Rapid Setup of Tissue Microarray and Tiled Area Imaging on the Multiplexed Ion Beam Imaging Microscope using the Tile/SED/Array Interface.

Multiplexed ion beam imaging (MIBI) is a next-generation mass spectrometry-based microscopy technique that generates 40+ plex images of protein expression in histologic tissues, enabling detailed dissection of cellular phenotypes and histoarchitectural organization. A key bottleneck in operation occurs when users select the physical locations on the tissue for imaging. As the scale and complexity of MIBI experiments have increased, the manufacturer-provided interface and third-party tools have become increasingly unwieldy for imaging large tissue microarrays and tiled tissue areas. Thus, a web-based, interactive, what-you-see-is-what-you-get (WYSIWYG) graphical interface layer - the tile/SED/array Interface (TSAI) - was developed for users to set imaging locations using familiar and intuitive mouse gestures such as drag-and-drop, click-and-drag, and polygon drawing. Written according to web standards already built into modern web browsers, it requires no installation of external programs, extensions, or compilers. Of interest to the hundreds of current MIBI users, this interface dramatically simplifies and accelerates the setup of large, complex MIBI runs.

A spatially resolved timeline of the human maternal-fetal interface.

Beginning in the first trimester, fetally derived extravillous trophoblasts (EVTs) invade the uterus and remodel its spiral arteries, transforming them into large, dilated blood vessels. Several mechanisms have been proposed to explain how EVTs coordinate with the maternal decidua to promote a tissue microenvironment conducive to spiral artery remodelling (SAR)1-3. However, it remains a matter of debate regarding which immune and stromal cells participate in these interactions and how this evolves with respect to gestational age. Here we used a multiomics approach, combining the strengths of spatial proteomics and transcriptomics, to construct a spatiotemporal atlas of the human maternal-fetal interface in the first half of pregnancy. We used multiplexed ion beam imaging by time-of-flight and a 37-plex antibody panel to analyse around 500,000 cells and 588 arteries within intact decidua from 66 individuals between 6 and 20 weeks of gestation, integrating this dataset with co-registered transcriptomics profiles. Gestational age substantially influenced the frequency of maternal immune and stromal cells, with tolerogenic subsets expressing CD206, CD163, TIM-3, galectin-9 and IDO-1 becoming increasingly enriched and colocalized at later time points. By contrast, SAR progression preferentially correlated with EVT invasion and was transcriptionally defined by 78 gene ontology pathways exhibiting distinct monotonic and biphasic trends. Last, we developed an integrated model of SAR whereby invasion is accompanied by the upregulation of pro-angiogenic, immunoregulatory EVT programmes that promote interactions with the vascular endothelium while avoiding the activation of maternal immune cells.

Proteomic Profiling of Hypoplastic Lungs Suggests an Underlying Inflammatory Response in the Pathogenesis of Abnormal Lung Development in Congenital Diaphragmatic Hernia.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To understand the underlying pathobiology, we compared the proteomic profiles of fetal rat lungs at the alveolar stage (E21) that were either exposed to nitrofen in utero (CDH lungs, n=5) or exposed to vehicle only (non-CDH control lungs, n=5). Pathway analysis of proteomic datasets showed significant enrichment in inflammatory response proteins associated with cytokine signaling and Epstein Barr Virus in nitrofen CDH lungs. Among the 218 significantly altered proteins between CDH and non-CDH control lungs were Tenascin C, CREBBP, LYN, and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen rat lungs and human CDH lungs, obtained from stillborn fetuses that did not receive pre- or postnatal treatment. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially rescued the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3-associated cytokines (IL-15, IL-9, andIL-2) are increased in fetal tracheal aspirates of CDH survivors compared with nonsurvivors after fetoscopic endoluminal tracheal occlusion. With our unbiased proteomics approach, we showed for the first time that downstream inflammatory processes are likely involved in the pathogenesis of abnormal lung development in CDH.

Characterization of sex-related differences in allergen house dust mite-challenged airway inflammation, in two different strains of mice.

Biological sex impacts disease prevalence, severity and response to therapy in asthma, however preclinical studies often use only one sex in murine models. Here, we detail sex-related differences in immune responses using a house dust mite (HDM)-challenge model of acute airway inflammation, in adult mice of two different strains (BALB/c and C57BL/6NJ). Female and male mice were challenged (intranasally) with HDM extract (~ 25 µg) for 2 weeks (N = 10 per group). Increase in serum HDM-specific IgE showed a female bias, which was statistically significant in BALB/c mice. We compared naïve and HDM-challenged mice to define immune responses in the lungs by assessing leukocyte accumulation in the bronchoalveolar lavage fluid (BALF), and profiling the abundance of 29 different cytokines in BALF and lung tissue lysates. Our results demonstrate specific sex-related and strain-dependent differences in airway inflammation. For example, HDM-driven accumulation of neutrophils, eosinophils and macrophages were significantly higher in females compared to males, in BALB/c mice. In contrast, HDM-mediated eosinophil accumulation was higher in males compared to females, in C57BL/6NJ mice. Differences in lung cytokine profiles indicated that HDM drives a T-helper (Th)17-biased response with higher IL-17 levels in female BALB/c mice compared to males, whereas female C57BL/6NJ mice elicit a mixed Th1/Th2-skewed response. Male mice of both strains showed higher levels of specific Th2-skewed cytokines, such as IL-21, IL-25 and IL-9, in response to HDM. Overall, this study details sex dimorphism in HDM-mediated airway inflammation in mice, which will be a valuable resource for preclinical studies in allergic airway inflammation and asthma.

Combination of IL-17A/F and TNF-α uniquely alters the bronchial epithelial cell proteome to enhance proteins that augment neutrophil migration.

BACKGROUND: The heterodimer interleukin (IL)-17A/F is elevated in the lungs in chronic respiratory disease such as severe asthma, along with the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Although IL-17A/F and TNF-α are known to functionally cooperate to exacerbate airway inflammation, proteins altered by their interaction in the lungs are not fully elucidated. RESULTS: We used Slow Off-rate Modified Aptamer-based proteomic array to identify proteins that are uniquely and/or synergistically enhanced by concurrent stimulation with IL-17A/F and TNF-α in human bronchial epithelial cells (HBEC). The abundance of 38 proteins was significantly enhanced by the combination of IL-17A/F and TNF-α, compared to either cytokine alone. Four out of seven proteins that were increased > 2-fold were those that promote neutrophil migration; host defence peptides (HDP; Lipocalin-2 (LCN-2) and Elafin) and chemokines (IL-8, GROα). We independently confirmed the synergistic increase of these four proteins by western blots and ELISA. We also functionally confirmed that factors secreted by HBEC stimulated with the combination of IL-17A/F and TNF-α uniquely enhances neutrophil migration. We further showed that PI3K and PKC pathways selectively control IL-17A/F + TNF-α-mediated synergistic production of HDPs LCN-2 and Elafin, but not chemokines IL-8 and GROα. Using a murine model of airway inflammation, we demonstrated enhancement of IL-17A/F, TNF-α, LCN-2 and neutrophil chemokine KC in the lungs, thus corroborating our findings in-vivo. CONCLUSION: This study identifies proteins and signaling mediated by concurrent IL-17A/F and TNF-α exposure in the lungs, relevant to respiratory diseases characterized by chronic inflammation, especially neutrophilic airway inflammation such as severe asthma.

Sex Dimorphism of Allergen-Induced Secreted Proteins in Murine and Human Lungs.

Biological sex influences disease severity, prevalence and response to therapy in allergic asthma. However, allergen-mediated sex-specific changes in lung protein biomarkers remain undefined. Here, we report sex-related differences in specific proteins secreted in the lungs of both mice and humans, in response to inhaled allergens. Female and male BALB/c mice (7-8 weeks) were intranasally challenged with the allergen house dust mite (HDM) for 2 weeks. Bronchoalveolar lavage fluid (BALF) was collected 24 hour after the last HDM challenge from allergen-naïve and HDM-challenged mice (N=10 per group, each sex). In a human study, adult participants were exposed to nebulized (2 min) allergens (based on individual sensitivity), BALF was obtained after 24 hour (N=5 each female and male). The BALF samples were examined in immunoblots for the abundance of 10 proteins shown to increase in response to allergen in both murine and human BALF, selected from proteomics studies. We showed significant sex-bias in allergen-driven increase in five out of the 10 selected proteins. Of these, increase in eosinophil peroxidase (EPX) was significantly higher in females compared to males, in both mice and human BALF. We also showed specific sex-related differences between murine and human samples. For example, allergen-driven increase in S100A8 and S100A9 was significantly higher in BALF of females compared to males in mice, but significantly higher in males compared to females in humans. Overall, this study provides sex-specific protein biomarkers that are enhanced in response to allergen in murine and human lungs, informing and motivating translational research in allergic asthma.

Rodent Lung Tissue Sample Preparation and Processing for Shotgun Proteomics.

Mass spectrometry (MS) is a routinely used approach to characterize global protein profile in various biological samples. Here we describe rodent lung tissue homogenization, sample preparation, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for shotgun proteomics.

Disrupting Tryptophan in the Central Hydrophobic Region Selectively Mitigates Immunomodulatory Activities of the Innate Defence Regulator Peptide IDR-1002.

Innate defense regulator (IDR) peptides show promise as immunomodulatory therapeutics. However, there is limited understanding of the relationship of IDR peptide sequence and/or structure with its immunomodulatory activity. We previously reported that an IDR peptide, IDR-1002, reduces airway hyperresponsiveness (AHR) and inflammation in a house dust mite (HDM)-challenged murine model of airway inflammation. Here, we examined the sequence-to-function relationship of IDR-1002 in HDM-challenged mice and human bronchial epithelial cells (HBEC). We demonstrated that the tryptophan (W8) in the central hydrophobic region of IDR-1002 is required for the peptide to (i) suppress the pro-inflammatory cytokine IL-33, and induce anti-inflammatory mediators IL-1RA and stanniocalcin-1 in HBEC, and (ii) reduce IL-33 abundance, and eosinophil and neutrophil infiltration, in the lungs of HDM-challenged mice, without affecting the capacity to improve AHR, suggesting multimodal activity in vivo. Findings from this study can be used to design IDR peptides with targeted impact on immunomodulation and pathophysiology in respiratory diseases.

Cathelicidin and Calprotectin Are Disparately Altered in Murine Models of Inflammatory Arthritis and Airway Inflammation.

Cationic host defense peptides (CHDP) are immunomodulatory molecules that control infections and contribute to immune homeostasis. CHDP such as cathelicidin and calprotectin expression is altered in the arthritic synovium, and in the lungs of asthma and COPD patients. Recent studies suggest a link between airway inflammation and the immunopathology of arthritis. Therefore, in this study we compared the abundance of mouse cathelicidin (CRAMP), defensins, and calprotectin subunits (S100A8 and S100A9) in murine models of collagen-induced arthritis (CIA) and allergen house dust mite (HDM)-challenged airway inflammation. CRAMP, S100A8, and S100A9 abundance were significantly elevated in the joint tissues of CIA mice, whereas these were decreased in the lung tissues of HDM-challenged mice, compared to naïve. We further compared the effects of administration of two different synthetic immunomodulatory peptides, IG-19 and IDR-1002, on cathelicidin and calprotectin abundance in the two models. Administration of IG-19, which controls disease progression and inflammation in CIA mice, significantly decreased CRAMP, S100A8, and S100A9 levels to baseline in the joints of the CIA mice, which correlated with the decrease in cellular influx in the joints. However, administration of IDR-1002, which suppresses HDM-induced airway inflammation, did not prevent the decrease in the levels of cathelicidin and calprotectin in the lungs of HDM-challenged mice. Cathelicidin and calprotectin levels did not correlate with leukocyte accumulation in the lungs of the HDM-challenged mice. Results of this study suggest that endogenous cathelicidin and calprotectin abundance are disparately altered, and may be differentially regulated, within local tissues in airway inflammation compared to arthritis.

Characterization of immune responses and the lung transcriptome in a murine model of IL-33 challenge.

IL-33 induces airway inflammation and hyper-responsiveness in respiratory diseases. Although defined as a therapeutic target, there are limited studies that have comprehensively investigated IL-33-mediated responses in the lungs in vivo. In this study, we characterized immunological and physiological responses induced by intranasal IL-33 challenge, in a mouse model. We identified specific cytokines, IL-4, IL-5, IL-6, IL-10, IP-10 and MIP1-α, that are increased in bronchoalveolar lavage and lung tissues by IL-33. Using transcriptomics (RNA-Seq) we demonstrated that 2279 transcripts were up-regulated and 1378 downregulated (≥ 2-fold, p < 0.01) in lung tissues, in response to IL-33. Bioinformatic interrogation of the RNA-Seq data was used to predict biological pathways and upstream regulators involved in IL-33-mediated responses. We showed that the mRNA and protein of STAT4, a predicted upstream regulator of IL-33-induced transcripts, was significantly enhanced in the lungs following IL-33 challenge. Overall, this study provides specific IL-33-induced molecular targets and endpoints that can be used as a resource for in vivo studies, e.g. in preclinical murine models examining novel interventions to target downstream effects of IL-33.

Cytokines IL-17, TNF and IFN-γ Alter the Expression of Antimicrobial Peptides and Proteins Disparately: A Targeted Proteomics Analysis using SOMAscan Technology.

Antimicrobial peptides, also known as host defence peptides, are immunomodulatory molecules required to resolve infections. Antimicrobial peptides and proteins (APPs) are important in the control of infections in the lungs. Despite evidence that APPs exhibit a wide range of immune functions and modulate inflammation, the effect of inflammatory cytokines on the expression of APPs is not completely defined. In this study, we profiled the expression of 39 different APPs in human bronchial epithelial cells (HBEC) using Slow Off-rate Modified Aptamer (SOMAmer)-based protein array, in the presence and absence of three different inflammatory cytokines (IL-17, TNF and IFN-γ). Expression of 13 different APPs was altered in response to IL-17, TNF or IFN-γ. Independent validations of selected proteins from the proteomics screen i.e., those that were significantly enhanced by >2-fold change (p < 0.01) using western blots conclusively demonstrated that inflammatory cytokines alter the expression of APPs differentially. For example, the abundance of cathepsin S was enhanced by only IFN-γ, whereas lipocalin-2 was increased by IL-17 alone. Abundance of elafin increased in presence of IL-17 or TNF, but decreased in response to IFN-γ. Whereas the abundance of cathepsin V decreased following stimulation with IL-17, TNF and IFN-γ. The results of this study demonstrate that inflammatory cytokines alter the expression of APPs disparately. This suggests that the composition of the inflammatory cytokine milieu may influence APPs abundance and thus alter the processes required for infection control and regulation of inflammation in the lungs.

Immunomodulatory innate defence regulator (IDR) peptide alleviates airway inflammation and hyper-responsiveness.

BACKGROUND: Exacerbation in asthma is associated with decreased expression of specific host defence peptides (HDPs) in the lungs. We examined the effects of a synthetic derivative of HDP, innate defence regulator (IDR) peptide IDR-1002, in house dust mite (HDM)-challenged murine model of asthma, in interleukin (IL)-33-challenged mice and in human primary bronchial epithelial cells (PBECs). METHODS: IDR-1002 (6 mg/kg per mouse) was administered (subcutaneously) in HDM-challenged and/or IL-33-challenged BALB/c mice. Lung function analysis was performed with increasing dose of methacholine by flexiVent small animal ventilator, cell differentials in bronchoalveolar lavage performed by modified Wright-Giemsa staining, and cytokines monitored by MesoScale Discovery assay and ELISA. PBECs stimulated with tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), with or without IDR-1002, were analysed by western blots. RESULTS: IDR-1002 blunted HDM challenge-induced airway hyper-responsiveness (AHR), and lung leucocyte accumulation including that of eosinophils and neutrophils, in HDM-challenged mice. Concomitantly, IDR-1002 suppressed HDM-induced IL-33 in the lungs. IFN-γ/TNF-α-induced IL-33 production was abrogated by IDR-1002 in PBECs. Administration of IL-33 in HDM-challenged mice, or challenge with IL-33 alone, mitigated the ability of IDR-1002 to control leucocyte accumulation in the lungs, suggesting that the suppression of IL-33 is essential for the anti-inflammatory activity of IDR-1002. In contrast, the peptide significantly reduced either HDM, IL-33 or HDM+IL-33 co-challenge-induced AHR in vivo. CONCLUSION: This study demonstrates that an immunomodulatory IDR peptide controls the pathophysiology of asthma in a murine model. As IL-33 is implicated in steroid-refractory severe asthma, our findings on the effects of IDR-1002 may contribute to the development of novel therapies for steroid-refractory severe asthma.

Inhaled diesel exhaust alters the allergen-induced bronchial secretome in humans.

Diesel exhaust (DE) is a paradigm for traffic-related air pollution. Human adaptation to DE is poorly understood and currently based on oversimplified models. DE promotes allergic responses, but protein expression changes mediated by this interaction have not been systematically investigated. The aim of this study was to define the effect of inhaled DE on allergen-induced proteins in the lung.We performed a randomised and blinded controlled human crossover exposure study. Participants inhaled filtered air or DE; thereafter, contralateral lung segments were challenged with allergen or saline. Using label-free quantitative proteomics, we comprehensively defined DE-mediated alteration of allergen-driven secreted proteins (secretome) in bronchoalveolar lavage. We further examined expression of proteins selected from the secretome data in independent validation experiments using Western blots, ELISA and immunohistochemistry.We identified protein changes unique to co-exposure (DE+allergen), undetected with mono-exposures (DE or allergen alone). Validation studies confirmed that specific proteins (e.g. the antimicrobial peptide cystatin-SA) were significantly enhanced with DE+allergen compared to either mono-exposure.This study demonstrates that common environmental co-exposures can uniquely alter protein responses in the lungs, illuminating biology that mono-exposures cannot. This study highlights the value of complex human in vivo models in detailing airway responses to inhaled pollution.

Biosignature for airway inflammation in a house dust mite-challenged murine model of allergic asthma.

House dust mite (HDM) challenge is commonly used in murine models of allergic asthma for preclinical pathophysiological studies. However, few studies define objective readouts or biomarkers in this model. In this study we characterized immune responses and defined molecular markers that are specifically altered after HDM challenge. In this murine model, we used repeated HDM challenge for two weeks which induced hallmarks of allergic asthma seen in humans, including airway hyper-responsiveness (AHR) and elevated levels of circulating total and HDM-specific IgE and IgG1. Kinetic studies showed that at least 24 h after last HDM challenge results in significant AHR along with eosinophil infiltration in the lungs. Histologic assessment of lung revealed increased epithelial thickness and goblet cell hyperplasia, in the absence of airway wall collagen deposition, suggesting ongoing tissue repair concomitant with acute allergic lung inflammation. Thus, this model may be suitable to delineate airway inflammation processes that precede airway remodeling and development of fixed airway obstruction. We observed that a panel of cytokines e.g. IFN-γ, IL-1ß, IL-4, IL-5, IL-6, KC, TNF-α, IL-13, IL-33, MDC and TARC were elevated in lung tissue and bronchoalveolar fluid, indicating local lung inflammation. However, levels of these cytokines remained unchanged in serum, reflecting lack of systemic inflammation in this model. Based on these findings, we further monitored the expression of 84 selected genes in lung tissues by quantitative real-time PCR array, and identified 31 mRNAs that were significantly up-regulated in lung tissue from HDM-challenged mice. These included genes associated with human asthma (e.g. clca3, ear11, il-13, il-13ra2, il-10, il-21, arg1 and chia1) and leukocyte recruitment in the lungs (e.g. ccl11, ccl12 and ccl24). This study describes a biosignature to enable broad and systematic interrogation of molecular mechanisms and intervention strategies for airway inflammation pertinent to allergic asthma that precedes and possibly potentiates airway remodeling and fibrosis.

The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed.

Human cathelicidin LL-37-derived peptide IG-19 confers protection in a murine model of collagen-induced arthritis.

Current therapies for autoimmune chronic inflammatory diseases e.g. rheumatoid arthritis (RA) include inhibitors of inflammatory cytokines. However, these therapies can result in increased risk of infections. There is a need to explore alternate strategies that can control inflammation without compromising the innate ability to resolve infections. In this study, we examined the effect of small peptides derived from endogenous cathelicidin peptides in a murine model of collagen-induced arthritis (CIA). Cathelicidins are immunomodulatory peptides known to control infections. We demonstrate that the administration of the peptide IG-19, which represents an internal segment of the human cathelicidin LL-37, decreased disease severity and significantly reduced the serum levels of antibodies against collagen type II in the CIA model. IG-19 peptide reduced cellular infiltration in joints, prevented cartilage degradation and suppressed pro-inflammatory cytokines in the CIA mice. We also showed that not all cathelicidin-derived peptides exhibit similar functions. A bovine cathelicidin-derived peptide IDR-1018 did not exhibit the beneficial effects observed with the human cathelicidin LL-37-derived peptide IG-19, in the same murine model of CIA. This is the first study to provide evidence demonstrating the ability of a peptide derived from the human cathelicidin LL-37 to alleviate the arthritic disease process in a murine model of RA. Our results has lead us to propose a new approach for controlling autoimmune chronic inflammatory disorders such as RA, by using specific synthetic derivatives of endogenous host defence peptides. Cathelicidin-derived peptides are particularly attractive for their dual antimicrobial and anti-inflammatory actions.

Conformational dynamics of a membrane transport protein probed by H/D exchange and covalent labeling: the glycerol facilitator.

Glycerol facilitator (GF) is a tetrameric membrane protein responsible for the selective permeation of glycerol and water. Each of the four GF subunits forms a transmembrane channel. Every subunit consists of six helices that completely span the lipid bilayer, as well as two half-helices (TM7 and TM3). X-ray crystallography has revealed that the selectivity of GF is due to its unique amphipathic channel interior. To explore the structural dynamics of GF, we employ hydrogen/deuterium exchange (HDX) and oxidative labeling with mass spectrometry (MS). HDX-MS reveals that transmembrane helices are generally more protected than extramembrane segments, consistent with data previously obtained for other membrane proteins. Interestingly, TM7 does not follow this trend. Instead, this half-helix undergoes rapid deuteration, indicative of a highly dynamic local structure. The oxidative labeling behavior of most GF residues is consistent with the static crystal structure. However, the side chains of C134 and M237 undergo labeling although they should be inaccessible according to the X-ray structure. In agreement with our HDX-MS data, this observation attests to the fact that TM7 is only marginally stable. We propose that the highly mobile nature of TM7 aids in the efficient diffusion of guest molecules through the channel ("molecular lubrication"). In the absence of such dynamics, host-guest molecular recognition would favor semipermanent binding of molecules inside the channel, thereby impeding transport. The current work highlights the complementary nature of HDX, covalent labeling, and X-ray crystallography for the characterization of membrane proteins.