Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

Exposing the molecular heterogeneity of glycosylated biotherapeutics.

The heterogeneity inherent in today's biotherapeutics, especially as a result of heavy glycosylation, can affect a molecule's safety and efficacy. Characterizing this heterogeneity is crucial for drug development and quality assessment, but existing methods are limited in their ability to analyze intact glycoproteins or other heterogeneous biotherapeutics. Here, we present an approach to the molecular assessment of biotherapeutics that uses proton-transfer charge-reduction with gas-phase fractionation to analyze intact heterogeneous and/or glycosylated proteins by mass spectrometry. The method provides a detailed landscape of the intact molecular weights present in biotherapeutic protein preparations in a single experiment. For glycoproteins in particular, the method may offer insights into glycan composition when coupled with a suitable bioinformatic strategy. We tested the approach on various biotherapeutic molecules, including Fc-fusion, VHH-fusion, and peptide-bound MHC class II complexes to demonstrate efficacy in measuring the proteoform-level diversity of biotherapeutics. Notably, we inferred the glycoform distribution for hundreds of molecular weights for the eight-times glycosylated fusion drug IL22-Fc, enabling correlations between glycoform sub-populations and the drug's pharmacological properties. Our method is broadly applicable and provides a powerful tool to assess the molecular heterogeneity of emerging biotherapeutics.

Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins.

Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca2+ ) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca2+ -mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca2+ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER-resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER-resident proteins are being secreted in a manner consistent with ER Ca2+ depletion. Antagonism of ER Ca2+ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER-resident proteins through ER Ca2+ depletion and describes a new facet of excitotoxicity.

Pharmacologic Manipulation of Complement Receptor 3 Prevents Dendritic Spine Loss and Cognitive Impairment After Acute Cranial Radiation.

PURPOSE: Cranial irradiation induces healthy tissue damage that can lead to neurocognitive complications, negatively affecting patient quality of life. One damage indicator associated with cognitive impairment is loss of neuronal spine density. We previously demonstrated that irradiation-mediated spine loss is microglial complement receptor 3 (CR3) and sex dependent. We hypothesized that these changes are associated with late-delayed cognitive deficits and amenable to pharmacologic intervention. METHODS AND MATERIALS: Our model of cranial irradiation (acute, 10 Gy gamma) used male and female CR3-wild type and CR3-deficient Thy-1 YFP mice of C57BL/6 background. Forty-five days after irradiation and behavioral testing, we quantified spine density and markers of microglial reactivity in the hippocampal dentate gyrus. In a separate experiment, male Thy-1 YFP C57BL/6 mice were treated with leukadherin-1, a modulator of CR3 function. RESULTS: We found that male mice demonstrate irradiation-mediated spine loss and cognitive deficits but that female and CR3 knockout mice do not. These changes were associated with greater reactivity of microglia in male mice. Pharmacologic manipulation of CR3 with LA1 prevented spine loss and cognitive deficits in irradiated male mice. CONCLUSIONS: This work improves our understanding of irradiation-mediated mechanisms and sex dependent responses and may help identify novel therapeutics to reduce irradiation-induced cognitive decline and improve patient quality of life.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Radiation and CNS effects: summary of evidence from a recent symposium of the Radiation Research Society.

This article summarizes a Symposium on 'Radiation risks of the central nervous system' held virtually at the 67th Annual Meeting of the Radiation Research Society, 3-6 October 2021. Repeated low-dose radiation exposure over a certain period could lead to reduced neuronal proliferation, altered neurogenesis, neuroinflammation and various neurological complications, including psychological consequences, necessitating further research in these areas. Four speakers from radiation biology, genetics and epidemiology presented the latest data from their studies seeking insights into this important topic. This symposium highlighted new and important directions for further research on mental health disorders, neurodegenerative conditions and cognitive impairment. Future studies will examine risks of mental and behavioral disorders and neurodegenerative diseases following protracted radiation exposures to better understand risks of occupational exposures as well as provide insights into risks from exposures to galactic cosmic rays.

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells.

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

Problem-oriented policing for reducing crime and disorder: An updated systematic review and meta-analysis.

Background: Herman Goldstein developed problem-oriented policing (POP) to focus police on more proactively addressing chronic problems, rather than using traditional reactive efforts. POP has been utilized to target a wide range of problems and has become commonly used in agencies across the United States and the world, although implementation is often uneven. POP interventions commonly use the SARA (scanning, analysis, response, assessment) model to identify problems, carefully analyze the conditions contributing to the problem, develop a tailored response to target these underlying factors, and evaluate outcome effectiveness. Objectives: To extend and update the findings of the original POP systematic review by synthesizing the findings of published and unpublished evaluations of POP through December 2018 to assess its overall impacts on crime and disorder. The review also examined impacts of POP on crime displacement, police financial costs, and noncrime outcomes. Search Methods: Searches using POP keywords of the Global Policing Database at the University of Queensland were conducted to identify published and unpublished evaluations between 2006 and 2018. We supplemented these searches with forward searches, hand searches of leading journals and the Center for Problem-Oriented Policing, and consultation with experts. Selection Criteria: Eligible studies had to include a target area or group that received a POP intervention AND a control area/group that received standard police services. The control condition could be either experimental or quasi-experimental. Units of analysis could be places or people. We defined POP as studies that generally followed the tenets of the SARA model. Data Collection and Analysis: We identified 39 new (published between 2006 and 2018) studies that met our eligibility criteria as an evaluation of POP. Twenty-four of these studies had sufficient data available to calculate an effect size. Along with the 10 studies from our initial systematic review of POP, these 34 studies are included in our meta-analytic review of POP. Nine of these studies were randomized experiments and 25 were quasi-experiments. We calculated effect sizes for each study using Cohen's D and relative incidence risk ratios and used random effects meta-analyses to synthesize studies. Results: Our meta-analyses suggest statistically significant impacts of POP. Our relative incident risk ratio analysis of mean effects suggests a 33.8% reduction in crime/disorder in the POP treatment areas/groups relative to the controls. We find no evidence of significant crime displacement as a result of POP and some evidence for a greater likelihood of a diffusion of crime control benefits. Few studies assessed noncrime outcomes, but our narrative review suggests POP is cost-effective, but has limited impacts on fear of crime, legitimacy, and collective efficacy. Authors' Conclusions: Our review provides strong and consistent evidence that POP is an effective strategy for reducing crime and disorder. There is a great deal of heterogeneity in the magnitude of effect sizes across factors such as study type, study rigor and crime type. Despite this heterogeneity, 31 out of 34 studies (91.2%) have effect sizes in favor of a treatment effect and the overall mean effect is positive and significant in all of our models.

Cranial irradiation mediated spine loss is sex-specific and complement receptor-3 dependent in male mice.

Cranial irradiation is the main therapeutic treatment for primary and metastatic malignancies in the brain. However, cranial radiation therapy produces long-term impairment in memory, information processing, and attention that contribute to a decline in quality of life. The hippocampal neural network is fundamental for proper storage and retrieval of episodic and spatial memories, suggesting that hippocampal signaling dysfunction could be responsible for the progressive memory deficits observed following irradiation. Previous rodent studies demonstrated that irradiation induces significant loss in dendritic spine number, alters spine morphology, and is associated with behavioral task deficits. Additionally, the literature suggests a common mechanism in which synaptic elimination via microglial-mediated phagocytosis is complement dependent and associated with cognitive impairment in aging as well as disease. We demonstrate sexual dimorphisms in irradiation-mediated alterations of microglia activation markers and dendritic spine density. Further, we find that the significant dendritic spine loss observed in male mice following irradiation is microglia complement receptor 3 (CR3)-dependent. By identifying sex-dependent cellular and molecular factors underlying irradiation-mediated spine loss, therapies can be developed to counteract irradiation-induced cognitive decline and improve patient quality of life.

Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor.

Accurate sequence characterization is essential for the development of therapeutic antibodies by the pharmaceutical industry. Presented here is a methodology to obtain comprehensive sequence analysis of a monoclonal antibody. An enzyme reactor of immobilized Aspergillopepsin I, a highly stable nonspecific protease, was used to cleave reduced antibody subunits into a peptide profile ranging from 1 to 20 kDa. Utilizing the Thermo Orbitrap Fusion's unique instrument architecture combined with state-of-the-art instrument control software allowed for dynamic instrument methods that optimally characterize eluting peptides based on their size and charge density. Using a data-dependent instrument method, both collisional dissociation and electron transfer dissociation were used to fragment the appropriate charge state of analyte peptides. The instrument layout also allowed for scans to be taken in parallel using both the ion trap and Orbitrap concurrently, thus allowing larger peptides to be analyzed in high resolution using the Orbitrap while simultaneously analyzing tryptic-like peptides using the ion trap. We harnessed these capabilities to develop a custom method to optimally fragment the eluting peptides based on their mass and charge density. Using this approach, we obtained 100% sequence coverage of the total antibody in a single chromatographic analysis, enabling unambiguous sequence assignment of all residues.

Microbubble gas volume: A unifying dose parameter in blood-brain barrier opening by focused ultrasound.

Focused ultrasound with microbubbles is being developed to transiently, locally and noninvasively open the blood-brain barrier (BBB) for improved pharmaceutical delivery. Prior work has demonstrated that, for a given concentration dose, microbubble size affects both the intravascular circulation persistence and extent of BBB opening. When matched to gas volume dose, however, the circulation half-life was found to be independent of microbubble size. In order to determine whether this holds true for BBB opening as well, we independently measured the effects of microbubble size (2 vs. 6 µm diameter) and concentration, covering a range of overlapping gas volume doses (1-40 µL/kg). We first demonstrated precise targeting and a linear dose-response of Evans Blue dye extravasation to the rat striatum for a set of constant microbubble and ultrasound parameters. We found that dye extravasation increased linearly with gas volume dose, with data points from both microbubble sizes collapsing to a single line. A linear trend was observed for both the initial sonication (R2=0.90) and a second sonication on the contralateral side (R2=0.68). Based on these results, we conclude that microbubble gas volume dose, not size, determines the extent of BBB opening by focused ultrasound (1 MHz, ~0.5 MPa at the focus). This result may simplify planning for focused ultrasound treatments by constraining the protocol to a single microbubble parameter - gas volume dose - which gives equivalent results for varying size distributions. Finally, using optimal parameters determined for Evan Blue, we demonstrated gene delivery and expression using a viral vector, dsAAV1-CMV-EGFP, one week after BBB disruption, which allowed us to qualitatively evaluate neuronal health.

The problem is not just sample size: the consequences of low base rates in policing experiments in smaller cities.

BACKGROUND: Hot spots policing is one of the most influential police innovations, with a strong body of experimental research showing it to be effective in reducing crime and disorder. However, most studies have been conducted in major cities, and we thus know little about whether it is effective in smaller cities, which account for a majority of police agencies. The lack of experimental studies in smaller cities is likely in part due to challenges designing statistically powerful tests in such contexts. OBJECTIVES: The current article explores the challenges of statistical power and "noise" resulting from low base rates of crime in smaller cities and provides suggestions for future evaluations to overcome these limitations. RESEARCH DESIGN: Data from a randomized experimental evaluation of broken windows policing in hot spots are used to illustrate the challenges that low base rates present for evaluating hot spots policing programs in smaller cities. RESULTS: Analyses show low base rates make it difficult to detect treatment effects. Very large effect sizes would be required to reach sufficient power, and random fluctuations around low base rates make detecting treatment effects difficult, irrespective of power, by masking differences between treatment and control groups. CONCLUSIONS: Low base rates present strong challenges to researchers attempting to evaluate hot spots policing in smaller cities. As such, base rates must be taken directly into account when designing experimental evaluations. The article offers suggestions for researchers attempting to expand the examination of hot spots policing and other microplace-based interventions to smaller jurisdictions.