Journal of Proteomics

Euglena longa is a heterotrophic model euglenid, of significant research interest as a unicellular eukaryote. To provide broad insight into the localisation of its proteome we used subcellular spatial proteomics (SSP), based on a label-free version of LOPIT-DC (Localisation of Organelle Proteins by Isotope Tagging after Differential Centrifugation), in which organelles are separated by differential centrifugation and each protein is assigned to a compartment from its abundance profile across the fractions. We detected 5,909 proteins, using protein abundance profiles across fractions to resolve 16 different subcellular compartments, such as the plastid, pellicle, and peroxisomes, and assigned high-confidence localisations to 2,955 of them. This study serves as a proteomic dataset to resolve protein compartmentalisation for this euglenid, enabling evolutionary, biological and functional insight and analysis. We are drafting manuscripts concerning the metabolic nature of the plastid, the evolutionary origins of the pellicle, and the functions of the peroxisome. Investigators interested in using this data prior to publication are encouraged to contact us to avoid overlap and to facilitate coordinated and collaborative use of this resource.

Lipid droplets (LDs) play a central role in cellular carbon and energy homeostasis, in particular through the roles played by numerous LD-associated proteins. A long-standing question concerns how these proteins are molecularly organized at the seemingly overcrowded LD surface. Delayed in TAG Hydrolysis 1 (DTH1) has been identified as a key protein for efficient LD degradation in Chlamydomonas reinhardtii, however the underlying molecular mechanism remains unknown. DTH1 contains several structural features including a coiled-coil domain, a phosphatidylethanolamine (PE) specific lipid-binding domain, intrinsically disordered regions, and an alpha-helical C-terminal domain, that make it a compelling candidate for acting as a molecular scaffold in protein recruitment and/or organelle tethering. In this study, we used the Chlamydomonas knock-out mutant dth1 as an entry point to begin dissecting the molecular organization of proteins at the LD surface. Comparative lipidomic and proteomic analyses of isolated LDs from both WT and dth1 mutants revealed several key features associated with LD degradation during nutrient recovery. First, PE emerged as an important lipid species involved in LD degradation. Second, active communication occurs among multiple subcellular organelles particularly between LDs, peroxisomes and mitochondria. Third, DTH1 was found to play a critical role in remodeling both the LD proteome and lipidome. Furthermore, detailed analyses of LD degradation during nitrogen recovery uncovered the formation of micro-LDs, a phenomenon not yet reported in microalgae. Collectively, these results highlight the tight coordination of metabolic activities across subcellular compartments and emphasize the importance of intracellular carbon trafficking during stress recovery.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by PAH gene defects and can cause irreversible neurological damage as early as the fetal period; however, non-invasive prenatal monitoring methods are lacking. In this study, Pah+/- female mice were mated with Pah+/-; male mice and wild-type male mice, respectively. Urine samples were collected longitudinally from pregnant mice at 10 time points during gestation from Day 1 to Day 19. Data-independent acquisition (DIA) quantitative proteomics combined with GO/KEGG analysis, WGCNA, and a dual-strategy LASSO-random forest approach was used to screen candidate biomarkers. The results showed that stable differentially expressed proteins between groups were detectable as early as Day 1, and the differences persisted throughout gestation and exhibited temporal dynamics. Permutation tests indicated that the probability of random generation of the differential protein combination was low (0.04-0.06), suggesting that it was not randomly generated. The intersection of the two strategies yielded five proteins, GINM1, PRG4, LY6A, NTM, and YIPF3, and after including GLO1, a total of six candidate biomarkers were obtained. The candidate proteins had interaction or co-expression associations with 14 proteins involved in PKU mechanisms, with GLO1 serving as a network hub. This study found that the maternal urine proteome can non-invasively reflect the systemic response induced by fetal-origin PKU, providing proof of concept and candidate biomarker resources for non-invasive early-pregnancy screening of PKU.

Proteolysis can be completed in a single-step, rapid (5-30 minutes) reaction using hyperthermoacidic archaeal (HTA) proteases. Protocol simplicity and stability of HTA proteases in ambient storage conditions removes many logistical barriers to widespread deployment of proteomic analysis. HTA protease cleavage selectivity is complicated but quantitatively reproducible and generates peptides that negligibly overlap with trypsin. Tryptic and HTA protease peptides emphasize different proteins and provide complementary coverage of amino acid sequence and post-translational modification (PTM) sites. Digestion of a sample's separate aliquot with HTA protease is a minor addition to trypsin-based workflows, but LC-MS analysis of both sets of peptides is not practical for large cohorts. This manuscript evaluates the identifications and reproducibility of LC-MS analysis of tryptic and HTA protease peptide mixtures. Adding HTA protease peptides to 20% increased total protein identifications by 24% on average in neat plasma and identified 216 additional non-artifact modifications compared to conventional trypsin-only bottom-up proteomic analysis. This approach is reproducible and does not increase sample preparation or analysis time. Peptides from each protease present independent opportunities to quantify overlapping proteins and a quantification model was developed in which the protease with the most complete data is selected for each protein while the second protease is used to infer missing values. This cross-protease inference is more accurate than the alternative minimum, mean, or nearest neighbor imputations. Finally, biomarkers discovered through HTA protease peptides are amenable to rapid HTA protease only proteomic workflows thus streamlining translation from discovery to fieldable applications.

ABSTRACT Background: The esophageal String Test (EST) is emerging as a minimally invasive alternative for assessing disease activity in eosinophilic esophagitis (EoE), yet the molecular composition of its protein content remains unknown. Objective: To comprehensively characterize the esophageal EST proteome, define an EoE-associated luminal protein signature, and evaluate its biological and clinical relevance. Methods: EST samples were obtained from patients with active EoE, healthy controls (C) and patients with gastroesophageal reflux disease (GERD). Label-free proteomic profiling was performed by liquid chromatography-tandem mass spectrometry. Differential protein accumulation, principal component analysis (PCA), pathway annotation, comparison with published esophageal tissue and salivary proteomes, receiver operating characteristic (ROC) analyses and correlations with clinical disease activity were performed. Results: More than 3,000 proteins were identified in esophageal EST samples. Global proteomic profiling clearly separated patients with EoE from both C and GERD, whereas only limited differences were observed between the latter two groups. This enabled the identification of an EoE-associated luminal protein signature comprising inflammatory, epithelial, and tissue remodeling proteins. Comparison with published proteomic datasets indicated that the EST samples a complex esophageal luminal protein microenvironment containing proteins represented in esophageal mucosa and saliva, together with inflammatory and extracellular components. Several individual proteins showed high discriminatory performance in this exploratory cohort for distinguishing EoE from non-EoE subjects, and multiple proteins correlated with eosinophil counts and endoscopic measures of disease activity. Conclusions: This study demonstrates that the EST provides access to a complex esophageal luminal protein microenvironment and establishes a molecular framework for its minimally invasive characterization in EoE. KEYWORDS: eosinophilic esophagitis, esophageal string test, esophageal luminal protein signature

Ubiquitination is a post-translational modification with well described roles in the regulation of immune cell function, such as dendritic cells (DCs). DCs are crucial initiators and regulators of T cell-mediated immune responses. In this study, we use activity-based probes and proteomics to systematically profile DUBs in a DC line. Using the lysate-based biotin(bio)-Ubiquitin(Ub)-propargylamide(Prg) probe and proteomics, the activity and abundance of deubiquitinases was measured in unstimulated and CpG-stimulated MutuDCs to simulate responses to bacteria. This analysis was complemented with profiling using the cell-permeable DUB activity-based probe IMP-2373. The combined analysis with bio-Ub-Prg and IMP-2373 provided insights into the dynamics of DUB activity and abundance in DCs following stimulation and identified several DUBs for further study.

Detecting low-abundance bacterial peptides in samples dominated by human proteins using liquid chromatography tandem mass spectrometry (LC MS/MS) is challenging. Bacterial peptides are typically far less abundant than host-derived peptides, and sequence similarity between bacterial and human peptides can complicate confident attribution to a bacterial origin. To address this problem, we developed and benchmarked an in silico spectral library protein identification workflow for detecting Staphylococcus aureus USA300 peptides against a background of A375 cells. Peptide and protein identifications obtained at a 1% peptide-level false discovery rate were compared between the in silico spectral library search and a conventional sequence database search across a defined bacterial dilution series containing 0, 1%, 2%, 10%, 25%, 50%, and 100% bacterial protein. A human sequence similarity filter was applied to identifications from both search strategies to reduce the likelihood of retaining human derived or human like peptide sequences. Both search strategies showed concentration dependent bacterial detection and shared a practical detection limit of approximately 1 to 2% bacterial protein but exhibited distinct performance profiles. The in silico spectral library search produced fewer bacterial-like background identifications in the human-only control, whereas MS2Rescore substantially increased spectral library identifications at higher bacterial concentrations, exceeding database search at 100% bacterial protein. The partially overlapping peptide sets identified by the two strategies indicate that they provide complementary coverage of the bacterial proteome.

Drug targets and vaccines for bacterial diseases are challenging to develop. Computational subtractive genomics and reverse vaccinology offer a viable substitute for conventional drug and vaccine development approaches. In recent years, Pasteurella multocida, a Gram-negative pathogen responsible for severe infections in humans along with major diseases in livestock and poultry, has developed resistance to multiple drugs. As a result, there is an urgent demand for research into reliable, efficient, highly potent, and cost-effective strategies to combat the rise of Pasteurella multocida infections. To address this, the present study integrated an in silico subtractive genomics and reverse vaccinology workflow to pinpoint proteins with potential value as both drug and vaccine targets. From the complete Pasteurella multocida Pm70 proteome, we identified 446 proteins that had no similarities with human proteins, out of which 129 were found to be essential for the pathogens survival. These 129 proteins are therefore potential candidates for drug development. Further metabolic pathway analysis, virulence potential, subcellular localization, and structural analyses revealed two outer membrane proteins, namely, 47 kDa outer membrane protein and lipopolysaccharide-assembly protein LptD, as promising candidates for therapeutic development. Subsequent antigenicity analysis revealed both outer membrane proteins as potential vaccine targets, exhibiting highly favorable antigenicity scores. Candidate B- and T-cell epitopes were systematically mapped from these two targets based on antigenicity, immunogenicity, toxicity, and allergenicity potential and only high-confidence epitopes were assembled using adjuvants and class-specific linkers to construct the final multi-epitope vaccine (MEV). The designed MEV construct was demonstrated to be highly antigenic, non-toxic and non-allergenic to humans, while possessing favorable structural and physicochemical properties, and exhibited strong binding interactions with human toll-like receptors with minimal molecular deformability. Although further experimental validation is recommended to fully assess their efficacy, the present study establishes a solid foundation for the development of novel therapeutics and vaccine candidates against P. multocida, ultimately contributing to the reduction of its rising infections.

Quantitative proteomics requires decisions about normalization, missing-data handling, and statistical modeling that can change the reported results. ProteoScopeR is an R package and Shiny application that connects these decisions in a traceable workflow and complements downstream exploration in xOmicsShiny. Side-by-side comparisons expose changes in distributions, feature retention, effect estimates, and selected protein sets. An optional external artificial-intelligence assistant reviews exported evidence and proposes settings for researcher approval; statistical calculations remain in R. A group-only case study used DIA-NN-derived aqueous humor data from 69 samples and 3,667 imported protein groups. Six normalization methods and seven missing-data strategies were screened, and the approved cyclic-loess analysis compared five differential workflows. For nAMD versus pmCNV, limma and proDA selected 54 and 37 protein groups, respectively, with 31 shared selections at adjusted P < 0.05 and absolute log2 fold change [&ge;] 0.5. Input routing, scientist decisions, locked settings, and an execution receipt preserve the analysis record. These comparisons describe sensitivity to analytical choices rather than identify a universally superior method.

Central carbon metabolism coordinates energy production and carbon skeleton allocation across photosynthetic and heterotrophic organisms. Here, we established an optimized targeted metabolomics workflow for Chlamydomonas reinhardtii and Arabidopsis thaliana using a single-run HILIC-MS/MS assay with alternating positive and negative ionization. Our method provides robust multi-class coverage across 21 amino acids, 11 carboxylic acids, 4 sugars, 3 adenine nucleotides, 2 nucleotide sugars, 18 phosphorylated compounds in CCM, and Acetyl-CoA in a 20-minute run using both ESI modes. An optimal concentration of medronic acid with a BEH Amide column at high pH prevented metal adsorption, and low quantification limits (10 to 50 nM) for labile intermediates such as PEP, ATP and RUBP were achieved. Retention time variations remained below 1.3%, and over 90% of analytes showed high linearity (R2 > 0.990) across dynamic ranges spanning two to three orders of magnitude (typically 0.01-0.5 uM up to 15-20 uM). For sample preparation, glass-fiber filters (GF/C) outperformed nitrocellulose for harvesting C. reinhardtii. For A. thaliana, it was proved that no further grinding with micro pestle and extended cooling were necessary. A two-phase chloroform/methanol/water extraction was essential, as a single-phase extraction co-extracted green chlorophyll, which heavily suppressed phosphorylated signals. In Arabidopsis, glutamate, fumaric acid and citric acid were the dominant primary pools, indicating the importance of dilution and column washing for preventing carryover, and sensitive intermediates were reliably quantified at physiological levels. Together, this unified analytical and extraction strategy enables robust, artifact-free, high-throughput profiling of central metabolism in photosynthetic organisms.

Endoplasmic reticulum (ER) stress is associated with many human diseases, but current understanding of how ER stress responses reshape the stressed proteome over time is still emerging. Specifically, how organellar protein quality control and clearance pathways coordinate to maintain proteostasis in early and prolonged ER stress is unclear. Here we describe a spatiotemporal proteomic strategy termed simultaneous proteome localization and turnover analysis with time resolution (SPLAT-TR) to interrogate the synthesis, degradation, and localization changes of over 4,000 proteins in early (1-4 hours) and prolonged (16-24 hours) ER stress. As ER stress progresses, each time point is distinguished by distinct protein translocation and clearance regulations, coupled to differential organellar proteostasis and usage of protein degradation pathways. Mitochondria show a bimodal response, with a shift from early activation of respiratory protein towards protein quality control pathways in prolonged stress, coinciding with energetics decline. In the Golgi, the increased clearance of collagen proteins is paradoxically coupled to an increased synthesis of secretory pathway components, suggesting removal by secretion. In the ER, the proteome remodels under prolonged stress via increased synthesis of UFMylation and ER-phagy-related proteins coupled to the selective degradation of ER membrane and microdomain proteins. Inhibition of UFMylation alters ER-phagy receptor usage and synergistically induces stress-induced cell death. These results present a systematic delineation of cell compartment proteostasis under unfolded protein response and highlight the utility of SPLAT-TR to investigate time-dependent cellular events.

Purpose. Neuroblastoma is the most common malignancy of infancy, and cancer cells strongly depend on iron metabolism for proliferation. Whether sub-cytotoxic iron load reprograms mitochondrial and redox metabolism, and how this relates to the malignant behavior of neuroblastoma cells, is poorly defined. We hypothesized that exposure of human neuroblastoma cells to a non-toxic iron overload perturbs mitochondrial and redox metabolism, thereby affecting cancer cell properties. Methods. SH-SY5Y neuroblastoma cells were exposed to 100 M iron sulfate for 24 h. The resulting changes were monitored using complementary methods, including cell viability, cytotoxicity, proliferation and migration assays, intracellular iron quantification by ICP-MS, confocal imaging of mitochondrial morphology and membrane potential ({Delta}{Psi}m), high-resolution respirometry with flux control ratios, probe-based oxidant and H2O2 measurements, and label-free quantitative proteomics with Ingenuity Pathway Analysis. Results. Non-toxic iron loading raised intracellular iron about four-fold, promoted cell proliferation and reduced migration. Iron induced mitochondrial fragmentation and {Delta}{Psi}m depolarization while preserving mitochondrial content and OXPHOS coupling efficiency. Among respiratory states, only proton-leak respiration increased, paralleled by a higher leak/ETS coupling ratio. Total cellular oxidant levels and H2O2 output were strongly reduced. The proteome showed downregulation of ribosomal proteins and translation/RNA-processing components and upregulation of histones and ferritin. Conclusion. Mild iron exposure reprograms mitochondrial and redox metabolism in neuroblastoma cells. The observed selective increase in proton leak lowers {Delta}{Psi}m to a range that biophysically limits oxidant production while OXPHOS coupling is preserved. Interfering with this adaptive, proton leak-dependent and preventive antioxidant program may offer a way to alter neuroblastoma progression.

In bottom-up proteomics, protease specificity determines peptide length and LC/MS/MS detectability; in N-terminomics, this is critical because each protein N-terminal peptide is unique and cannot be substituted by other peptides. CHromatographic AMplification of Protein N-terminal peptides (CHAMP-N) combines LysargiNase digestion with StageTip-based strong cation exchange chromatography (SCX), removing internal peptides by exploiting their newly generated N-terminal Lys/Arg residues and thereby selectively isolating protein N-terminal peptides. However, LysargiNase cleavage near the protein N-terminus can generate excessively short peptides. Here, we developed two complementary CHAMP-N approaches that modurate N-terminal peptide length while retaining the charge-based SCX separation principle. LysN digestion avoided cleavage at N-terminal-proximal Arg residues, whereas D3-acetylation blocked Lys cleavage by LysargiNase to produce ArgN-like specificity. Both approaches extended otherwise short N-terminal peptides while preserving N-terminal basic residues on internal peptides. Conventional LysargiNase, LysN, and ArgN-like CHAMP-N generated complementary sets of N-terminal peptides. Integration of the three workflows identified 2,552 protein N-termini, approximately twice the number identified by conventional CHAMP-N alone. The median relative standard deviations of N-terminal peptide intensities were approximately 15% for all three workflows, indicating comparable technical reproducibility. These results demonstrate that coordinating protease cleavage specificity with SCX selectivity reduces sequence-dependent losses and expands N-terminome coverage while preserving the simplicity and reproducibility of CHAMP-N.

Dictyostelium discoideum is a genetically tractable amoebozoan model organism widely used to study conserved eukaryotic cellular processes, yet a comprehensive map of its subcellular proteome has been lacking. Here, we combined subcellular fractionation with label-free mass spectrometry to resolve a global protein localization map for D. discoideum. We detected 6,337 proteins (~50% of the predicted proteome) and generated protein abundance profiles across 10 subcellular fractions. Using a curated marker set and a support vector machine classifier with a median cutoff, we assigned high-confidence localizations to 3,169 proteins across 17 subcellular compartments. Independent validation using sequence-based targeting predictions strongly supported the compartment assignments. Together, this study provides the first global subcellular localization map of the Dictyostelium proteome, establishing a foundational resource for functional, cell biological, and evolutionary analyses. We are currently preparing manuscripts that further analyze (i) the membrane trafficking system of Dictyostelium, including detailed investigation of the contractile vacuole, Jotnarlogs, and patchy proteins, and (ii) the composition of the Dictyostelium peroxisome, with a focus on the subcellular localization of sterol biosynthesis enzymes. Investigators interested in using these data are encouraged to contact us prior to publication to avoid overlap and to facilitate coordinated and collaborative use of this resource.

Opening of the mitochondrial permeability transition (PT) pore is an important step in necrotic cell death in various pathologic conditions such as ischemia-reperfusion injury. Methylglyoxal (MGO) is a byproduct of glycolysis that covalently modifies proteins (i.e., glycation) and is widely regarded as toxic (glycative stress), contributing to the pathology of diabetes. Paradoxically, MGO has been shown to inhibit PT pore opening, but the mechanism of this effect is poorly understood. Herein we show that glycation is elevated in various cardioprotective scenarios. Furthermore, building on a serendipitous finding of the ability of phosphate to block MGO's effect on the PT pore, we used proteomics to map potential sites mediating this effect. From this analysis, mitochondrial creatine kinase (CK) emerged as a phosphate-sensitive MGO target with links to the PT pore. Although MGO inhibited CK activity, this did not appear to be important for its effect on the PT pore. Rather, MGO modified sites on CK that are important for its membrane binding, which likely mediates the combined effects of MGO and phosphate on the PT pore. Furthermore, a consensus sequence for MGO modifications in mitochondria is reported.

Mass spectrometry-based proteomics increasingly supports study designs with repeated measurements, such as longitudinal sampling and spatial tissue profiling. These designs offer rich biological insights but introduce statistical challenges due to within-subject correlations and variance heterogeneity across time points or anatomical locations. Despite the growing use, best practices for analyzing repeated-measures proteomics data remain limited. We systematically compare commonly applied methods, including paired t-tests and linear mixed-effects models with correlation induced by random effects, to more flexible covariance-pattern models with unstructured covariance. Through simulation studies, we evaluate false-positive rates and statistical power under varying conditions of covariance heterogeneity. Our results demonstrate substantial limitations of conventional approaches and highlight the robustness and superior performance of unstructured covariance pattern models for repeated measures proteomics. This approach accommodates diverse study designs supporting accurate inference and reproducibility. We demonstrate that conventional F-tests can be severely biased, not just for small sample sizes but also moderately large ones, while using pairwise t-tests or applying parametric bootstrapping to evaluate the p-value of the F-tests mitigates the problem. We provide real world data examples showing how to perform valid and informative statistical analyses for temporal and spatial proteomics studies with repeated measurements.

While absolute blood biomarkers have long guided diagnosis, their rate of change often provides greater diagnostic value. For the Limb Girdle Muscular Dystrophies studied here, increased muscle weakness is associated with abnormal sarcolemma repair. Time course analyses were made to changes in muscle-derived plasma enzymes after modest physical activity of ambulatory inpatients. Blood samples at 20 selected time points at variable intervals were obtained at rest, and before and after routine morning activities and motor function testing. After these activities, plasma enzyme levels increased in a similar way, suggesting damage and leakage from skeletal muscle. The levels then decreased in a similar way, suggesting that muscle cells stopped leaking and enzymes were subsequently cleared from the blood. The complex temporal changes were modeled using both continuous time and data features to reduce the number of parameters required to describe the irregularly sampled, interrupted, time series data. Unlike individuals with cardiac muscle ischemia or following intense exercise, there was no extended post-injury rising phase of enzyme levels. In contradistinction, levels fell once activity ceased. Time series analyses of blood biomarkers most likely provide an assay and index of sarcolemma fragility and by extension skeletal muscle damage.

Background: Glioblastoma, isocitrate dehydrogenase wildtype, central nervous system World Health Organization grade 4, is the most common primary malignant brain tumor in adults and remains associated with poor prognosis despite aggressive multimodal treatment. The gut-brain axis has been proposed as a modulator of glioblastoma biology, but human data are scarce and the gut mycobiome has not been characterized in these patients. We characterized both bacteria and fungi at diagnosis as well as the intratumoral myeloid compartment and survival. Methods: In a prospective case-control study, we sequenced bacterial (16S rRNA gene) and fungal (internal transcribed spacer, ITS) communities from rectal swabs of 23 patients with newly diagnosed glioblastoma and 23 healthy controls. Additionally, we quantified the intratumoral myeloid compartment by immunofluorescence and performed survival analysis. Results: Both bacterial and fungal communities were more diverse in patients than in controls (Shannon, 16S p < 0.001; ITS p = 0.004), and even more diverse in patients with shorter survival. Community structure itself was unchanged, locating the difference at the level of individual taxa. Fourteen of 218 bacterial and archaeal taxa and four of 102 fungal species differed at nominal, uncorrected p [&le;] 0.05. Members of the Lachnospiraceae and Ruminococcaceae were reduced in glioblastoma patients, while the methanogenic archaeon Methanobrevibacter and Nakaseomyces glabratus (formerly Candida glabrata) were enriched. Survival was also addressed at the tissue level. In the tumor, a higher density of ionized calcium-binding adapter molecule 1-positive myeloid cells was associated with longer survival, independently of age, performance status and extent of resection (hazard ratio 0.82 per percentage point, 95% confidence interval 0.67-0.99). Conclusions: This study characterizes the gut bacteriome and mycobiome of newly diagnosed glioblastoma patients and identifies candidate taxa, among them several butyrate- and propionate producers within the depleted Lachnospiraceae and Ruminococcaceae. Combining these observations with the established role of short-chain fatty acids in microglial maturation, we hypothesize that the myeloid compartment is the interface at which the gut-level and tissue-level findings converge.

The acquisition of sufficient nitrogen for growth, as well as the disposal of highly toxic nitrogen-containing degradation products, represent serious metabolic challenges in all multicellular organisms and microbial ecosystems. Understanding these processes requires a broadly applicable system to accurately quantify the concentration of a wide spectrum of biological amines and inorganic nitrogen-containing compounds. To date, this has been challenging due to limited specificity, selectivity, breadth and/or sensitivity of assays. Here we present a simple, optimized method based on liquid chromatography and high-resolution mass spectrometry, with an accompanying analysis pipeline, for the accurate quantification of more than 30 of these compounds, including ammonia. We demonstrate the effectiveness of this method in quantifying relevant nitrogen-containing compounds in mouse and human gut content, bacterial cultures, algal symbiosis in a cnidarian and freshwater ecosystem samples. As a proof-of-concept, we applied the method to study the influence of the gut microbiome on cecal nitrogen distribution in mice with different gnotobiotic microbiomes. This revealed a profound influence of gut microbiota composition on amino acid, ammonia, and amine concentrations in the upper large intestine. Overall, we provide a broadly applicable approach that can be used across microbial ecology fields to generate novel insights into the biology of nitrogen assimilation, exchange, and disposal.

Retinal neurons rely on continuous oxygen supply to maintain function, yet some species, like goldfish, withstand prolonged hypoxia due to adaptations that remain unclear. Horizontal cells (HCs) are inhibitory interneurons of the retina that express ionotropic glutamate receptors (iGluRs) and secrete gama-aminobutyric acid (GABA), allowing them to shape visual signalling by feeding back to photoreceptors. We investigated whether iGluRs in dissociated goldfish HCs are suppressed during hypoxia, and whether this process is mediated by oxygen-sensitive activation of mitochondrial ATP-sensitive K+ (mKATP) channels. Through perforated patch-clamp electrophysiology, we determined that 20 min of hypoxia reduced peak inward current density associated with glutamate application by ~25% (P = 0.0059). When mKATP channels were selectively blocked with 100 microM glibenclamide, hypoxic suppression of iGluRs was abolished. Inhibiting mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter with ruthenium red, or blocking Ca2+ release through ryanodine receptors in the endoplasmic reticulum, also abolished the hypoxia-induced suppression. Similarly, inhibition of calmodulin with calmidazolium, or of protein phosphatase 1 and 2A (PP1/PP2A) activity with calyculin A, prevented the hypoxia-induced suppression of iGluRs. Taken together, these results indicate that mKATP activation is required to mediate an intracellular Ca2+ signal that downregulates iGluR activity during hypoxia. Through the reduction of iGluR activity, HCs may lower their metabolic demand, allowing them to maintain cell function during hypoxia. Additionally, GABA release from HCs, as measured by mass spectrometry, was not affected by hypoxia, and we propose that downregulation of iGluR may account for hypoxic suppression of the retina in hypoxia-tolerant organisms.