Metabolite-driven mechanisms reveal chemical ecology of Lehmann Lovegrass (Eragrostis lehmanniana) invasion in North American semi-arid ecosystems
Not provided.
Life Sciences & Biomedicine - Other Topics↗
Engineering topics
Veličković, Dušan
Publications and source records attributed to Veličković, Dušan.
Spatially structured bacterial interactions alter algal carbon flow to bacteria
Phytoplankton account for nearly half of global photosynthetic carbon fixation, and the fate of that carbon is regulated in large part by microbial food web processing. We currently lack a mechanistic understanding of how interactions among heterotrophic bacteria impact the fate of photosynthetically fixed carbon. Here, we used a set of bacterial isolates capable of growing on exudates from the diatom Phaeodactylum tricornutum to investigate how bacteria-bacteria interactions affect the balance between exudate remineralization and incorporation into biomass. With exometabolomics and genome-scale metabolic modeling, we estimated the degree of resource competition between bacterial pairs. In a sequential spent media experiment, we found that pairwise interactions were more beneficial than predicted based on resource competition alone, and 30% exhibited facilitative interactions. To link this to carbon fate, we used single-cell isotope tracing in a custom cultivation system to compare the impact of different "primary" bacterial strains in close proximity to live P. tricornutum on a distal "secondary" strain. We found that a primary strain with a high degree of competition decreased secondary strain carbon drawdown by 51% at the single-cell level, providing a quantitative metric for the "cost" of competition on algal carbon fate. Additionally, a primary strain classified as facilitative based on sequential interactions increased total algal-derived carbon assimilation by 7.6 times, integrated over all members, compared to the competitive primary strain. Our findings suggest that the degree of interaction between bacteria along a spectrum from competitive to facilitative is directly linked to algal carbon drawdown.
genome-scale metabolic model↗
Protein N -Glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease
Diabetes is expected to directly affect renal glycosylation; yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). Here, we used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in formalin-fixed paraffin-embedded sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single-nucleus RNA sequencing (snRNAseq) data were used to inform on transcript levels of glycosylation machinery in different cell types and states. We detected 120 N-glycans, and among them, we identified 12 of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that were common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and N-acetyllactosamine synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.
60 APPLIED LIFE SCIENCES↗
RhizoMAP: a comprehensive, nondestructive, and sensitive platform for metabolic imaging of the rhizosphere
Elucidating the intricate structural organization and spatial gradients of biomolecular composition within the rhizosphere is critical to understanding important biogeochemical processes, which include the mechanisms of root-microbe interactions for maintaining sustainable plant ecosystem services. While various analytical methods have been developed to assess the spatial heterogeneity within the rhizosphere, a comprehensive view of the fine distribution of metabolites within the root-soil interface has remained a significant challenge. This is primarily due to the difficulty of maintaining the original spatial organization during sample preparation without compromising its molecular content.
59 BASIC BIOLOGICAL SCIENCES↗
Coupling Microdroplet-Based Sample Preparation, Multiplexed Isobaric Labeling, and Nanoflow Peptide Fractionation for Deep Proteome Profiling of the Tissue Microenvironment
There is increasing interest in developing in-depth proteomic approaches for mapping tissue heterogeneity in a cell-type-specific manner to better understand and predict the function of complex biological systems such as human organs. Existing spatially resolved proteomics technologies cannot provide deep proteome coverage due to limited sensitivity and poor sample recovery. Herein, we seamlessly combined laser capture microdissection with a low-volume sample processing technology that includes a microfluidic device named microPOTS (microdroplet processing in one pot for trace samples), multiplexed isobaric labeling, and a nanoflow peptide fractionation approach. The integrated workflow allowed us to maximize proteome coverage of laser-isolated tissue samples containing nanogram levels of proteins. We demonstrated that the deep spatial proteomics platform can quantify more than 5000 unique proteins from a small-sized human pancreatic tissue pixel (∼60,000 μm2) and differentiate unique protein abundance patterns in pancreas. Furthermore, the use of the microPOTS chip eliminated the requirement for advanced microfabrication capabilities and specialized nanoliter liquid handling equipment, making it more accessible to proteomic laboratories.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Spatiotemporal metabolic responses to water deficit stress in distinct leaf cell-types of poplar
The impact of water-deficit (WD) stress on plant metabolism has been predominantly studied at the whole tissue level. However, plant tissues are made of several distinct cell types with unique and differentiated functions, which limits whole tissue ‘omics’-based studies to determine only an averaged molecular signature arising from multiple cell types. Advancements in spatial omics technologies provide an opportunity to understand the molecular mechanisms underlying plant responses to WD stress at distinct cell-type levels. Here, we studied the spatiotemporal metabolic responses of two poplar ( Populus tremula× P. alba ) leaf cell types -palisade and vascular cells- to WD stress using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). We identified unique WD stress-mediated metabolic shifts in each leaf cell type when exposed to early and prolonged WD stresses and recovery from stress. During water-limited conditions, flavonoids and phenolic metabolites were exclusively accumulated in leaf palisade cells. However, vascular cells mainly accumulated sugars and fatty acids during stress and recovery conditions, respectively, highlighting the functional divergence of leaf cell types in response to WD stress. By comparing our MALDI-MSI metabolic data with whole leaf tissue gas chromatography-mass spectrometry (GC-MS)-based metabolic profile, we identified only a few metabolites including monosaccharides, hexose phosphates, and palmitic acid that showed a similar accumulation trend at both cell-type and whole leaf tissue levels. Overall, this work highlights the potential of the MSI approach to complement the whole tissue-based metabolomics techniques and provides a novel spatiotemporal understanding of plant metabolic responses to WD stress. This will help engineer specific metabolic pathways at a cellular level in strategic perennial trees like poplars to help withstand future aberrations in environmental conditions and to increase bioenergy sustainability.
59 BASIC BIOLOGICAL SCIENCES↗
Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization
Aliphatic carboxylic acids, aldehydes, and ketones play diverse roles in microbial adaptation to their microenvironment, from excretion as toxins to adaptive metabolites for membrane fluidity. However, the spatial distribution of these molecules throughout biofilms and how microbes in these environments exchange these molecules remain elusive for many of these bioactive species due to inefficient molecular imaging strategies. Herein, we apply on-tissue chemical derivatization (OTCD) using 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) on a co-culture of a soil bacterium (Bacillus subtilis NCIB 3610) and fungus (Fusarium sp. DS 682) grown on agar as our model system. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we spatially resolved more than 300 different metabolites containing carbonyl groups within this model system. Various spatial patterns are observable in these species, which indicate possible extracellular or intercellular processes of the metabolites and their up- or downregulation during microbial interaction. The unique chemistry of our approach allowed us to bring additional confidence in accurate carbonyl identification, especially when multiple isomeric candidates were possible, and this provided the ability to generate hypotheses about the potential role of some aliphatic carbonyls in this B. subtilis/Fusarium sp. interaction. The results shown here demonstrate the utility of 4-ABEBA-based OTCD MALDI-MSI in probing interkingdom interactions directly from microbial co-cultures, and these methods will enable future microbial interaction studies with expanded metabolic coverage.
4-APEBA↗
Spatial Proteomics toward Subcellular Resolution by Coupling Deep Ultraviolet Laser Ablation with Nanodroplet Sample Preparation
Not Available
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Expanded Coverage of Phytocompounds by Mass Spectrometry Imaging Using On-Tissue Chemical Derivatization by 4-APEBA
Probing the entirety of any species metabolome is an analytical grand challenge, especially at a cellular scale. Where spatial metabolomics, completed primarily by matrix-assisted laser desorption/ionization (MALDI), has limited molecular coverage for several reasons. To expand the scope of spatial metabolomics, we developed an on-tissue chemical derivatization (OTCD) workflow using 4-APEBA for confident identification of several dozen elusive phytocompounds, including several phytohormones, which have various roles within stress responses and cellular communication. Superiority of 4-APEBA is established in comparison to other derivatization agents with (1) broad specificity towards carbonyls, (2) low background, and (3) introduction of bromine isotopes, where the latter two facilitate confident bioinformatics. In conclusion, the outlined workflow trailblazes a path towards spatial hormonomics within plant samples, enhancing detection of carboxylates, aldehydes, ketones, and plausibly phenols.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
IsoMatchMS : Open-Source Software for Automated Annotation and Visualization of High Resolution MALDI-MS Spectra
Due to its speed, accuracy, and adaptability to various sample types, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has become a popular method to identify molecular isotope profiles from biological samples. Often MALDI-MS data do not include tandem MS fragmentation data, and thus the identification of compounds in samples requires external databases so that the accurate mass of detected signals can be matched to known molecular compounds. Most relevant MALDI-MS software tools developed to confirm compound identifications are focused on small molecules (e.g., metabolites, lipids) and cannot be easily adapted to protein data due to their more complex isotopic distributions. Here, we present an R package called IsoMatchMS for the automated annotation of MALDI-MS data for multiple datatypes: intact proteins, peptides, and glycans. This tool accepts already derived molecular formulas or, for proteomics applications, can derive molecular formulas from a list of input peptides or proteins including proteins with post-translational modifications. In conclusion, visualization of all matched isotopic profiles is provided in a highly accessible HTML format called a trelliscope display, which allows users to filter and sort by several parameters such as match scores and the number of peaks matched. IsoMatchMS simplifies the annotation and visualization of MALDI-MS data for downstream analyses.
47 OTHER INSTRUMENTATION↗
Experimental Assessment of Mammalian Lipidome Complexity Using Multimodal 21 T FTICR Mass Spectrometry Imaging
Herein, we assess the complementarity and complexity of data that can be detected within mammalian lipidome mass spectrometry imaging (MSI) via matrix-assisted laser desorption ionization (MALDI) and nanospray desorption electrospray ionization (nano-DESI). We do so by employing 21 T Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with absorption mode FT processing in both cases, allowing unmatched mass resolving power per unit time (≥613k at m/z 760, 1.536 s transients). While our results demonstrated that molecular coverage and dynamic range capabilities were greater in MALDI analysis, nano-DESI provided superior mass error, and all annotations for both modes had sub-ppm error. Taken together, these experiments highlight the coverage of 1676 lipids and serve as a functional guide for expected lipidome complexity within nano-DESI-MSI and MALDI-MSI. To further assess the lipidome complexity, mass splits (i.e., the difference in mass between neighboring peaks) within single pixels were collated across all pixels from each respective MSI experiment. The spatial localization of these mass splits was powerful in informing whether the observed mass splits were biological or artificial (e.g., matrix related). Mass splits down to 2.4 mDa were observed (i.e., sodium adduct ambiguity) in each experiment, and both modalities highlighted comparable degrees of lipidome complexity. Further, we highlight the persistence of certain mass splits (e.g., 8.9 mDa; double bond ambiguity) independent of ionization biases. Here, we also evaluate the need for ultrahigh mass resolving power for mass splits ≤4.6 mDa (potassium adduct ambiguity) at m/z > 1000, which may only be resolved by advanced FTICR-MS instrumentation.
21T-FTICR-MS↗
Initiation of fatty acid biosynthesis in Pseudomonas putida $\mathrm{KT2440}$
Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
09 BIOMASS FUELS↗
Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform
Conventional proteomics measures the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of significant changes in subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot precisely infer the abundance changes of intact proteins, which are presented as proteoforms. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantification directly on thin tissue sections. The spatial TDP platform consisted of a nanoPOTS (nanodroplet Processing in One pot for Trace Samples)-based sample preparation system and an LCM (laser capture microdissection)-based cell isolation system. We improved the nanoPOTS sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ~200 cultured cells as model samples, the improved approach increased proteoform identifications from 493 to 700; newly identified proteoforms primarily corresponded to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed LCM-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 426 proteoforms by combining identifications from TopPIC and TDPortal with the quantitation from ProMex. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential PTM-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.
59 BASIC BIOLOGICAL SCIENCES↗
Proteomic Profiling of Intra-Islet Features Reveals Substructure-Specific Protein Signatures
Despite their diminutive size, islets of Langerhans play a large role in maintaining systemic energy balance in the body. New technologies have enabled us to go from studying the whole pancreas, to isolated whole islets, to partial islet sections, and now to islet substructures isolated from within the islet. Using a microfluidic nanodroplet-based proteomics platform coupled with laser capture microdissection (LCM), we present an in-depth investigation of protein profiles specific to regions within the islet. These regions studied include the vascular tissue containing interface boundary, micro-vascular tissue internal to the islet, isolated endocrine cells, islet sections with vasculature intact, and finally acinar tissue from around the islet. Unique protein signatures observed in the inner vasculature potentially indicate increased innervation and intra-islet neuron-like crosstalk compared to external vasculature. We also demonstrate the utility of these data for isolating localized structure-specific drug-target interactions using existing protein/drug binding databases.
59 BASIC BIOLOGICAL SCIENCES↗
Discovery top-down proteomics in symbiotic soybean root nodules
Proteomic methods have been widely used to study proteins in complex biological samples to understand biological molecular mechanisms. Most well-established methods (known as bottom-up proteomics, BUP) employ an enzymatic digestion step to cleave intact proteins into smaller peptides for liquid chromatography (LC) mass spectrometry (MS) detection. In contrast, top-down proteomics (TDP) directly characterizes intact proteins including all possible post-translational modifications (PTMs), thus offering unique insights into proteoform biology where combinations of individual PTMs may play important roles. We performed TDP on soybean root nodules infected by the symbiotic Bradyrhizobium japonicum in both the wildtype bacterium and a nifH- mutant, which lacks the ability to fix nitrogen in the soybean root nodule. TDP captured 1648 proteoforms derived from 313 bacterial genes and 178 soybean genes. Leghemoglobin, the most abundant protein in the sample, existed in many truncated proteoforms. Interestingly, these truncated proteoforms were considerably more abundant in the wildtype relative to the nifH- mutant, implicating protease activity as an important factor in nitrogen fixation. Proteoforms with various PTMs and combinations thereof were identified using an unrestricted open modification search. This included less common PTMs such as myristoylation, palmitoylation, cyanylation, and sulfation. In parallel, we collected high resolution MS imaging (MSI) data of intact proteins and biopolymers (<20 kDa due to current technical limitations) from sections of the soybean root nodules using matrix-assisted laser desorption/ionization (MALDI) coupled to high resolution Orbitrap. Several detected proteoforms exhibited unique spatial distributions inside the infection zone and cortex, suggesting functional compartmentalization in these regions. A subset of peaks from the MALDI-MSI were assigned to proteoforms detected in TDP LCMS data based on matching accurate masses. Many of the proteins detected in both LCMS and MALDI-MSI are currently uncharacterized in UniProt: the PTM and spatial information presented here will be valuable in understanding their biological functions. Taken together, our study demonstrates how untargeted TDP approach can provide unique insights into plant proteoform biology. On-going technology developments are expected to further improve TDP coverage for more comprehensive high-throughput analysis of proteoforms.
59 BASIC BIOLOGICAL SCIENCES↗
Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection
Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles within the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS) based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific Q-Exactive HF Orbitrap MS upgraded with ultra-high mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass measurements of proteoforms directly from tissue using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and a vastly improved duty cycle over other Fourier transform mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. Further, the use of MALDI-MS imaging (MSI) for proteoform mapping demonstrates several steps towards high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Novel metabolic interactions and environmental conditions mediate the boreal peatmoss-cyanobacteria mutualism
Abstract Interactions between Sphagnum (peat moss) and cyanobacteria play critical roles in terrestrial carbon and nitrogen cycling processes. Knowledge of the metabolites exchanged, the physiological processes involved, and the environmental conditions allowing the formation of symbiosis is important for a better understanding of the mechanisms underlying these interactions. In this study, we used a cross-feeding approach with spatially resolved metabolite profiling and metatranscriptomics to characterize the symbiosis between Sphagnum and Nostoc cyanobacteria. A pH gradient study revealed that the Sphagnum–Nostoc symbiosis was driven by pH, with mutualism occurring only at low pH. Metabolic cross-feeding studies along with spatially resolved matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) identified trehalose as the main carbohydrate source released by Sphagnum, which were depleted by Nostoc along with sulfur-containing choline-O-sulfate, taurine and sulfoacetate. In exchange, Nostoc increased exudation of purines and amino acids. Metatranscriptome analysis indicated that Sphagnum host defense was downregulated when in direct contact with the Nostoc symbiont, but not as a result of chemical contact alone. The observations in this study elucidated environmental, metabolic, and physiological underpinnings of the widespread plant–cyanobacterial symbioses with important implications for predicting carbon and nitrogen cycling in peatland ecosystems as well as the basis of general host-microbe interactions.
59 BASIC BIOLOGICAL SCIENCES↗
Toward Revealing Microcystin Distribution in Mouse Liver Tissue Using MALDI-MS Imaging
Cyanotoxins can be found in water and air during cyanobacterial harmful algal blooms (cHABs) in lakes and rivers. Therefore, it is very important to monitor their potential uptake by animals and humans as well as their health effects and distribution in affected organs. Herein, the distribution of hepatotoxic peptide microcystin-LR (MC-LR) is investigated in liver tissues of mice gavaged with this most common MC congener. Preliminary matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging experiments performed using a non-automated MALDI matrix deposition device and a MALDI-time-of-flight (TOF) mass spectrometer yielded ambiguous results in terms of MC-LR distribution in liver samples obtained from MC-LR-gavaged mice. The tissue preparation for MALDI-MS imaging was improved by using an automated sprayer for matrix deposition, and liver sections were imaged using an Nd:YAG MALDI laser coupled to a 15 Tesla Fourier-transform ion cyclotron resonance (FT-ICR)-mass spectrometer. MALDI-FT-ICR-MS imaging provided unambiguous detection of protonated MC-LR (calculated m/z 995.5560, z = +1) and the sodium adduct of MC-LR (m/z 1017.5380, z = +1) in liver sections from gavaged mice with great mass accuracy and ultra-high mass resolution. Since both covalently bound and free MC-LR can be found in liver of mice exposed to this toxin, the present results indicate that the distribution of free microcystins in tissue sections from affected organs, such as liver, can be monitored with high-resolution MALDI-MS imaging.
59 BASIC BIOLOGICAL SCIENCES↗