ChaMP-CMD: A Phenotype-Blinded, Randomized Controlled, Cross-Over Trial.

BACKGROUND: Angina with nonobstructive coronary arteries is a common condition for which no effective treatment has been established. We hypothesized that the measurement of coronary flow reserve (CFR) allows identification of patients with angina with nonobstructive coronary arteries who would benefit from anti-ischemic therapy. METHODS: Patients with angina with nonobstructive coronary arteries underwent blinded invasive CFR measurement and were randomly assigned to receive 4 weeks of amlodipine or ranolazine. After a 1-week washout, they crossed over to the other drug for 4 weeks; final assessment was after the cessation of study medication for another 4 weeks. The primary outcome was change in treadmill exercise time, and the secondary outcome was change in Seattle Angina Questionnaire summary score in response to anti-ischemic therapy. Analysis was on a per protocol basis according to the following classification: coronary microvascular disease (CMD group) if CFR<2.5 and reference group if CFR≥2.5. The study protocol was registered before the first patient was enrolled (International Standard Randomised Controlled Trial Number: ISRCTN94728379). RESULTS: Eighty-seven patients (61±8 years of age; 62% women) underwent random assignment (57 CMD group and 30 reference group). Baseline exercise time and Seattle Angina Questionnaire summary scores were similar between groups. The CMD group had a greater increment (delta) in exercise time than the reference group in response to both amlodipine (difference in delta, 82 s [95% CI, 37-126 s]; P<0.001) and ranolazine (difference in delta, 68 s [95% CI, 21-115 s]; P=0.005). The CMD group reported a greater increment (delta) in Seattle Angina Questionnaire summary score than the reference group in response to ranolazine (difference in delta, 7 points [95% CI, 0-15]; P=0.048), but not to amlodipine (difference in delta, 2 points [95% CI, -5 to 8]; P=0.549). CONCLUSIONS: Among phenotypically similar patients with angina with nonobstructive coronary arteries, only those with an impaired CFR derive benefit from anti-ischemic therapy. These findings support measurement of CFR to diagnose and guide management of this otherwise heterogeneous patient group.

Revealing the Fates of Proteins in the Gas Phase.

The ability to observe intact proteins by native mass spectrometry allows measurements of size, oligomeric state, numbers and types of ligands and post translational modifications bound, among many other characteristics. These studies have the potential to, and in some cases are, advancing our understanding of the role of structure in protein biology and biochemistry. However, there are some long-unresolved questions about to what extent solution-like structures persist without solvent in the vacuum of the mass spectrometer. Strong evidence from multiple sources over the years has demonstrated that well-folded proteins maintain native-like states if care is taken during sample preparation, ionization, and transmission through the gas phase. For partially unfolded states, dynamic and disordered proteins, and other important landmarks along the protein folding/unfolding pathway, caution has been urged in the interpretation of the results of native ion mobility/mass spectrometric data. New gas-phase tools allow us to provide insight into these questions with in situ, in vacuo labeling reactions delivered through ion/ion chemistry. This Young Scientist Perspective demonstrates the robustness of these tools in describing native-like structure as well as possible deviations from native-like structure during native ion mobility/mass spectrometry. This Perspective illustrates some of the changes in structure produced by the removal of solvent and details some of the challenges and potential of the field.

Intravascular lithotripsy in the treatment of coronary artery calcification in a high-risk real world population.

BACKGROUND: The DISRUPT-CAD study series demonstrated feasibility and safety of intravascular lithotripsy (IVL) in selected patients, but applicability across a broad range of clinical scenarios remains unclear. AIMS: This study aims to evaluate the procedural and clinical outcomes of IVL in a high-risk real-world cohort, compared to a regulatory approval cohort. METHODS: Consecutive patients treated with IVL and percutaneous coronary intervention at our center from May 2016 to April 2020 were included. Comparison was made between those enrolled in the DISRUPT-CAD series of studies to those with calcified lesions but an exclusion criteria. RESULTS: Among 177 patients treated with IVL, 142 were excluded from regulatory trials due to acute coronary syndrome presentation (47.2%), left ventricular ejection fraction <40% (22.5%), chronic renal failure (12.0%), or use of mechanical circulatory support (8.5%). This clinical cohort had a higher SYNTAX score (22.6 ± 12.1 vs. 17.4 ± 9.9, p = 0.019), and more treated ACC/AHA C lesions (56.3% vs. 37.1%, p = 0.042). Rates of device success (93.7% vs. 100.0%, p = 0.208), procedural success (96.5% vs. 100.0%, p = 0.585), and minimal lumen area gain (221.2 ± 93.7% vs. 198.6 ± 152.0%, p = 0.807) were similar in both groups. The DISRUPT-CAD cohort had no in-hospital mortality, 30-day major adverse cardiac events (MACE), or 30-day target vessel revascularization (TVR). The clinical cohort had an in-hospital mortality of 4.2%, 30-day MACE of 7.8%, and 30-day TVR of 1.5%. There was no difference in 12-month TVR (2.9% vs. 2.2%; p = 0.825). Twelve-month MACE was higher in the clinical cohort (21.1% vs. 8.6%, p = 0.03). CONCLUSION: IVL use remains associated with high clinical efficacy, procedural success, and low complication rates in a real-world population previously excluded from regulatory approving trials.

The role of solvation on the conformational landscape of α-synuclein.

Native ion mobility mass spectrometry has been used extensively to characterize ensembles of intrinsically disordered protein (IDP) conformers, but the extent to which the gaseous measurements provide realistic pictures of the solution conformations for such flexible proteins remains unclear. Therefore, we systematically studied the relationship between the solution and gaseous structural ensembles by measuring electrospray charge state and collision cross section (CCS) distributions for cationic and anionic forms of α-synuclein (αSN), an anionic protein in solution, as well as directly probed gas phase residue to residue distances via ion/ion reactions between gaseous α-synuclein cations and disulfonic acid linkers that form strong electrostatic bonds. We also combined results from in-solution protein crosslinking identified from native tandem mass spectrometry (MS/MS) with an initial αSN ensemble generated computationally by IDPConformerGenerator to generate an experimentally restrained solution ensemble of αSN. CCS distributions were directly calculated for the solution ensembles determined by NMR and compared to predicted gaseous conformers. While charge state and collision cross section distributions are useful for qualitatively describing the relative structural dynamics of proteins and major conformational changes induced by changes to solution states, the predicted and measured gas phase conformers include subpopulations that are significantly different than those expected from completely "freezing" solution conformations and preserving them in the gas phase. However, insights were gained on the various roles of solvent in stabilizing various conformers for extremely dynamic proteins like α-synuclein.

Online protein unfolding characterized by ion mobility electron capture dissociation mass spectrometry: cytochrome C from neutral and acidic solutions.

Electrospray ionization mass spectrometry (ESI-MS) experiments, including ion mobility spectrometry mass spectrometry (ESI-IMS-MS) and electron capture dissociation (ECD) of proteins ionized from aqueous solutions, have been used for the study of solution-like structures of intact proteins. By mixing aqueous proteins with denaturants online before ESI, the amount of protein unfolding can be precisely controlled and rapidly analyzed, permitting the characterization of protein folding intermediates in protein folding pathways. Herein, we mixed various pH solutions online with aqueous cytochrome C for unfolding and characterizing its unfolding intermediates with ESI-MS charge state distribution measurements, IMS, and ECD. The presence of folding intermediates and unfolded cytochrome c structures were detected from changes in charge states, arrival time distributions (ATDs), and ECD. We also compared structures from nondenaturing and denaturing solution mixtures measured under "gentle" (i.e., low energy) ion transmission conditions with structures measured under "harsh" (i.e., higher energy) transmission. This work confirms that when using "gentle" instrument conditions, the gas-phase cytochrome c ions reflect attributes of the various solution-phase structures. However, "harsh" conditions that maximize ion transmission produce extended structures that no longer correlate with changes in solution structure.

Multiplexed Conformationally Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation.

In this article, we present an approach for conformationally multiplexed, localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-IM-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. Gas-phase HDX with ECD fragmentation for exchange site localization was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed many regions with protection from HDX, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed high levels of deuterium incorporation throughout most of the protein sequence. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with unstructured regions of the protein relative to interior, structured regions, especially those involved in heme binding. With careful tuning and attention to deuterium scrambling, our approach holds promise for determining region-specific information on a conformer-selected basis for gas-phase protein structures, including localized characterizations of ligand, epitope, and protein-protein binding.

Application of Multiple Length Cross-linkers to the Characterization of Gaseous Protein Structure.

The speed, sensitivity, and tolerance of heterogeneity, as well as the kinetic trapping of solution-like states during electrospray, make native mass spectrometry an attractive method to study protein structure. Increases in the resolution of ion mobility measurements and in mass resolving power and range are leading to the increase of the information content of intact protein measurements and an expanded role of mass spectrometry in structural biology. Herein, a suite of different length noncovalent (sulfonate to positively charged side chain) cross-linkers was introduced via gas-phase ion/ion chemistry and used to determine distance restraints of kinetically trapped gas-phase structures of native-like cytochrome c ions. Electron capture dissociation allowed for the identification of cross-linked sites. Different length linkers resulted in distinct pairs of side chains being linked, supporting the ability of gas-phase cross-linking to be structurally specific. The gas-phase lengths of the cross-linkers were determined by conformational searches and density functional theory, allowing for the interpretation of the cross-links as distance restraints. These distance restraints were used to model gas-phase structures with molecular dynamics simulations, revealing a mixture of structures with similar overall shape/size but distinct features, thereby illustrating the kinetic trapping of multiple native-like solution structures in the gas phase.

AutoCCS: automated collision cross-section calculation software for ion mobility spectrometry-mass spectrometry.

MOTIVATION: Ion mobility spectrometry (IMS) separations are increasingly used in conjunction with mass spectrometry (MS) for separation and characterization of ionized molecular species. Information obtained from IMS measurements includes the ion's collision cross section (CCS), which reflects its size and structure and constitutes a descriptor for distinguishing similar species in mixtures that cannot be separated using conventional approaches. Incorporating CCS into MS-based workflows can improve the specificity and confidence of molecular identification. At present, there is no automated, open-source pipeline for determining CCS of analyte ions in both targeted and untargeted fashion, and intensive user-assisted processing with vendor software and manual evaluation is often required. RESULTS: We present AutoCCS, an open-source software to rapidly determine CCS values from IMS-MS measurements. We conducted various IMS experiments in different formats to demonstrate the flexibility of AutoCCS for automated CCS calculation: (i) stepped-field methods for drift tube-based IMS (DTIMS), (ii) single-field methods for DTIMS (supporting two calibration methods: a standard and a new enhanced method) and (iii) linear calibration for Bruker timsTOF and non-linear calibration methods for traveling wave based-IMS in Waters Synapt and Structures for Lossless Ion Manipulations. We demonstrated that AutoCCS offers an accurate and reproducible determination of CCS for both standard and unknown analyte ions in various IMS-MS platforms, IMS-field methods, ionization modes and collision gases, without requiring manual processing. AVAILABILITY AND IMPLEMENTATION: https://github.com/PNNL-Comp-Mass-Spec/AutoCCS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. Demo datasets are publicly available at MassIVE (Dataset ID: MSV000085979).

SCAI cardiogenic shock classification after out of hospital cardiac arrest and association with outcome.

OBJECTIVES: We aimed to validate the Society for Cardiovascular Angiography and Interventions (SCAI) classification to evaluate association with outcome in a real-world population and effect of invasive therapies. BACKGROUND: Cardiogenic shock is common after Out of Hospital Cardiac Arrest (OOHCA) but is often multifactorial and challenging to stratify. METHODS: The SCAI shock grade was applied to an observational registry of OOHCA patients on admission to our center between 2012 and 2017. The primary end-point was 30-day mortality and secondary end-points were mode of death and 12-month mortality. Provision of early CAG and mechanical circulatory support (MCS) was evaluated by SCAI shock grade using logistic regression. RESULTS: Three hundred and ninety-three patients (median age 64.3 years (24.9% females) were included. One hundred and seven patients (27.2%) were in Grade A, 94 (23.9%) in Grade B, 66 (16.8%) in Grade C, 91 (23.2%) in Grade D, and 35 (8.9%) in Grade E. There was a step-wise significant increase in 30-day mortality with increasing shock grade (A 28.9% vs. B 33.0% vs. C 54.5% vs. D 59.3% vs. E 82.9%; p < .0001). With worsening shock grade, requirement for renal replacement therapy and mortality from multiorgan dysfunction syndrome and cardiogenic causes increased. Early CAG was performed equally in all groups but was significantly associated with reduced mortality in SCAI grade D only (OR 0.26 [CI 0.08-0.91], p = .036). CONCLUSIONS: Increasing SCAI shock grade after OOHCA is associated with 30-day mortality, requirement for renal replacement therapy and mortality attributed to multiorgan dysfunction syndrome and cardiac etiology death.

Gas-Phase Ion/Ion Chemistry for Structurally Sensitive Probes of Gaseous Protein Ion Structure: Electrostatic and Electrostatic to Covalent Cross-Linking.

Intramolecular interactions within a protein are key in maintaining protein tertiary structure and understanding how proteins function. Ion mobility-mass spectrometry (IM-MS) has become a widely used approach in structural biology since it provides rapid measurements of collision cross sections (CCS), which inform on the gas-phase conformation of the biomolecule under study. Gas-phase ion/ion reactions target amino acid residues with specific chemical properties and the modified sites can be identified by MS. In this study, electrostatically reactive, gas-phase ion/ion chemistry and IM-MS are combined to characterize the structural changes between ubiquitin electrosprayed from aqueous and denaturing conditions. The electrostatic attachment of sulfo-NHS acetate to ubiquitin via ion/ion reactions and fragmentation by electron-capture dissociation (ECD) provide the identification of the most accessible protonated sites within ubiquitin as the sulfonate group forms an electrostatic complex with accessible protonated side chains. The protonated sites identified by ECD from the different solution conditions are distinct and, in some cases, reflect the disruption of interactions such as salt bridges that maintain the native protein structure. This agrees with previously published literature demonstrating that a high methanol concentration at low pH causes the structure of ubiquitin to change from a native (N) state to a more elongated A state. Results using gas-phase, electrostatic cross-linking reagents also point to similar structural changes and further confirm the role of methanol and acid in favoring a more unfolded conformation. Since cross-linking reagents have a distance constraint for the two reactive sites, the data is valuable in guiding computational structures generated by molecular dynamics. The research presented here describes a promising strategy that can detect subtle changes in the local environment of targeted amino acid residues to inform on changes in the overall protein structure.

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden.

SLIM Ultrahigh Resolution Ion Mobility Spectrometry Separations of Isotopologues and Isotopomers Reveal Mobility Shifts due to Mass Distribution Changes.

We report on separations of ion isotopologues and isotopomers using ultrahigh-resolution traveling wave-based Structures for Lossless Ion Manipulations with serpentine ultralong path and extended routing ion mobility spectrometry coupled to mass spectrometry (SLIM SUPER IMS-MS). Mobility separations of ions from the naturally occurring ion isotopic envelopes (e.g., [M], [M+1], [M+2], ... ions) showed the first and second isotopic peaks (i.e., [M+1] and [M+2]) for various tetraalkylammonium ions could be resolved from their respective monoisotopic ion peak ([M]) after SLIM SUPER IMS with resolving powers of ∼400-600. Similar separations were obtained for other compounds (e.g., tetrapeptide ions). Greater separation was obtained using argon versus helium drift gas, as expected from the greater reduced mass contribution to ion mobility described by the Mason-Schamp relationship. To more directly explore the role of isotopic substitutions, we studied a mixture of specific isotopically substituted (15N, 13C, and 2H) protonated arginine isotopologues. While the separations in nitrogen were primarily due to their reduced mass differences, similar to the naturally occurring isotopologues, their separations in helium, where higher resolving powers could also be achieved, revealed distinct additional relative mobility shifts. These shifts appeared correlated, after correction for the reduced mass contribution, with changes in the ion center of mass due to the different locations of heavy atom substitutions. The origin of these apparent mass distribution-induced mobility shifts was then further explored using a mixture of Iodoacetyl Tandem Mass Tag (iodoTMT) isotopomers (i.e., each having the same exact mass, but with different isotopic substitution sites). Again, the observed mobility shifts appeared correlated with changes in the ion center of mass leading to multiple monoisotopic mobilities being observed for some isotopomers (up to a ∼0.04% difference in mobility). These mobility shifts thus appear to reflect details of the ion structure, derived from the changes due to ion rotation impacting collision frequency or momentum transfer, and highlight the potential for new approaches for ion structural characterization.

Rapid Ion Mobility Separations of Bile Acid Isomers Using Cyclodextrin Adducts and Structures for Lossless Ion Manipulations.

Bile acids (BAs) constitute an important class of steroid metabolites often displaying changes associated with disease states and other health conditions. Current analyses for these structurally similar compounds are limited by a lack of sensitivity and long separation times with often poor isomeric resolution. To overcome these challenges and provide rapid analyses for the BA isomers, we utilized cyclodextrin adducts in conjunction with novel ion mobility (IM) separation capabilities provided by structures for lossless ion manipulations (SLIM). Cyclodextrin was found to interact with both the tauro- and glyco-conjugated BA isomers studied, forming rigid noncovalent host-guest inclusion complexes. Without the use of cyclodextrin adducts, the BA isomers were found to be nearly identical in their respective mobilities and thus unable to be baseline resolved. Each separation of the cyclodextrin-bile acid host-guest inclusion complex was performed in less than 1 s, providing a much more rapid alternative to current liquid chromatography-based separations. SLIM provided capabilities for the accumulation of larger ion populations and IM peak compression that resulted in much higher resolution separations and increased signal intensities for the BA isomers studied.

Improved Sensitivity and Separations for Phosphopeptides using Online Liquid Chromotography Coupled with Structures for Lossless Ion Manipulations Ion Mobility-Mass Spectrometry.

Phosphoproteomics greatly augments proteomics and holds tremendous potential for insights into the modulation of biological systems for various disease states. However, numerous challenges hinder conventional methods in terms of measurement sensitivity, throughput, quantification, and capabilities for confident phosphopeptide and phosphosite identification. In this work, we report the first example of integrating structures for lossless ion manipulations ion mobility-mass spectrometry (SLIM IM-MS) with online reversed-phase liquid chromatography (LC) to evaluate its potential for addressing the aforementioned challenges. A mixture of 51 heavy-labeled phosphopeptides was analyzed with a SLIM IM module having integrated ion accumulation and long-path separation regions. The SLIM IM-MS provided limits of detection as low as 50-100 pM (50-100 amol/µL) for several phosphopeptides, with the potential for significant further improvements. In addition, conventionally problematic phosphopeptide isomers could be resolved following an 18 m SLIM IM separation. The 2-D LC-IM peak capacity was estimated as ∼9000 for a 90 min LC separation coupled to an 18 m SLIM IM separation, considerably higher than LC alone and providing a basis for both improved identification and quantification, with additional gains projected with the future use of longer path SLIM IM separations. Thus, LC-SLIM IM-MS offers great potential for improving the sensitivity, separation, and throughput of phosphoproteomics analyses.

Distinguishing enantiomeric amino acids with chiral cyclodextrin adducts and structures for lossless ion manipulations.

Enantiomeric molecular evaluations remain an enormous challenge for current analytical techniques. To date, derivatization strategies and long separation times are generally required in these studies, and the development and implementation of new approaches are needed to increase speed and distinguish currently unresolvable compounds. Herein, we describe a method using chiral cyclodextrin adducts and structures for lossless ion manipulations (SLIM) and serpentine ultralong path with extended routing (SUPER) ion mobility (IM) to achieve rapid, high resolution separations of d and l enantiomeric amino acids. In the analyses, a chiral cyclodextrin is added to each sample. Two cyclodextrins were found to complex each amino acid molecule (i.e. potentially sandwiching the amino acid in their cavities) and forming host-guest noncovalent complexes that were distinct for each d and l amino acid pair studied and thus separable with IM in SLIM devices. The SLIM was also used to accumulate much larger ion populations than previously feasible for evaluation and therefore allow enantiomeric measurements of higher sensitivity, with gains in resolution from our ultralong path separation capabilities, than previously reported by any other IM-based approach.

Ventral Tegmental Area Dopamine Cell Activation during Male Rat Sexual Behavior Regulates Neuroplasticity and d-Amphetamine Cross-Sensitization following Sex Abstinence.

UNLABELLED: Experience with sexual behavior causes cross-sensitization of amphetamine reward, an effect dependent on a period of sexual reward abstinence. We previously showed that ΔFosB in the nucleus accumbens (NAc) is a key mediator of this cross-sensitization, potentially via dopamine receptor activation. However, the role of mesolimbic dopamine for sexual behavior or cross-sensitization between natural and drug reward is unknown. This was tested using inhibitory designer receptors exclusively activated by designer drugs in ventral tegmental area (VTA) dopamine cells. rAAV5/hSvn-DIO-hm4D-mCherry was injected into the VTA of TH::Cre adult male rats. Males received clozapine N-oxide (CNO) or vehicle injections before each of 5 consecutive days of mating or handling. Following an abstinence period of 7 d, males were tested for amphetamine conditioned place preference (CPP). Next, males were injected with CNO or vehicle before mating or handling for analysis of mating-induced cFos, sex experience-induced ΔFosB, and reduction of VTA dopamine soma size. Results showed that CNO did not affect mating behavior. Instead, CNO prevented sexual experience-induced cross-sensitization of amphetamine CPP, ΔFosB in the NAc and medial prefrontal cortex, and decreases in VTA dopamine soma size. Expression of hm4D-mCherry was specific to VTA dopamine cells and CNO blocked excitation and mating-induced cFos expression in VTA dopamine cells. These findings provide direct evidence that VTA dopamine activation is not required for initiation or performance of sexual behavior. Instead, VTA dopamine directly contributes to increased vulnerability for drug use following loss of natural reward by causing neuroplasticity in the mesolimbic pathway during the natural reward experience. SIGNIFICANCE STATEMENT: Drugs of abuse act on the neural pathways that mediate natural reward learning and memory. Exposure to natural reward behaviors can alter subsequent drug-related reward. Specifically, experience with sexual behavior, followed by a period of abstinence from sexual behavior, causes increased reward for amphetamine in male rats. This study demonstrates that activation of ventral tegmental area dopamine neurons during sexual experience regulates cross-sensitization of amphetamine reward. Finally, ventral tegmental area dopamine cell activation is essential for experience-induced neural adaptations in the nucleus accumbens, prefrontal cortex, and ventral tegmental area. These findings demonstrate a role of mesolimbic dopamine in the interaction between natural and drug rewards, and identify mesolimbic dopamine as a key mediator of changes in vulnerability for drug use after loss of natural reward.

Compression Ratio Ion Mobility Programming (CRIMP) Accumulation and Compression of Billions of Ions for Ion Mobility-Mass Spectrometry Using Traveling Waves in Structures for Lossless Ion Manipulations (SLIM).

We report on the implementation of a traveling wave (TW) based compression ratio ion mobility programming (CRIMP) approach within structures for lossless ion manipulations (SLIM) that enables both greatly enlarged trapped ion charge capacities and also efficient ion population compression for use in ion mobility (IM) separations. Ion accumulation is conducted in a SLIM serpentine ultralong path with extended routing (SUPER) region after which CRIMP compression allows the large ion populations to be "squeezed". The SLIM SUPER IM module has two regions, one operating with conventional traveling waves (i.e., traveling trap; TT region) and the second having an intermittently pausing or "stuttering" TW (i.e., stuttering trap; ST region). When a stationary voltage profile was used in the ST region, ions are blocked at the TT-ST interface and accumulated in the TT region and then can be released by resuming a conventional TW in the ST region. The population can also be compressed using CRIMP by the repetitive merging of ions distributed over multiple TW bins in the TT region into a single TW bin in the ST region. Ion accumulation followed by CRIMP compression provides the basis for the use of larger ion populations for IM separations. We show that over 109 ions can be accumulated with high efficiency in the present device and that the extent of subsequent compression is only limited by the space charge capacity of the trapping region. Approximately 5 × 109 charges introduced from an electrospray ionization source were trapped for a 40 s accumulation period, more than 2 orders of magnitude greater than the previously reported charge capacity of an ion funnel trap. Importantly, we show that extended ion accumulation in conjunction with CRIMP compression and multiple passes through the serpentine path provides the basis for a highly desirable combination of ultrahigh sensitivity and SLIM SUPER high-resolution IM separations.

Serpentine Ultralong Path with Extended Routing (SUPER) High Resolution Traveling Wave Ion Mobility-MS using Structures for Lossless Ion Manipulations.

Ion mobility (IM) separations have a broad range of analytical applications, but insufficient resolution often limits their utility. Here, we report on ion mobility separations in a structures for lossless ion manipulations (SLIM) serpentine ultralong path with extended routing (SUPER) traveling wave (TW) ion mobility (IM) module in conjunction with mass spectrometry (MS). Ions were confined in the SLIM by rf fields in conjunction with a DC guard bias, enabling essentially lossless TW transmission over greatly extended paths. The extended routing utilized multiple passes (e.g., ∼1094 m over 81 passes through the 13.5 m serpentine path) and was facilitated by the introduction of a lossless ion switch that allowed ions to be directed to either the MS detector or for another pass through the serpentine separation region, allowing theoretically unlimited IM path lengths. The multipass SUPER IM-MS provided resolution approximately proportional to the square root of the number of passes (or total path length). More than 30-fold higher IM resolution (∼340 vs ∼10) for Agilent tuning mix m/z 622 and 922 ions was achieved for 40 passes compared to commercially available drift tube IM and other TWIM-based platforms. An initial evaluation of the isomeric sugars lacto-N-hexaose and lacto-N-neohexaose showed the isomeric structures to be baseline resolved, and a new conformational feature for lacto-N-neohexaose was revealed after 9 passes. The new SLIM SUPER high resolution TWIM platform has broad utility in conjunction with MS and is expected to enable a broad range of previously challenging or intractable separations.

New frontiers for mass spectrometry based upon structures for lossless ion manipulations.

Structures for lossless ion manipulations (SLIM) provide a new paradigm for efficient, complex and extended gas phase ion manipulations. SLIM are created from electric fields generated by the application of DC and RF potentials to arrays of electrodes patterned on two parallel surfaces. The electric fields provide lossless ion manipulations, including effective ion transport and storage. SLIM modules have been developed using both constant and oscillatory electric fields (e.g. traveling waves) to affect the ion motion. Ion manipulations demonstrated to date with SLIM include: extended trapping, ion selection, ion dissociation, and ion mobility spectrometry (IMS) separations achieving unprecedented ultra high resolution. SLIM thus provide the basis for previously impractical manipulations, such as very long path length ion mobility separations where ions traverse a serpentine path multiple times, as well as new capabilities that extend the utility of these developments based on temporal and spatial compression of ion mobility separations and other ion distributions. The evolution of SLIM devices developed over the last three years is reviewed and we provide examples of various ion manipulations performed, and briefly discuss potential applications and new directions.