Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism.

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Distinguishing the molecular diversity, nutrient content, and energetic potential of exometabolomes produced by macroalgae and reef-building corals.

Metabolites exuded by primary producers comprise a significant fraction of marine dissolved organic matter, a poorly characterized, heterogenous mixture that dictates microbial metabolism and biogeochemical cycling. We present a foundational untargeted molecular analysis of exudates released by coral reef primary producers using liquid chromatography-tandem mass spectrometry to examine compounds produced by two coral species and three types of algae (macroalgae, turfing microalgae, and crustose coralline algae [CCA]) from Mo'orea, French Polynesia. Of 10,568 distinct ion features recovered from reef and mesocosm waters, 1,667 were exuded by producers; the majority (86%) were organism specific, reflecting a clear divide between coral and algal exometabolomes. These data allowed us to examine two tenets of coral reef ecology at the molecular level. First, stoichiometric analyses show a significantly reduced nominal carbon oxidation state of algal exometabolites than coral exometabolites, illustrating one ecological mechanism by which algal phase shifts engender fundamental changes in the biogeochemistry of reef biomes. Second, coral and algal exometabolomes were differentially enriched in organic macronutrients, revealing a mechanism for reef nutrient-recycling. Coral exometabolomes were enriched in diverse sources of nitrogen and phosphorus, including tyrosine derivatives, oleoyl-taurines, and acyl carnitines. Exometabolites of CCA and turf algae were significantly enriched in nitrogen with distinct signals from polyketide macrolactams and alkaloids, respectively. Macroalgal exometabolomes were dominated by nonnitrogenous compounds, including diverse prenol lipids and steroids. This study provides molecular-level insights into biogeochemical cycling on coral reefs and illustrates how changing benthic cover on reefs influences reef water chemistry with implications for microbial metabolism.

Venomics and Peptidomics of Palearctic Vipers: A Clade-Wide Analysis of Seven Taxa of the Genera Vipera, Montivipera, Macrovipera, and Daboia across Türkiye.

Snake venom variations are a crucial factor to understand the consequences of snakebite envenoming worldwide, and therefore it is important to know about toxin composition alterations between taxa. Palearctic vipers of the genera Vipera, Montivipera, Macrovipera, and Daboia have high medical impacts across the Old World. One hotspot for their occurrence and diversity is Türkiye, located on the border between continents, but many of their venoms remain still understudied. Here, we present the venom compositions of seven Turkish viper taxa. By complementary mass spectrometry-based bottom-up and top-down workflows, the venom profiles were investigated on proteomics and peptidomics level. This study includes the first venom descriptions of Vipera berus barani, Vipera darevskii, Montivipera bulgardaghica albizona, and Montivipera xanthina, as well as the first snake venomics profiles of Turkish Macrovipera lebetinus obtusa, and Daboia palaestinae, including an in-depth reanalysis of M. bulgardaghica bulgardaghica venom. Additionally, we identified the modular consensus sequence pEXW(PZ)1-2P(EI)/(KV)PPLE for bradykinin-potentiating peptides in viper venoms. For better insights into variations and potential impacts of medical significance, the venoms were compared against other Palearctic viper proteomes, including the first genus-wide Montivipera venom comparison. This will help the risk assessment of snakebite envenoming by these vipers and aid in predicting the venoms' pathophysiology and clinical treatments.

Leptochelins A-C, Cytotoxic Metallophores Produced by Geographically Dispersed Leptothoe Strains of Marine Cyanobacteria.

Metals are important cofactors in the metabolic processes of cyanobacteria, including photosynthesis, cellular respiration, DNA replication, and the biosynthesis of primary and secondary metabolites. In adaptation to the marine environment, cyanobacteria use metallophores to acquire trace metals when necessary as well as to reduce potential toxicity from excessive metal concentrations. Leptochelins A-C were identified as structurally novel metallophores from three geographically dispersed cyanobacteria of the genus Leptothoe. Determination of the complex structures of these metabolites presented numerous challenges, but they were ultimately solved using integrated data from NMR, mass spectrometry and deductions from the biosynthetic gene cluster. The leptochelins are comprised of halogenated linear NRPS-PKS hybrid products with multiple heterocycles that have potential for hexadentate and tetradentate coordination with metal ions. The genomes of the three leptochelin producers were sequenced, and retrobiosynthetic analysis revealed one candidate biosynthetic gene cluster (BGC) consistent with the structure of leptochelin. The putative BGC is highly homologous in all three Leptothoe strains, and all possess genetic signatures associated with metallophores. Postcolumn infusion of metals using an LC-MS metabolomics workflow performed with leptochelins A and B revealed promiscuous binding of iron, copper, cobalt, and zinc, with greatest preference for copper. Iron depletion and copper toxicity experiments support the hypothesis that leptochelin metallophores may play key ecological roles in iron acquisition and in copper detoxification. In addition, the leptochelins possess significant cytotoxicity against several cancer cell lines.

Connecting metabolome and phenotype: recent advances in functional metabolomics tools for the identification of bioactive natural products.

Covering: 1995 to 2023Advances in bioanalytical methods, particularly mass spectrometry, have provided valuable molecular insights into the mechanisms of life. Non-targeted metabolomics aims to detect and (relatively) quantify all observable small molecules present in a biological system. By comparing small molecule abundances between different conditions or timepoints in a biological system, researchers can generate new hypotheses and begin to understand causes of observed phenotypes. Functional metabolomics aims to investigate the functional roles of metabolites at the scale of the metabolome. However, most functional metabolomics studies rely on indirect measurements and correlation analyses, which leads to ambiguity in the precise definition of functional metabolomics. In contrast, the field of natural products has a history of identifying the structures and bioactivities of primary and specialized metabolites. Here, we propose to expand and reframe functional metabolomics by integrating concepts from the fields of natural products and chemical biology. We highlight emerging functional metabolomics approaches that shift the focus from correlation to physical interactions, and we discuss how this allows researchers to uncover causal relationships between molecules and phenotypes.

Identifying Quinones in Complex Aqueous Environmental Media (Biochar Extracts) through Tagging with Cysteine and Cysteine-Contained Peptides and High Resolution Mass Spectrometry Analysis.

Quinones are among the most important components in natural organic matter (NOM) for redox reactions; however, no quinones in complex environmental media have been identified. To aid the identification of quinone-containing molecules in ultracomplex environmental samples, we developed a chemical tagging method that makes use of a Michael addition reaction between quinones and thiols (-SH) in cysteine (Cys) and cysteine-contained peptides (CCP). After the tagging, candidates of quinones in representative aqueous environmental samples (water extractions of biochar) were identified through high-resolution mass spectrometry (HRMS) analysis. The MS and UV spectra analysis showed rapid reactions between Cys/CCP and model quinones with ß-carbon from the same benzene ring available for Michael addition. The tagging efficiency was not influenced by other co-occurring nonquinone representative compounds, including caffeic acid, cinnamic acid, and coumaric acid. Cys and CCP were used to tag quinones in water extractions of biochars, and possible candidates of quinones (20 and 53 based on tagging with Cys and CCP, respectively) were identified based on the HRMS features for products of reactions with Cys/CCP. This study has successfully demonstrated that such a Michael addition reaction can be used to tag quinones in complex environmental media and potentially determine their identities. The method will enable an in-depth understanding of the redox chemistry of NOM and its critical chemical compositions and structures.

High-Resolution Tandem Mass Spectrometry-Based Analysis of Model Lignin-Iron Complexes: Novel Pipeline and Complex Structures.

Understanding the chemical nature of soil organic carbon (SOC) with great potential to bind iron (Fe) minerals is critical for predicting the stability of SOC. Organic ligands of Fe are among the top candidates for SOCs able to strongly sorb on Fe minerals, but most of them are still molecularly uncharacterized. To shed insights into the chemical nature of organic ligands in soil and their fate, this study developed a protocol for identifying organic ligands using ultrahigh-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and metabolomic tools. The protocol was used for investigating the Fe complexes formed by model compounds of lignin-derived organic ligands, namely, caffeic acid (CA), p-coumaric acid (CMA), vanillin (VNL), and cinnamic acid (CNA). Isotopologue analysis of 54/56Fe was used to screen out the potential UHPLC-HRMS (m/z) features for complexes formed between organic ligands and Fe, with multiple features captured for CA, CMA, VNL, and CNA when 35/37Cl isotopologue analysis was used as supplementary evidence for the complexes with Cl. MS/MS spectra, fragment analysis, and structure prediction with SIRIUS were used to annotate the structures of mono/bidentate mono/biligand complexes. The analysis determined the structures of monodentate and bidentate complexes of FeLxCly (L: organic ligand, x = 1-4, y = 0-3) formed by model compounds. The protocol developed in this study can be used to identify unknown organic ligands occurring in complex environmental samples and shed light on the molecular-level processes governing the stability of the SOC.

Two-Dimensional Liquid Chromatography Tandem Mass Spectrometry Untangles the Deep Metabolome of Marine Dissolved Organic Matter.

Dissolved organic matter (DOM) is an ultracomplex mixture that plays a central role in global biogeochemical cycles. Despite its importance, DOM remains poorly understood at the molecular level. Over the last decades, significant efforts have been made to decipher the chemical composition of DOM by high-resolution mass spectrometry (HR-MS) and liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Yet, the complexity and high degree of nonresolved isomers still hamper the full structural analysis of DOM. To address this challenge, we developed an offline two-dimensional (2D) LC approach using two reversed-phase dimensions with orthogonal pH levels, followed by MS/MS data acquisition and molecular networking. 2D-LC-MS/MS reduced the complexity of DOM, enhancing the quality of MS/MS spectra and increasing spectral annotation rates. Applying our approach to analyze coastal-surface DOM from Southern California (USA) and open-ocean DOM from the central North Pacific (Hawaii), we annotated in total more than 600 structures via MS/MS spectrum matching, which was up to 90% more than that in iterative 1D LC-MS/MS analysis with the same total run time. Our data offer unprecedented insights into the molecular composition of marine DOM and highlight the potential of 2D-LC-MS/MS approaches to decipher the chemical composition of ultracomplex samples.

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides.

The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V1A receptor, a member of the G protein-coupled receptor superfamily.

Decoding the molecular interplay in the central dogma: An overview of mass spectrometry-based methods to investigate protein-metabolite interactions.

With the emergence of next-generation nucleotide sequencing and mass spectrometry-based proteomics and metabolomics tools, we have comprehensive and scalable methods to analyze the genes, transcripts, proteins, and metabolites of a multitude of biological systems. Despite the fascinating new molecular insights at the genome, transcriptome, proteome and metabolome scale, we are still far from fully understanding cellular organization, cell cycles and biology at the molecular level. Significant advances in sensitivity and depth for both sequencing as well as mass spectrometry-based methods allow the analysis at the single cell and single molecule level. At the same time, new tools are emerging that enable the investigation of molecular interactions throughout the central dogma of molecular biology. In this review, we provide an overview of established and recently developed mass spectrometry-based tools to probe metabolite-protein interactions-from individual interaction pairs to interactions at the proteome-metabolome scale.

Spatial Venomics─Cobra Venom System Reveals Spatial Differentiation of Snake Toxins by Mass Spectrometry Imaging.

Among venomous animals, toxic secretions have evolved as biochemical weapons associated with various highly specialized delivery systems on many occasions. Despite extensive research, there is still limited knowledge of the functional biology of most animal toxins, including their venom production and storage, as well as the morphological structures within sophisticated venom producing tissues that might underpin venom modulation. Here, we report on the spatial exploration of a snake venom gland system by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), in combination with standard proteotranscriptomic approaches, to enable in situ toxin mapping in spatial intensity maps across a venom gland sourced from the Egyptian cobra (Naja haje). MALDI-MSI toxin visualization on the elapid venom gland reveals a high spatial heterogeneity of different toxin classes at the proteoform level, which may be the result of physiological constraints on venom production and/or storage that reflects the potential for venom modulation under diverse stimuli.

Evaluation of Data-Dependent MS/MS Acquisition Parameters for Non-Targeted Metabolomics and Molecular Networking of Environmental Samples: Focus on the Q Exactive Platform.

Non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widely used tool for metabolomics analysis, enabling the detection and annotation of small molecules in complex environmental samples. Data-dependent acquisition (DDA) of product ion spectra is thereby currently one of the most frequently applied data acquisition strategies. The optimization of DDA parameters is central to ensuring high spectral quality, coverage, and number of compound annotations. Here, we evaluated the influence of 10 central DDA settings of the Q Exactive mass spectrometer on natural organic matter samples from ocean, river, and soil environments. After data analysis with classical and feature-based molecular networking using MZmine and GNPS, we compared the total number of network nodes, multivariate clustering, and spectrum quality-related metrics such as annotation and singleton rates, MS/MS placement, and coverage. Our results show that automatic gain control, microscans, mass resolving power, and dynamic exclusion are the most critical parameters, whereas collision energy, TopN, and isolation width had moderate and apex trigger, monoisotopic selection, and isotopic exclusion minor effects. The insights into the data acquisition ergonomics of the Q Exactive platform presented here can guide new users and provide them with initial method parameters, some of which may also be transferable to other sample types and MS platforms.

Feature-based molecular networking in the GNPS analysis environment.

Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present feature-based molecular networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. FBMN enables quantitative analysis and resolution of isomers, including from ion mobility spectrometry.

ReDU: a framework to find and reanalyze public mass spectrometry data.

We present ReDU ( https://redu.ucsd.edu/ ), a system for metadata capture of public mass spectrometry-based metabolomics data, with validated controlled vocabularies. Systematic capture of knowledge enables the reanalysis of public data and/or co-analysis of one's own data. ReDU enables multiple types of analyses, including finding chemicals and associated metadata, comparing the shared and different chemicals between groups of samples, and metadata-filtered, repository-scale molecular networking.

Mass spectrometry DDA parameters and global coverage of the metabolome: Spectral molecular networks of momordica cardiospermoides plants.

INTRODUCTION: Molecular networking (MN) has emerged as a key strategy to organize and annotate untargeted tandem mass spectrometry (MS/MS) data generated using either data independent- or dependent acquisition (DIA or DDA). The latter presents a time-efficient approach where full scan (MS1) and MS2 spectra are obtained with shorter cycle times. However, there are limitations related to DDA parameters, some of which are (i) intensity threshold and (ii) collision energy. The former determines ion prioritization for fragmentation, and the latter defines the fragmentation of selected ions. These DDA parameters inevitably determine the coverage and quality of spectral data, which would affect the outputs of MN methods. OBJECTIVES: This study assessed the extent to which the quality of the tandem spectral data relates to MN topology and subsequent implications in the annotation of metabolites and chemical classification relative to the different DDA parameters employed. METHODS: Herein, characterising the metabolome of Momordica cardiospermoides plants, we employ classical MN performance indicators to investigate the effects of collision energies and intensity thresholds on the topology of generated MN and propagated annotations. RESULTS: We demonstrated that the lowest predefined intensity thresholds and collision energies result in comprehensive molecular networks. Comparatively, higher intensity thresholds and collision energies resulted in fewer MS2 spectra acquisition, subsequently fewer nodes, and a limited exploration of the metabolome through MN. CONCLUSION: Contributing to ongoing efforts and conversations on improving DDA strategies, this study proposes a framework in which multiple DDA parameters are utilized to increase the coverage of ions acquired and improve the global coverage of MN, propagated annotations, and the chemical classification performed.

Bacterial and Chemical Evidence of Coastal Water Pollution from the Tijuana River in Sea Spray Aerosol.

Roughly half of the human population lives near the coast, and coastal water pollution (CWP) is widespread. Coastal waters along Tijuana, Mexico, and Imperial Beach (IB), USA, are frequently polluted by millions of gallons of untreated sewage and stormwater runoff. Entering coastal waters causes over 100 million global annual illnesses, but CWP has the potential to reach many more people on land via transfer in sea spray aerosol (SSA). Using 16S rRNA gene amplicon sequencing, we found sewage-associated bacteria in the polluted Tijuana River flowing into coastal waters and returning to land in marine aerosol. Tentative chemical identification from non-targeted tandem mass spectrometry identified anthropogenic compounds as chemical indicators of aerosolized CWP, but they were ubiquitous and present at highest concentrations in continental aerosol. Bacteria were better tracers of airborne CWP, and 40 tracer bacteria comprised up to 76% of the bacteria community in IB air. These findings confirm that CWP transfers in SSA and exposes many people along the coast. Climate change may exacerbate CWP with more extreme storms, and our findings call for minimizing CWP and investigating the health effects of airborne exposure.

Listeria monocytogenes exposed to antimicrobial peptides displays differential regulation of lipids and proteins associated to stress response.

With the onset of Listeria monocytogenes resistance to the bacteriocin nisin, the search for alternative antimicrobial treatments is of fundamental importance. In this work, we set out to investigate proteins and lipids involved in the resistance mechanisms of L. monocytogenes against the antimicrobial peptides (AMPs) nisin and fengycin. The effect of sub-lethal concentrations of nisin and lipopeptide fengycin secreted by Bacillus velezensis P34 on L. monocytogenes was investigated by mass spectrometry-based lipidomics and proteomics. Both AMPs caused a differential regulation of biofilm formation, confirming the promotion of cell attachment and biofilm assembling after treatment with nisin, whereas growth inhibition was observed after fengycin treatment. Anteiso branched-chain fatty acids were detected in higher amounts in fengycin-treated samples (46.6%) as compared to nisin-treated and control samples (39.4% and 43.4%, respectively). In addition, a higher relative abundance of 30:0, 31:0 and 32:0 phosphatidylglycerol species was detected in fengycin-treated samples. The lipidomics data suggest the inhibition of biofilm formation by the fengycin treatment, while the proteomics data revealed downregulation of important cell wall proteins involved in the building of biofilms, such as the lipoteichoic acid backbone synthesis (Lmo0927) and the flagella-related (Lmo0718) proteins among others. Together, these results provide new insights into the modification of lipid and protein profiles and biofilm formation in L. monocytogenes upon exposure to antimicrobial peptides.

Illuminating the dark metabolome of Pseudo-nitzschia-microbiome associations.

The exchange of metabolites mediates algal and bacterial interactions that maintain ecosystem function. Yet, while thousands of metabolites are produced, only a few molecules have been identified in these associations. Using the ubiquitous microalgae Pseudo-nitzschia sp., as a model, we employed an untargeted metabolomics strategy to assign structural characteristics to the metabolites that distinguished specific diatom-microbiome associations. We cultured five species of Pseudo-nitzschia, including two species that produced the toxin domoic acid, and examined their microbiomes and metabolomes. A total of 4826 molecular features were detected by tandem mass spectrometry. Only 229 of these could be annotated using available mass spectral libraries, but by applying new in silico annotation tools, characterization was expanded to 2710 features. The metabolomes of the Pseudo-nitzschia-microbiome associations were distinct and distinguished by structurally diverse nitrogen compounds, ranging from simple amines and amides to cyclic compounds such as imidazoles, pyrrolidines and lactams. By illuminating the dark metabolomes, this study expands our capacity to discover new chemical targets that facilitate microbial partnerships and uncovers the chemical diversity that underpins algae-bacteria interactions.

Mass Difference Matching Unfolds Hidden Molecular Structures of Dissolved Organic Matter.

Ultrahigh-resolution Fourier transform mass spectrometry (FTMS) has revealed unprecedented details of natural complex mixtures such as dissolved organic matter (DOM) on a molecular formula level, but we lack approaches to access the underlying structural complexity. We here explore the hypothesis that every DOM precursor ion is potentially linked with all emerging product ions in FTMS2 experiments. The resulting mass difference (Δm) matrix is deconvoluted to isolate individual precursor ion Δm profiles and matched with structural information, which was derived from 42 Δm features from 14 in-house reference compounds and a global set of 11 477 Δm features with assigned structure specificities, using a dataset of ∼18 000 unique structures. We show that Δm matching is highly sensitive in predicting potential precursor ion identities in terms of molecular and structural composition. Additionally, the approach identified unresolved precursor ions and missing elements in molecular formula annotation (P, Cl, F). Our study provides first results on how Δm matching refines structural annotations in van Krevelen space but simultaneously demonstrates the wide overlap between potential structural classes. We show that this effect is likely driven by chemodiversity and offers an explanation for the observed ubiquitous presence of molecules in the center of the van Krevelen space. Our promising first results suggest that Δm matching can both unfold the structural information encrypted in DOM and assess the quality of FTMS-derived molecular formulas of complex mixtures in general.