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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

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↗
DOE OSTI2023

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↗
DOE OSTI2023

193 nm Ultraviolet Photodissociation for the Characterization of Singly Charged Proteoforms Generated by MALDI

MALDI imaging allows for the near-cellular profiling of proteoforms directly from microbial, plant, and mammalian samples. Despite detecting hundreds of proteoforms, identification of unknowns with only intact mass information remains a distinct challenge, even with high mass resolving power and mass accuracy. To this end, many supplementary methods have been used to create experimental databases for accurate mass matching, including bulk or spatially resolved bottom-up and/or top-down proteomics. Herein, we describe the application of 193 nm ultraviolet photodissociation (UVPD) for fragmentation of quadrupole isolated singly charged ubiquitin (m/z 8565) by MALDI-UVPD on a UHMR HF Orbitrap. This platform permitted the high-resolution accurate mass measurement of not just terminal fragments but also large internal fragments. Finally, the outlined workflow demonstrates the feasibility of top-down analyses of isolated MALDI protein ions and the potential toward more comprehensive characterization of proteoforms in MALDI imaging applications.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Unimodal Imaging of Monovalent Metal-Chelator Complexes and Lipids by MALDI Imaging Mass Spectrometry

Careful regulation of monovalent metal ions (M + ) is necessary to maintain a functional cellular system. Of these ions, appropriate sodium (Na + ) and potassium (K + ) concentrations are particularly integral for electrochemical signaling, as well as the secondary transport of nutrients and waste. Dysregulation of M + homeostasis can disrupt these mechanisms, potentially influencing the metabolism of downstream biomolecules such as lipids. Thus, the relationship between M + abundances and related biomolecular distributions must be elucidated to better understand the physiology of healthy and disordered tissues. Traditional techniques for imaging biological metal distributions include SIMS, LA-ICP-MS, and XRF; however, these capabilities are limited to elemental analysis or the analysis of molecular fragments and must be paired with other modalities to visualize distributions of more complex biomolecules within the same or similar samples. Conversely, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful tool often used for mapping such biomolecular distributions, but current methods are unable to detect metals within tissue. This study illustrates a novel methodology that adds metal detection to the MALDI IMS repertoire through which the simultaneous detection of M + metals and lipids is achievable. Using a robotic sprayer for homogeneous application, on-tissue deposition of the chelator deferiprone (DEF) enables subsequent detection of the ionizable metal-chelator complex by MALDI without hindering lipid detection. Our work provides proof-of-concept data for the simultaneous detection of K + , Na + , and intact lipids using MALDI IMS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Enhanced Spatial Proteomics and Metabolomics from a Single Tissue Section Using MALDI-MSI and LCM-microPOTS Platforms

Spatially resolved mass spectrometry (MS)-based multi-omics workflows are becoming more utilized for revealing the complex biology that occurs within tissues. However, these approaches commonly require multiple independent tissue sections to analyze the metabolite and protein compositions of these samples. This poses a significant challenge in preserving cell- or region-specific molecular fidelity, as variations between tissue sections can compromise the accurate correlation of molecular data. Here, in this study, we developed workflows for comprehensive multi-omics profiling from a single tissue section (STS) using different MS modalities. We enhanced the functionality of an electrically insulated substrate by employing metal-assisted approaches that enabled both MS-based untargeted spatial metabolomics and proteomics from STS. This allowed metabolite imaging using matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI), without compromising it for subsequent proteome profiling with laser capture microdissection (LCM)-based technology. Specifically, implementing copper tape as a backing for polyethylene naphthalate (PEN) slides enabled the detection of >140 metabolites across a poplar root tissue section using MALDI-trapped ion mobility spectrometry time of flight (timsTOF)-MS. Afterwards, we detected 6,571 unique proteins from two distinct root regions by leveraging LCM technology coupled to our microdroplet based sample preparation approach. We also developed an alternative workflow utilizing gold-coated PEN substrates for imaging with MALDI-Fourier-transform ion cyclotron resonance (FTICR)-MS, which permitted the profiling of >170 metabolites and the identification of 6,542 unique proteins across a single poplar root tissue section. These results were comparable to using each assay independently without modifications. These approaches offer new opportunities for high-resolution molecular profiling of multiple omics-levels across biological tissues.

Veličković, Marija [Pacific Northwest National Lab↗
DOE OSTI2024

Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured Trichodesmium morphotypes

Trichodesmium, a globally significant N 2 -fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of Trichodesmium species that form distinct colony morphotypes and are inhabited by diverse microorganisms. Trichodesmium colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct Trichodesmium colonies collected from the Red Sea. We also compared these results with two morphotypes of the cultivable Trichodesmium strain IMS101. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) coupled with liquid extraction surface analysis (LESA) tandem mass spectrometry (MS2), we identified and localized a wide range of small metabolites associated with single-colony Trichodesmium morphotypes. Our untargeted MALDI-MSI approach revealed 80 unique features (metabolites) shared between Trichodesmium morphotypes. Discrimination analysis showed spatial variations in 57 shared metabolites, accounting for 62% of the observed variation between morphotypes. The greatest variations in metabolite abundance were observed between the cultured morphotypes compared to the natural colony morphotypes, suggesting substantial differences in metabolite production between the cultivable strain IMS101 and the naturally occurring colony morphotypes that the cultivable strain is meant to represent. This study highlights the variations in metabolite abundance between natural and cultured Trichodesmium morphotypes and provides valuable insights into metabolites common to morphologically distinct Trichodesmium colonies, offering a foundation for future targeted metabolomic investigations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Using MALDI-FTICR-MS Imaging to Track Low-Molecular-Weight Aromatic Derivatives of Fungal Decayed Wood

Low-molecular-weight (LMW) aromatics are crucial in meditating fungal processes for plant biomass decomposition. Some LMW compounds are employed as electron donors for oxidative degradation in brown rot (BR), an efficient wood-degrading strategy in fungi that selectively degrades carbohydrates but leaves modified lignins. Previous understandings of LMW aromatics were primarily based on “bulk extraction”, an approach that cannot fully reflect their real-time functions during BR. Here, we applied an optimized molecular imaging method that combines matrix-assisted laser desorption ionization (MALDI) with Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to directly measure the temporal profiles of BR aromatics as Rhodonia placenta decayed a wood wafer. We found that some phenolics were pre-existing in wood, while some (e.g., catechin-methyl ether and dihydroxy-dimethoxyflavan) were generated immediately after fungal activity. These pinpointed aromatics might be recruited to drive early BR oxidative mechanisms by generating Fenton reagents, Fe2+ and H2O2. As BR progressed, ligninolytic products were accumulated and then modified into various aromatic derivatives, confirming that R. placenta depolymerizes lignin. Together, this work confirms aromatic patterns that have been implicated in BR fungi, and it demonstrates the use of MALDI-FTICR-MS imaging as a new approach to monitor the temporal changes of LMW aromatics during wood degradation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Whole‐Cell MALDI‐ToF MS Coupled with Untargeted Metabolomics Facilitates Investigations of Microbial Chemical Interactions

Abstract The emergence of drug‐resistant pathogens necessitates the development of new countermeasures. In this regard, the introduction of probiotics to directly attack or competitively exclude pathogens presents a useful strategy. Application of this approach requires an understanding of how a probiotic and its target pathogen interact. A key means of probiotic‐pathogen interaction involves the production of small molecules called natural products (NPs). Here, we report the use of whole‐cell matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐ToF) mass spectrometry to characterize NP production by candidate probiotics (mouse airway microbiome isolates) when co‐cultured with the respiratory pathogen Burkholderia . We found that a Bacillus velezensis strain inhibits growth of and elicits NP production by Burkholderia thailandensis . Dereplication of known NPs detected in the metabolome of this B. velezensis strain suggests that a previously unannotated bioactive compound is involved. Thus, we present the use of whole‐cell MALDI as a broadly applicable method for screening the NP composition of microbial co‐cultures; this can be combined with other ‐omics methods to characterize probiotic‐pathogen and other microbe‐microbe interactions.

Aiosa, Nicole↗
DOE OSTI2022

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↗
DOE OSTI2024

Applying MALDI-TOF MS to resolve morphologic and genetic similarities between two Dermacentor tick species of public health importance

Abstract Hard ticks (Acari: Ixodidae) have been historically identified by morphological methods which require highly specialized expertise and more recently by DNA-based molecular assays that involve high costs. Although both approaches provide complementary data for tick identification, each method has limitations which restrict their use on large-scale settings such as regional or national tick surveillance programs. To overcome those obstacles, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced as a cost-efficient method for the identification of various organisms, as it balances performance, speed, and high data output. Here we describe the use of this technology to validate the distinction of two closely relatedDermacentortick species based on the development of the first nationwide MALDI-TOF MS reference database described to date. The dataset obtained from this protein-based approach confirms that tick specimens collected from United States regions west of the Rocky Mountains and identified previously asDermacentor variabilisare the recently described species,Dermacentor similis. Therefore, we propose that this integrative taxonomic tool can facilitate vector and vector-borne pathogen surveillance programs in the United States and elsewhere.

Science & Technology - Other Topics↗
DOE OSTI2024

Characterization of Humanized Mouse Model of Organophosphate Poisoning and Detection of Countermeasures via MALDI-MSI

Organophosphoate (OP) chemicals are known to inhibit the enzyme acetylcholinesterase (AChE). Studying OP poisoning is difficult because common small animal research models have serum carboxylesterase, which contributes to animals’ resistance to OP poisoning. Historically, guinea pigs have been used for this research; however, a novel genetically modified mouse strain (KIKO) was developed with nonfunctional serum carboxylase (Es1 KO) and an altered acetylcholinesterase (AChE) gene, which expresses the amino acid sequence of the human form of the same protein (AChE KI). KIKO mice were injected with 1xLD50 of an OP nerve agent or vehicle control with or without atropine. After one to three minutes, animals were injected with 35 mg/kg of the currently fielded Reactivator countermeasure for OP poisoning. Postmortem brains were imaged on a Bruker RapifleX ToF/ToF instrument. Data confirmed the presence of increased acetylcholine in OP-exposed animals, regardless of treatment or atropine status. More interestingly, we detected a small amount of Reactivator within the brain of both exposed and unexposed animals; it is currently debated if reactivators can cross the blood–brain barrier. Further, we were able to simultaneously image acetylcholine, the primary affected neurotransmitter, as well as determine the location of both Reactivator and acetylcholine in the brain. This study, which utilized sensitive MALDI-MSI methods, characterized KIKO mice as a functional model for OP countermeasure development.

2-PAM↗
DOE OSTI2024

Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Elucidating Drought-Tolerance Mechanisms in Plant Roots through 1H NMR Metabolomics in Parallel with MALDI-MS, and NanoSIMS Imaging Techniques

As direct mediators between plants and soil, roots play an important role in metabolic responses to environmental stresses such as drought, yet these responses are vastly uncharacterized on a plant-specific level, especially for co-occurring species. Here, we aim to examine the effects of drought on root metabolic profiles and carbon allocation pathways of three tropical rainforest species by combining cutting-edge metabolomic and imaging technologies in an in situ position-specific 13C-pyruvate root-labeling experiment. Further, washed (rhizosphere-depleted) and unwashed roots were examined to test the impact of microbial presence on root metabolic pathways. Drought had a species-specific impact on the metabolic profiles and spatial distribution in Piper sp. and Hibiscus rosa sinensis roots, signifying different defense mechanisms; Piper sp. enhanced root structural defense via recalcitrant compounds including lignin, while H. rosa sinensis enhanced biochemical defense via secretion of antioxidants and fatty acids. In contrast, Clitoria fairchildiana, a legume tree, was not influenced as much by drought but rather by rhizosphere presence where carbohydrate storage was enhanced, indicating a close association with symbiotic microbes. This study demonstrates how multiple techniques can be combined to identify how plants cope with drought through different drought-tolerance strategies and the consequences of such changes on below-ground organic matter composition.

Drought, Carbon cycle, MALDI-MSI, Metabolomics, Na↗
DOE OSTI2023

MALDI-MS screening of microbial colonies with isomer resolution to select fatty acid desaturase variants

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid MS assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Lastly, we used the fabA deficient chassis to concomitantly express a non-native acyl- ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Data for MALDI-MS Screening of Microbial Colonies With Isomer Resolution to Select Fatty Acid Desaturase Variants

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid mass spectrometry (MS) assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Finally, we used the fabA deficient chassis to concomitantly express a non-native acyl-ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

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