memory engrams. Extensive studies have been performed to elucidate molecular mechanisms of memory traces formation; however, the identity of plasticity related proteins is still evasive. In this study, we investigated protein turnover in mouse hippocampus during long-term spatial memory formation using the reference memory version of radial arm maze (RAM) paradigm. We identified 1592 proteins, which exhibited a complex picture of expression changes during spatial memory formation. Variable linear decomposition reduced significantly data dimensionality and enriched three principal factors responsible for variance of memory-related protein levels at (1) the initial phase of memory acquisition (165 proteins), (2) during the steep learning improvement (148 proteins), and (3) the final phase of the learning curve (123 proteins). Gene ontology and signaling pathways analysis revealed a clear correlation between memory improvement and learning phase-curbed expression profiles of proteins belonging to specific functional categories. We found differential enrichment of (1) neurotrophic factors signaling pathways, proteins regulating synaptic transmission, and actin microfilament during the first day of the learning curve; (2) transcription and translation machinery, protein trafficking, enhancement of metabolic activity, and Wnt signaling pathway during the steep phase of memory formation; and (3) cytoskeleton organization proteins. Taken together, this study clearly demonstrates dynamic assembly and disassembly of protein-protein interaction networks depending on the stage of memory formation engrams.

Synapsin IIb as a functional marker of submissive behavior.

Dominance and submissiveness are important functional elements of the social hierarchy. By employing selective breeding based on a social interaction test, we developed mice with strong and stable, inheritable features of dominance and submissiveness. In order to identify candidate genes responsible for dominant and submissive behavior, we applied transcriptomic and proteomic studies supported by molecular, behavioral and pharmacological approaches. We clearly show here that the expression of Synapsin II isoform b (Syn IIb) is constitutively upregulated in the hippocampus and striatum of submissive mice in comparison to their dominant and wild type counterparts. Moreover, the reduction of submissive behavior achieved after mating and delivery was accompanied by a marked reduction of Syn IIb expression. Since submissiveness has been shown to be associated with depressive-like behavior, we applied acute SSRI (Paroxetine) treatment to reduce submissiveness in studied mice. We found that reduction of submissive behavior evoked by Paroxetine was paired with significantly decreased Syn IIb expression. In conclusion, our findings indicate that submissiveness, known to be an important element of depressive-like behavioral abnormalities, is strongly linked with changes in Syn IIb expression.

Early onset of cognitive impairment is associated with altered synaptic plasticity and enhanced hippocampal GluA1 expression in a mouse model of depression.

Memory deficit is a common manifestation of age-related cognitive impairment, of which depression is a frequently occurring comorbidity. Previously, we developed a submissive (Sub) mouse line, validated as a model of depressive-like behavior. Using learning paradigms testing hippocampus-dependent spatial and nonspatial memory, we demonstrate here that Sub mice developed cognitive impairments at earlier age (3 months), compared with wild-type mice. Furthermore, acute hippocampal slices from Sub animals failed to display paired-pulse facilitation, whereas primed burst stimulation elicited significantly enhanced long-term potentiation in region CA1, relative to control mice. Changes in synaptic plasticity were accompanied by markedly reduced hippocampal messenger RNA expression of insulin-like growth factor and brain-derived neurotrophic factor. Finally, we identified markedly elevated protein levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 in the hippocampi of Sub mice, which was exacerbated with age. Taken together, the results point to a linkage between depressive-like behavior and the susceptibility to develop age-related cognitive impairment, potentially by hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated glutamatergic signaling.

StimDuino: an Arduino-based electrophysiological stimulus isolator.

BACKGROUND: Electrical stimulus isolator is a widely used device in electrophysiology. The timing of the stimulus application is usually automated and controlled by the external device or acquisition software; however, the intensity of the stimulus is adjusted manually. Inaccuracy, lack of reproducibility and no automation of the experimental protocol are disadvantages of the manual adjustment. To overcome these shortcomings, we developed StimDuino, an inexpensive Arduino-controlled stimulus isolator allowing highly accurate, reproducible automated setting of the stimulation current. NEW METHOD: The intensity of the stimulation current delivered by StimDuino is controlled by Arduino, an open-source microcontroller development platform. The automatic stimulation patterns are software-controlled and the parameters are set from Matlab-coded simple, intuitive and user-friendly graphical user interface. The software also allows remote control of the device over the network. RESULTS: Electrical current measurements showed that StimDuino produces the requested current output with high accuracy. In both hippocampal slice and in vivo recordings, the fEPSP measurements obtained with StimDuino and the commercial stimulus isolators showed high correlation. COMPARISON WITH EXISTING METHODS: Commercial stimulus isolators are manually managed, while StimDuino generates automatic stimulation patterns with increasing current intensity. The pattern is utilized for the input-output relationship analysis, necessary for assessment of excitability. In contrast to StimuDuino, not all commercial devices are capable for remote control of the parameters and stimulation process. CONCLUSIONS: StimDuino-generated automation of the input-output relationship assessment eliminates need for the current intensity manually adjusting, improves stimulation reproducibility, accuracy and allows on-site and remote control of the stimulation parameters.

Syntaxin13 expression is regulated by mammalian target of rapamycin (mTOR) in injured neurons to promote axon regeneration.

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.

A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1.

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.

Gas-phase compaction and unfolding of protein structures.

Ion-mobility mass spectrometry is emerging as a powerful tool for studying the structures of less established protein assemblies. The method provides simultaneous measurement of the mass and size of intact protein assemblies, providing information not only on the subunit composition and network of interactions but also on the overall topology and shape of protein complexes. However, how the experimental parameters affect the measured collision cross-sections remains elusive. Here, we present an extensive systematic study on a range of proteins and protein complexes with differing sizes, structures, and oligomerization states. Our results indicate that the experimental parameters, T-wave height and velocity, influence the determined collision cross-section independently and in opposite directions. Increasing the T-wave height leads to compaction of the protein structures, while higher T-wave velocities lead to their expansion. These different effects are attributed to differences in energy transmission and dissipation rates. Moreover, by analyzing proteins in their native and denatured states, we could identify the lower and upper boundaries of the collision cross-section, which reflect the "maximally packed" and "ultimately unfolded" states. Together, our results provide grounds for selecting optimal experimental parameters that will enable preservation of the nativelike conformation, providing structural information on uncharacterized protein assemblies.

T-wave ion mobility-mass spectrometry: basic experimental procedures for protein complex analysis.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N(2;)) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (Omega). Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as Omega is related to the overall shape and topology of the ion. The correlation between the measured drift time values and Omega is calculated using a calibration curve generated from calibrant proteins with defined cross-sections(1). The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions(1). Several recent IM studies of both individual proteins(2,3) and non-covalent protein complexes(4-9), successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)(10). We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical Omega values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.