Publications by authors named "Christopher R Anderton"

40 Publications

Optical Microscopy-Guided Laser Ablation Electrospray Ionization Ion Mobility Mass Spectrometry: Ambient Single Cell Metabolomics with Increased Confidence in Molecular Identification.

Metabolites 2021 Mar 27;11(4). Epub 2021 Mar 27.

Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352, USA.

Single cell analysis is a field of increasing interest as new tools are continually being developed to understand intercellular differences within large cell populations. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is an emerging technique for single cell metabolomics. Over the years, it has been validated that this ionization technique is advantageous for probing the molecular content of individual cells in situ. Here, we report the integration of a microscope into the optical train of the LAESI source to allow for visually informed ambient in situ single cell analysis. Additionally, we have coupled this 'LAESI microscope' to a drift-tube ion mobility mass spectrometer to enable separation of isobaric species and allow for the determination of ion collision cross sections in conjunction with accurate mass measurements. This combined information helps provide higher confidence for structural assignment of molecules ablated from single cells. Here, we show that this system enables the analysis of the metabolite content of epidermal cells with high confidence structural identification together with their spatial locations within a tissue.
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http://dx.doi.org/10.3390/metabo11040200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065410PMC
March 2021

Spatially Resolved Mass Spectrometry at the Single Cell: Recent Innovations in Proteomics and Metabolomics.

J Am Soc Mass Spectrom 2021 Apr 3;32(4):872-894. Epub 2021 Mar 3.

Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, United States.

Biological systems are composed of heterogeneous populations of cells that intercommunicate to form a functional living tissue. Biological function varies greatly across populations of cells, as each single cell has a unique transcriptome, proteome, and metabolome that translates to functional differences within single species and across kingdoms. Over the past decade, substantial advancements in our ability to characterize omic profiles on a single cell level have occurred, including in multiple spectroscopic and mass spectrometry (MS)-based techniques. Of these technologies, spatially resolved mass spectrometry approaches, including mass spectrometry imaging (MSI), have shown the most progress for single cell proteomics and metabolomics. For example, reporter-based methods using heavy metal tags have allowed for targeted MS investigation of the proteome at the subcellular level, and development of technologies such as laser ablation electrospray ionization mass spectrometry (LAESI-MS) now mean that dynamic metabolomics can be performed in situ. In this Perspective, we showcase advancements in single cell spatial metabolomics and proteomics over the past decade and highlight important aspects related to high-throughput screening, data analysis, and more which are vital to the success of achieving proteomic and metabolomic profiling at the single cell scale. Finally, using this broad literature summary, we provide a perspective on how the next decade may unfold in the area of single cell MS-based proteomics and metabolomics.
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http://dx.doi.org/10.1021/jasms.0c00439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033567PMC
April 2021

Draft Genome Sequence of sp. Strain DS 682, a Novel Fungal Isolate from the Grass Rhizosphere.

Microbiol Resour Announc 2021 Jan 7;10(1). Epub 2021 Jan 7.

Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington, USA

The novel fungal strain, sp. strain DS 682, was isolated from the rhizosphere of the perennial grass, , at the Konza Prairie Biological Station in Kansas. This fungal strain is common across North American grasslands and is resilient to environmental fluctuations. The draft genome is estimated to be 97.2% complete.
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http://dx.doi.org/10.1128/MRA.00884-20DOI Listing
January 2021

A multimodal and integrated approach to interrogate human kidney biopsies with rigor and reproducibility: guidelines from the Kidney Precision Medicine Project.

Physiol Genomics 2021 01 16;53(1):1-11. Epub 2020 Nov 16.

Washington University in Saint Louis School of Medicine, St. Louis, Missouri.

Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and personalized therapies for diseases. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate three-dimensional (3-D) molecular atlases of healthy and diseased kidney biopsies by using multiple state-of-the-art omics and imaging technologies across several institutions. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single-cell level or in 3-D space is a significant challenge that can be a futile exercise if not well controlled. We describe a "follow the tissue" pipeline for generating a reliable and authentic single-cell/region 3-D molecular atlas of human adult kidney. Our approach emphasizes quality assurance, quality control, validation, and harmonization across different omics and imaging technologies from sample procurement, processing, storage, shipping to data generation, analysis, and sharing. We established benchmarks for quality control, rigor, reproducibility, and feasibility across multiple technologies through a pilot experiment using common source tissue that was processed and analyzed at different institutions and different technologies. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before their being approved to interrogate clinical biopsy specimens. The process established economizes the use of valuable biopsy tissue for multiomics and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and serves as a model for precision medicine projects across laboratories, institutions and consortia.
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http://dx.doi.org/10.1152/physiolgenomics.00104.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847045PMC
January 2021

Activity-Based Protein Profiling of Chitin Catabolism.

Chembiochem 2021 Feb 17;22(4):717-723. Epub 2020 Nov 17.

The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, 1505 NE Stadium Way, Pullman, WA 99164, USA.

The microbial catabolism of chitin, an abundant and ubiquitous environmental organic polymer, is a fundamental cog in terrestrial and aquatic carbon and nitrogen cycles. Despite the importance of this critical bio-geochemical function, there is a limited understanding of the synergy between the various hydrolytic and accessory enzymes involved in chitin catabolism. To address this deficit, we synthesized activity-based probes (ABPs) designed to target active chitinolytic enzymes by modifying the chitin subunits N-acetyl glucosamine and chitotriose. The ABPs were used to determine the active complement of chitinolytic enzymes produced over time by the soil bacterium Cellvibrio japonicus treated with various C substrates. We demonstrate the utility of these ABPs in determining the synergy between various enzymes involved in chitin catabolism. The strategy can be used to gain molecular-level insights that can be used to better understand microbial roles in soil bio-geochemical cycling in the face of a changing climate.
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http://dx.doi.org/10.1002/cbic.202000616DOI Listing
February 2021

Modelling kidney disease using ontology: insights from the Kidney Precision Medicine Project.

Nat Rev Nephrol 2020 11 16;16(11):686-696. Epub 2020 Sep 16.

Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, MI, USA.

An important need exists to better understand and stratify kidney disease according to its underlying pathophysiology in order to develop more precise and effective therapeutic agents. National collaborative efforts such as the Kidney Precision Medicine Project are working towards this goal through the collection and integration of large, disparate clinical, biological and imaging data from patients with kidney disease. Ontologies are powerful tools that facilitate these efforts by enabling researchers to organize and make sense of different data elements and the relationships between them. Ontologies are critical to support the types of big data analysis necessary for kidney precision medicine, where heterogeneous clinical, imaging and biopsy data from diverse sources must be combined to define a patient's phenotype. The development of two new ontologies - the Kidney Tissue Atlas Ontology and the Ontology of Precision Medicine and Investigation - will support the creation of the Kidney Tissue Atlas, which aims to provide a comprehensive molecular, cellular and anatomical map of the kidney. These ontologies will improve the annotation of kidney-relevant data, and eventually lead to new definitions of kidney disease in support of precision medicine.
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http://dx.doi.org/10.1038/s41581-020-00335-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8012202PMC
November 2020

Storage Conditions of Human Kidney Tissue Sections Affect Spatial Lipidomics Analysis Reproducibility.

J Am Soc Mass Spectrom 2020 Dec 25;31(12):2538-2546. Epub 2020 Sep 25.

Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, Washington99352, United States.

Lipids often are labile, unstable, and tend to degrade overtime, so it is of the upmost importance to study these molecules in their most native state. We sought to understand the optimal storage conditions for spatial lipidomic analysis of human kidney tissue sections. Specifically, we evaluated human kidney tissue sections on several different days throughout the span of a week using our established protocol for elucidating lipids using high mass resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We studied kidney tissue sections stored under five different conditions: open stored at -80 °C, vacuumed sealed and stored at -80 °C, with matrix preapplied before storage at -80 °C, under a nitrogen atmosphere and stored at -80 °C, and at room temperature in a desiccator. Results were compared to data obtained from kidney tissue sections that were prepared and analyzed immediately after cryosectioning. Data was processed using METASPACE. After a week of storage, the sections stored at room temperature showed the largest amount of lipid degradation, while sections stored under nitrogen and at -80 °C retained the greatest number of overlapping annotations in relation to freshly cut tissue. Overall, we found that molecular degradation of the tissue sections was unavoidable over time, regardless of storage conditions, but storing tissue sections in an inert gas at low temperatures can curtail molecular degradation within tissue sections.
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http://dx.doi.org/10.1021/jasms.0c00256DOI Listing
December 2020

Deep learning predicts microbial interactions from self-organized spatiotemporal patterns.

Comput Struct Biotechnol J 2020 29;18:1259-1269. Epub 2020 May 29.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.

Microbial communities organize into spatial patterns that are largely governed by interspecies interactions. This phenomenon is an important metric for understanding community functional dynamics, yet the use of spatial patterns for predicting microbial interactions is currently lacking. Here we propose supervised deep learning as a new tool for network inference. An agent-based model was used to simulate the spatiotemporal evolution of two interacting organisms under diverse growth and interaction scenarios, the data of which was subsequently used to train deep neural networks. For small-size domains (100 µm × 100 µm) over which interaction coefficients are assumed to be invariant, we obtained fairly accurate predictions, as indicated by an average R value of 0.84. In application to relatively larger domains (450 µm × 450 µm) where interaction coefficients are varying in space, deep learning models correctly predicted spatial distributions of interaction coefficients without any additional training. Lastly, we evaluated our model against real biological data obtained using and co-cultures treated with polymeric chitin or N-acetylglucosamine, the hydrolysis product of chitin. While can utilize both substrates for growth, lacked the ability to degrade chitin. Consistent with our expectations, our model predicted context-dependent interactions across two substrates, i.e., degrader-cheater relationship on chitin polymers and competition on monomers. The combined use of the agent-based model and machine learning algorithm successfully demonstrates how to infer microbial interactions from spatially distributed data, presenting itself as a useful tool for the analysis of more complex microbial community interactions.
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http://dx.doi.org/10.1016/j.csbj.2020.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298420PMC
May 2020

Nitrogen Source Governs Community Carbon Metabolism in a Model Hypersaline Benthic Phototrophic Biofilm.

mSystems 2020 Jun 9;5(3). Epub 2020 Jun 9.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA

Increasing anthropogenic inputs of fixed nitrogen are leading to greater eutrophication of aquatic environments, but it is unclear how this impacts the flux and fate of carbon in lacustrine and riverine systems. Here, we present evidence that the form of nitrogen governs the partitioning of carbon among members in a genome-sequenced, model phototrophic biofilm of 20 members. Consumption of NO as the sole nitrogen source unexpectedly resulted in more rapid transfer of carbon to heterotrophs than when NH was also provided, suggesting alterations in the form of carbon exchanged. The form of nitrogen dramatically impacted net community nitrogen, but not carbon, uptake rates. Furthermore, this alteration in nitrogen form caused very large but focused alterations to community structure, strongly impacting the abundance of only two species within the biofilm and modestly impacting a third member species. Our data suggest that nitrogen metabolism may coordinate coupled carbon-nitrogen biogeochemical cycling in benthic biofilms and, potentially, in phototroph-heterotroph consortia more broadly. It further indicates that the form of nitrogen inputs may significantly impact the contribution of these communities to carbon partitioning across the terrestrial-aquatic interface. Anthropogenic inputs of nitrogen into aquatic ecosystems, and especially those of agricultural origin, involve a mix of chemical species. Although it is well-known in general that nitrogen eutrophication markedly influences the metabolism of aquatic phototrophic communities, relatively little is known regarding whether the specific chemical form of nitrogen inputs matter. Our data suggest that the nitrogen form alters the rate of nitrogen uptake significantly, whereas corresponding alterations in carbon uptake were minor. However, differences imposed by uptake of divergent nitrogen forms may result in alterations among phototroph-heterotroph interactions that rewire community metabolism. Furthermore, our data hint that availability of other nutrients (i.e., iron) might mediate the linkage between carbon and nitrogen cycling in these communities. Taken together, our data suggest that different nitrogen forms should be examined for divergent impacts on phototrophic communities in fluvial systems and that these anthropogenic nitrogen inputs may significantly differ in their ultimate biogeochemical impacts.
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http://dx.doi.org/10.1128/mSystems.00260-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289588PMC
June 2020

Sequential Ammonia and Carbon Dioxide Adsorption on Pyrolyzed Biomass to Recover Waste Stream Nutrients.

ACS Sustain Chem Eng 2020 May 15;8(18):7121-7131. Epub 2020 Apr 15.

Soil and Crop Sciences, College of Agriculture and Life Sciences, Cornell University, 306 Tower Road, Ithaca, New York 14853, United States.

The amine-rich surfaces of pyrolyzed human solid waste (py-HSW) can be "primed" or "regenerated" with carbon dioxide (CO) to enhance their adsorption of ammonia (NH) for use as a soil amendment. To better understand the mechanism by which CO exposure facilitates NH adsorption to py-HSW, we artificially enriched a model sorbent, pyrolyzed, oxidized wood (py-ox wood) with amine functional groups through exposure to NH. We then exposed these N-enriched materials to CO and then resorbed NH. The high heat of CO adsorption () on py-HSW, 49 kJ mol, at low surface coverage, 0.4 mmol CO g, showed that the naturally occurring N compounds in py-HSW have a high affinity for CO. The of CO on py-ox wood also increased after exposure to NH, reaching 50 kJ mol at 0.7 mmol CO g, demonstrating that the incorporation of N-rich functional groups by NH adsorption is favorable for CO uptake. Adsorption kinetics of py-ox wood revealed continued, albeit diminishing NH uptake after each CO treatment, averaging 5.9 mmol NH g for the first NH exposure event and 3.5 and 2.9 mmol NH g for the second and third; the electrophilic character of CO serves as a Lewis acid, enhancing surface affinity for NH uptake. Furthermore, penetration of NH and CO measured by NanoSIMS reached over 7 μm deep into both materials, explaining the large NH capture. We expected similar NH uptake in py-HSW sorbed with CO and py-ox wood because both materials, py-HSW and py-ox wood sorbed with NH, had similar N contents and similarly high CO uptake. Yet NH sorption in py-HSW was unexpectedly low, apparently from potassium (K) bicarbonate precipitation, reducing interactions between NH and sorbed CO; 2-fold greater surface K in py-HSW was detected after exposure to CO and NH than before gas exposure. We show that amine-rich pyrolyzed waste materials have high CO affinity, which facilitates NH uptake. However, high ash contents as found in py-HSW hinder this mechanism.
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http://dx.doi.org/10.1021/acssuschemeng.0c01427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218926PMC
May 2020

Metabolomic profiling of wild-type and mutant soybean root nodules using laser-ablation electrospray ionization mass spectrometry reveals altered metabolism.

Plant J 2020 08 21;103(5):1937-1958. Epub 2020 Jun 21.

Divisions of Plant Sciences and Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO, 65211, USA.

The establishment of the nitrogen-fixing symbiosis between soybean and Bradyrhizobium japonicum is a complex process. To document the changes in plant metabolism as a result of symbiosis, we utilized laser ablation electrospray ionization-mass spectrometry (LAESI-MS) for in situ metabolic profiling of wild-type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl-acyl carrier protein desaturase (sacpd-c) gene, which were previously shown to have an altered nodule ultrastructure. The results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid, correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix ) and ineffective (nifH mutant, fix ) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd-c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. The present study demonstrates the utility of LAESI-MS for high-throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.
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http://dx.doi.org/10.1111/tpj.14815DOI Listing
August 2020

Single-Cell Metabolic Profiling: Metabolite Formulas from Isotopic Fine Structures in Heterogeneous Plant Cell Populations.

Anal Chem 2020 05 4;92(10):7289-7298. Epub 2020 May 4.

Department of Chemistry, George Washington University, Washington D.C. 20052, United States.

Characterization of the metabolic heterogeneity in cell populations requires the analysis of single cells. Most current methods in single-cell analysis rely on cell manipulation, potentially altering the abundance of metabolites in individual cells. A small sample volume and the chemical diversity of metabolites are additional challenges in single-cell metabolomics. Here, we describe the combination of fiber-based laser ablation electrospray ionization (f-LAESI) with 21 T Fourier transform ion cyclotron resonance mass spectrometry (21TFTICR-MS) for single-cell metabolic profiling in plant tissue. Single plant cells infected by bacteria were selected and sampled directly from the tissue without cell manipulation through mid-infrared ablation with a fine optical fiber tip for ionization by f-LAESI. Ultrahigh performance 21T-FTICR-MS enabled the simultaneous capture of isotopic fine structures (IFSs) for 47 known and 11 unknown compounds, thus elucidating their elemental compositions from single cells and providing information on metabolic heterogeneity in the cell population.
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http://dx.doi.org/10.1021/acs.analchem.0c00936DOI Listing
May 2020

Response Surface Methodology As a New Approach for Finding Optimal MALDI Matrix Spraying Parameters for Mass Spectrometry Imaging.

J Am Soc Mass Spectrom 2020 Mar 6;31(3):508-516. Epub 2020 Feb 6.

Kidney Precision Medicine Project (KPMP), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, United States.

Automated spraying devices have become ubiquitous in laboratories employing matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), in part because they permit control of a number of matrix application parameters that can easily be reproduced for intra- and interlaboratory studies. Determining the optimal parameters for MALDI matrix application, such as temperature, flow rate, spraying velocity, number of spraying cycles, and solvent composition for matrix application, is critical for obtaining high-quality MALDI-MSI data. However, there are no established approaches for optimizing these multiple parameters simultaneously. Instead optimization is performed iteratively (i.e., one parameter at a time), which is time-consuming and can lead to overall nonoptimal settings. In this report, we demonstrate the use a novel experimental design and the response surface methodology to optimize five parameters of MALDI matrix application using a robotic sprayer. Thirty-two combinations of MALDI matrix spraying conditions were tested, which allowed us to elucidate relationships between each of the application parameters as determined by MALDI-MS (specifically, using a 15 T Fourier transform ion cyclotron resonance mass spectrometer). As such, we were able to determine the optimal automated spraying parameters that minimized signal delocalization and enabled high MALDI sensitivity. We envision this optimization strategy can be utilized for other matrix application approaches and MALDI-MSI analyses of other molecular classes and tissue types.
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http://dx.doi.org/10.1021/jasms.9b00074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293970PMC
March 2020

Visualizing Microbial Community Dynamics via a Controllable Soil Environment.

mSystems 2020 Feb 11;5(1). Epub 2020 Feb 11.

Environmental Molecular Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA

Understanding the basic biology that underpins soil microbiome interactions is required to predict the metaphenomic response to environmental shifts. A significant knowledge gap remains in how such changes affect microbial community dynamics and their metabolic landscape at microbially relevant spatial scales. Using a custom-built SoilBox system, here we demonstrated changes in microbial community growth and composition in different soil environments (14%, 24%, and 34% soil moisture), contingent upon access to reservoirs of nutrient sources. The SoilBox emulates the probing depth of a common soil core and enables determination of both the spatial organization of the microbial communities and their metabolites, as shown by confocal microscopy in combination with mass spectrometry imaging (MSI). Using chitin as a nutrient source, we used the SoilBox system to observe increased adhesion of microbial biomass on chitin islands resulting in degradation of chitin into -acetylglucosamine (NAG) and chitobiose. With matrix-assisted laser desorption/ionization (MALDI)-MSI, we also observed several phospholipid families that are functional biomarkers for microbial growth on the chitin islands. Fungal hyphal networks bridging different chitin islands over distances of 27 mm were observed only in the 14% soil moisture regime, indicating that such bridges may act as nutrient highways under drought conditions. In total, these results illustrate a system that can provide unprecedented spatial information about interactions within soil microbial communities as a function of changing environments. We anticipate that this platform will be invaluable in spatially probing specific intra- and interkingdom functional relationships of microbiomes within soil. Microbial communities are key components of the soil ecosystem. Recent advances in metagenomics and other omics capabilities have expanded our ability to characterize the composition and function of the soil microbiome. However, characterizing the spatial metabolic and morphological diversity of microbial communities remains a challenge due to the dynamic and complex nature of soil microenvironments. The SoilBox system, demonstrated in this work, simulates an ∼12-cm soil depth, similar to a typical soil core, and provides a platform that facilitates imaging the molecular and topographical landscape of soil microbial communities as a function of environmental gradients. Moreover, the nondestructive harvesting of soil microbial communities for the imaging experiments can enable simultaneous multiomics analysis throughout the depth of the SoilBox. Our results show that by correlating molecular and optical imaging data obtained using the SoilBox platform, deeper insights into the nature of specific soil microbial interactions can be achieved.
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http://dx.doi.org/10.1128/mSystems.00645-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018529PMC
February 2020

DESI-MSI and METASPACE indicates lipid abnormalities and altered mitochondrial membrane components in diabetic renal proximal tubules.

Metabolomics 2020 01 10;16(1):11. Epub 2020 Jan 10.

Center for Renal Precision Medicine, Division of Nephrology, Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.

Introduction: Diabetic kidney disease (DKD) is the most prevalent complication in diabetic patients, which contributes to high morbidity and mortality. Urine and plasma metabolomics studies have been demonstrated to provide valuable insights for DKD. However, limited information on spatial distributions of metabolites in kidney tissues have been reported.

Objectives: In this work, we employed an ambient desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) coupled to a novel bioinformatics platform (METASPACE) to characterize the metabolome in a mouse model of DKD.

Methods: DESI-MSI was performed for spatial untargeted metabolomics analysis in kidneys of mouse models (F1 C57BL/6J-Ins2Akita male mice at 17 weeks of age) of type 1 diabetes (T1D, n = 5) and heathy controls (n = 6).

Results: Multivariate analyses (i.e., PCA and PLS-DA (a 2000 permutation test: P < 0.001)) showed clearly separated clusters for the two groups of mice on the basis of 878 measured m/z's in kidney cortical tissues. Specifically, mice with T1D had increased relative abundances of pseudouridine, accumulation of free polyunsaturated fatty acids (PUFAs), and decreased relative abundances of cardiolipins in cortical proximal tubules when compared with healthy controls.

Conclusion: Results from the current study support potential key roles of pseudouridine and cardiolipins for maintaining normal RNA structure and normal mitochondrial function, respectively, in cortical proximal tubules with DKD. DESI-MSI technology coupled with METASPACE could serve as powerful new tools to provide insight on fundamental pathways in DKD.
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http://dx.doi.org/10.1007/s11306-020-1637-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301343PMC
January 2020

In-Situ Metabolomic Analysis of Roots Colonized by Beneficial Endophytic Bacteria.

Mol Plant Microbe Interact 2020 Feb 6;33(2):272-283. Epub 2019 Dec 6.

Divisions of Plant Sciences and Biochemistry, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, U.S.A.

Over the past decades, crop yields have risen in parallel with increasing use of fossil fuel-derived nitrogen (N) fertilizers but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic, and endophytic plant growth-promoting bacteria (PGPB) are known to stimulate plant growth. However, compared with the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species upon endophytic colonization by SmR1 (fix) or a fix mutant strain (SmR54) compared with uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole-root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in-situ laser ablation electrospray ionization mass spectrometry to sample only those root segments at or adjacent to the sites of bacterial colonization. Metabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants, while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix strain, perhaps reflecting a plant defense response.
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http://dx.doi.org/10.1094/MPMI-06-19-0174-RDOI Listing
February 2020

An approach for visualizing the spatial metabolome of an entire plant root system inspired by the Swiss-rolling technique.

J Mass Spectrom 2020 Apr 14;55(4):e4363. Epub 2019 May 14.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington.

The spatial configuration and morphology of roots are commonly monitored for a better understanding of plant health and development. However, this approach provides minimal details about the biochemistry regulating the observable traits. Therefore, the ability to metabolically map the entire root structure would be of major value. Here, we developed a sample preparation approach that enables imaging of the entire root within a restricted space (width of microscope slide), which was influenced by the Swiss-rolling technique. We were able to image and confidently identify molecules along the entire root structure from rolled-root tissue sections using multiple spatially resolved mass spectrometry approaches.
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http://dx.doi.org/10.1002/jms.4363DOI Listing
April 2020

Spatiotemporal Transformation in the Alkaloid Profile of Pinus Roots in Response to Mycorrhization.

J Nat Prod 2019 05 22;82(5):1382-1386. Epub 2019 Apr 22.

Environmental Molecular Sciences Laboratory, Earth and Biological Sciences Directorate , Pacific Northwest National Laboratory , 902 Battelle Boulevard , Richland , Washington 99354 , United States.

Root alkaloids remain highly unexplored in ectomycorrhizae development studies. By employing ultrahigh mass resolution mass spectrometry imaging techniques, we showed substantial relocation and transformation of piperidine alkaloids in pine root tips in response to Suillus mycorrhization. We imaged, in the time frame of ectomycorrhizae formation, a completely different alkaloid profile in Pinus strobus, where basidiospores of Suillus spraguei induce morphogenesis of symbiotic tissues, than in Pinus taeda, where such interaction fails to induce morphogenesis. On the basis of spatial colocalization studies, we proposed some alternative routes for biosynthesis of these alkaloids that supplement existing literature data.
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http://dx.doi.org/10.1021/acs.jnatprod.8b01050DOI Listing
May 2019

Ambient Metabolic Profiling and Imaging of Biological Samples with Ultrahigh Molecular Resolution Using Laser Ablation Electrospray Ionization 21 Tesla FTICR Mass Spectrometry.

Anal Chem 2019 04 12;91(8):5028-5035. Epub 2019 Mar 12.

Department of Chemistry , The George Washington University , Washington , D.C. 20052 , United States.

Mass spectrometry (MS) is an indispensable analytical tool to capture the array of metabolites within complex biological systems. However, conventional MS-based metabolomic workflows require extensive sample processing and separation resulting in limited throughput and potential alteration of the native molecular states in these systems. Ambient ionization methods, capable of sampling directly from tissues, circumvent some of these issues but require high-performance MS to resolve the molecular complexity within these samples. Here, we demonstrate a unique combination of laser ablation electrospray ionization (LAESI) coupled with a 21 tesla Fourier transform ion cyclotron resonance (21T-FTICR) for direct MS analysis and imaging applications. This analytical platform provides isotopic fine structure information directly from biological tissues, enabling the rapid assignment of molecular formulas and delivering a higher degree of confidence for molecular identification.
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http://dx.doi.org/10.1021/acs.analchem.8b05084DOI Listing
April 2019

Insights into the histology of planarian flatworm Phagocata gracilis based on location specific, intact lipid information provided by GCIB-ToF-SIMS imaging.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 05 4;1864(5):733-743. Epub 2019 Feb 4.

NESACBIO, University of Washington, Seattle, WA, United States of America; Department of Bioengineering, University of Washington, Seattle, WA, United States of America. Electronic address:

Planarian flatworms are known as the masters of regeneration, re-growing an entire organism from as little as 1/279th part of their body. While the proteomics of these processes has been studied extensively, the planarian lipodome remains relatively unknown. In this study we investigate the lipid profile of planarian tissue sections with imaging Time-of-Flight - Secondary-Ion-Mass-Spectrometry (ToF-SIMS). ToF-SIMS is a label-free technique capable of gathering intact, location specific lipid information on a cellular scale. Lipid identities are confirmed using LC-MS/MS. Our data shows that different organ structures within planarians have unique lipid profiles. The 22-carbon atom poly unsaturated fatty acids (PUFAs) which occur in unusually high amounts in planarians are found to be mainly located in the testes. Additionally, we observe that planarians contain various odd numbered fatty acid species, that are usually found in bacteria, localized in the reproductive and ectodermal structures of the planarian. An abundance of poorly understood ether fatty acids and ether lipids were found in unique areas in planarians as well as a new, yet unidentified class of potential lipids in planarian intestines. Identifying the location of these lipids in the planarian body provides insights into their bodily functions and, in combination with knowledge about their diet and their genome, enables drawing conclusions about planarian fatty acid processing.
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http://dx.doi.org/10.1016/j.bbalip.2019.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506226PMC
May 2019

Towards resolving the spatial metabolome with unambiguous molecular annotations in complex biological systems by coupling mass spectrometry imaging with structures for lossless ion manipulations.

Chem Commun (Camb) 2019 Jan;55(3):306-309

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA.

We demonstrate the coupling of liquid extraction surface analysis (LESA) to structures for lossless ion manipulations in conjunction with serpentine ultralong path with extending routing (SLIM SUPER) ion mobility-mass spectrometry (IM-MS) for the unambiguous annotation of important isomeric glycoforms in carbon-fixing communities.
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http://dx.doi.org/10.1039/c8cc07482hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6537888PMC
January 2019

Metabolic Noise and Distinct Subpopulations Observed by Single Cell LAESI Mass Spectrometry of Plant Cells .

Front Plant Sci 2018 15;9:1646. Epub 2018 Nov 15.

Department of Chemistry, The George Washington University, Washington, DC, United States.

Phenotypic variations and stochastic expression of transcripts, proteins, and metabolites in biological tissues lead to cellular heterogeneity. As a result, distinct cellular subpopulations emerge. They are characterized by different metabolite expression levels and by associated metabolic noise distributions. To capture these biological variations unperturbed, highly sensitive analytical techniques are needed that can sample tissue embedded single cells with minimum sample preparation. Optical fiber-based laser ablation electrospray ionization mass spectrometry (f-LAESI-MS) is a promising tool for metabolic profiling of single cells under ambient conditions. Integration of this MS-based platform with fluorescence and brightfield microscopy provides the ability to target single cells of specific type and allows for the selection of rare cells, e.g., excretory idioblasts. Analysis of individual leaf blade cells ( = 103) by f-LAESI-MS revealed significant differences between the prespecified subpopulations of epidermal cells ( = 97) and excretory idioblasts ( = 6) that otherwise would have been masked by the population average. Primary metabolites, e.g., malate, aspartate, and ascorbate, as well as several glucosides were detected in higher abundance in the epidermal cells. The idioblasts contained lipids, e.g., PG(16:0/18:2), and triterpene saponins, e.g., medicoside I and azukisaponin I, and their isomers. Metabolic noise for the epidermal cells were compared to results for soybean () root nodule cells ( = 60) infected by rhizobia (). Whereas some primary metabolites showed lower noise in the latter, both cell types exhibited higher noise for secondary metabolites. grouping of epidermal and root nodule cells, based on the abundance distributions for certain metabolites (e.g., malate), enabled the discovery of cellular subpopulations characterized by different mean abundance values, and the magnitudes of the corresponding metabolic noise. Comparison of prespecified populations from epidermal cells of the closely related ( = 20) and ( = 20) revealed significant differences, e.g., higher sugar content in the former and higher levels of ascorbate in the latter, and the presence of species-specific metabolites. These results demonstrate that the f-LAESI-MS single cell analysis platform has the potential to explore cellular heterogeneity and metabolic noise for hundreds of tissue-embedded cells.
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http://dx.doi.org/10.3389/fpls.2018.01646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250120PMC
November 2018

Optimizing colormaps with consideration for color vision deficiency to enable accurate interpretation of scientific data.

PLoS One 2018 1;13(7):e0199239. Epub 2018 Aug 1.

Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington, United States of America.

Color vision deficiency (CVD) affects more than 4% of the population and leads to a different visual perception of colors. Though this has been known for decades, colormaps with many colors across the visual spectra are often used to represent data, leading to the potential for misinterpretation or difficulty with interpretation by someone with this deficiency. Until the creation of the module presented here, there were no colormaps mathematically optimized for CVD using modern color appearance models. While there have been some attempts to make aesthetically pleasing or subjectively tolerable colormaps for those with CVD, our goal was to make optimized colormaps for the most accurate perception of scientific data by as many viewers as possible. We developed a Python module, cmaputil, to create CVD-optimized colormaps, which imports colormaps and modifies them to be perceptually uniform in CVD-safe colorspace while linearizing and maximizing the brightness range. The module is made available to the science community to enable others to easily create their own CVD-optimized colormaps. Here, we present an example CVD-optimized colormap created with this module that is optimized for viewing by those without a CVD as well as those with red-green colorblindness. This colormap, cividis, enables nearly-identical visual-data interpretation to both groups, is perceptually uniform in hue and brightness, and increases in brightness linearly.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199239PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070163PMC
December 2018

Observed metabolic asymmetry within soybean root nodules reflects unexpected complexity in rhizobacteria-legume metabolite exchange.

ISME J 2018 09 13;12(9):2335-2338. Epub 2018 Jun 13.

Environmental Molecular Sciences Laboratory, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA, 99354, USA.

In this study, the three-dimensional spatial distributions of a number of metabolites involved in regulating symbiosis and biological nitrogen fixation (BNF) within soybean root nodules were revealed using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While many metabolites exhibited distinct spatial compartmentalization, some metabolites were asymmetrically distributed throughout the nodule (e.g., S-adenosylmethionine). These results establish a more complex metabolic view of plant-bacteria symbiosis (and BNF) within soybean nodules than previously hypothesized. Collectively these findings suggest that spatial perspectives in metabolic regulation should be considered to unravel the overall complexity of interacting organisms, like those relating to associations of nitrogen-fixing bacteria with host plants.
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http://dx.doi.org/10.1038/s41396-018-0188-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092352PMC
September 2018

Multimodal MSI in Conjunction with Broad Coverage Spatially Resolved MS Increases Confidence in Both Molecular Identification and Localization.

Anal Chem 2018 01 19;90(1):702-707. Epub 2017 Dec 19.

Biosciences Division, Oak Ridge National Laboratory , Oak Ridge, Tennessee 37830, United States.

One critical aspect of mass spectrometry imaging (MSI) is the need to confidently identify detected analytes. While orthogonal tandem MS (e.g., LC-MS) experiments from sample extracts can assist in annotating ions, the spatial information about these molecules is lost. Accordingly, this could cause mislead conclusions, especially in cases where isobaric species exhibit different distributions within a sample. In this Technical Note, we employed a multimodal imaging approach, using matrix assisted laser desorption/ionization (MALDI)-MSI and liquid extraction surface analysis (LESA)-MSI, to confidently annotate and localize a broad range of metabolites involved in a tripartite symbiosis system of moss, cyanobacteria, and fungus. We found that the combination of these two imaging modalities generated very congruent ion images, providing the link between highly accurate structural information onfered by LESA and high spatial resolution attainable by MALDI. These results demonstrate how this combined methodology could be very useful in differentiating metabolite routes in complex systems.
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http://dx.doi.org/10.1021/acs.analchem.7b04319DOI Listing
January 2018

NanoSIMS for biological applications: Current practices and analyses.

Biointerphases 2017 Sep 27;13(3):03B301. Epub 2017 Sep 27.

Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington 99354.

Secondary ion mass spectrometry (SIMS) has become an increasingly utilized tool in biologically relevant studies. Of these, high lateral resolution methodologies using the NanoSIMS 50/50L have been especially powerful within many biological fields over the past decade. Here, the authors provide a review of this technology, sample preparation and analysis considerations, examples of recent biological studies, data analyses, and current outlooks. Specifically, the authors offer an overview of SIMS and development of the NanoSIMS. The authors describe the major experimental factors that should be considered prior to NanoSIMS analysis and then provide information on best practices for data analysis and image generation, which includes an in-depth discussion of appropriate colormaps. Additionally, the authors provide an open-source method for data representation that allows simultaneous visualization of secondary electron and ion information within a single image. Finally, the authors present a perspective on the future of this technology and where they think it will have the greatest impact in near future.
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http://dx.doi.org/10.1116/1.4993628DOI Listing
September 2017

Laser-ablation electrospray ionization mass spectrometry with ion mobility separation reveals metabolites in the symbiotic interactions of soybean roots and rhizobia.

Plant J 2017 Jul 23;91(2):340-354. Epub 2017 May 23.

Environmental Molecular Sciences Laboratory, Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA, 99354, USA.

Technologies enabling in situ metabolic profiling of living plant systems are invaluable for understanding physiological processes and could be used for rapid phenotypic screening (e.g., to produce plants with superior biological nitrogen-fixing ability). The symbiotic interaction between legumes and nitrogen-fixing soil bacteria results in a specialized plant organ (i.e., root nodule) where the exchange of nutrients between host and endosymbiont occurs. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is a method that can be performed under ambient conditions requiring minimal sample preparation. Here, we employed LAESI-MS to explore the well characterized symbiosis between soybean (Glycine max L. Merr.) and its compatible symbiont, Bradyrhizobium japonicum. The utilization of ion mobility separation (IMS) improved the molecular coverage, selectivity, and identification of the detected biomolecules. Specifically, incorporation of IMS resulted in an increase of 153 differentially abundant spectral features in the nodule samples. The data presented demonstrate the advantages of using LAESI-IMS-MS for the rapid analysis of intact root nodules, uninfected root segments, and free-living rhizobia. Untargeted pathway analysis revealed several metabolic processes within the nodule (e.g., zeatin, riboflavin, and purine synthesis). Compounds specific to the uninfected root and bacteria were also detected. Lastly, we performed depth profiling of intact nodules to reveal the location of metabolites to the cortex and inside the infected region, and lateral profiling of sectioned nodules confirmed these molecular distributions. Our results established the feasibility of LAESI-IMS-MS for the analysis and spatial mapping of plant tissues, with its specific demonstration to improve our understanding of the soybean-rhizobial symbiosis.
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http://dx.doi.org/10.1111/tpj.13569DOI Listing
July 2017

Constant-Distance Mode Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Biological Samples with Complex Topography.

Anal Chem 2017 01 28;89(2):1131-1137. Epub 2016 Dec 28.

Physical Sciences Division and ‡Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory , Richland, Washington, United States.

A new approach for constant-distance mode mass spectrometry imaging (MSI) of biological samples using nanospray desorption electrospray ionization (nano-DESI) was developed by integrating a shear-force probe with the nano-DESI probe. The technical concept and basic instrumental setup, as well as the general operation of the system are described. Mechanical dampening of resonant oscillations due to the presence of shear forces between the probe and the sample surface enabled the constant-distance imaging mode via a computer-controlled closed-feedback loop. The capability of simultaneous chemical and topographic imaging of complex biological samples is demonstrated using living Bacillus subtilis ATCC 49760 colonies on agar plates. The constant-distance mode nano-DESI MSI enabled imaging of many metabolites, including nonribosomal peptides (surfactin, plipastatin, and iturin) on the surface of living bacterial colonies, ranging in diameter from 10 to 13 mm, with height variations up to 0.8 mm above the agar plate. Co-registration of ion images to topographic images provided higher-contrast images. Based on this effort, constant-mode nano-DESI MSI proved to be ideally suited for imaging biological samples of complex topography in their native states.
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http://dx.doi.org/10.1021/acs.analchem.6b03293DOI Listing
January 2017

Secondary Ion Mass Spectrometry Imaging of Tissues, Cells, and Microbial Systems.

Micros Today 2016 Mar 18;24(2):24-31. Epub 2016 Mar 18.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Boulevard, Richland, WA 99352, USA.

Secondary ion mass spectrometry (SIMS) is a technique capable of imaging tissues, single cells, and microbes revealing chemical species with sub-micrometer spatial resolution. The recently developed Fourier transform ion cyclotron resonance (FTICR) SIMS instrument provides high mass resolving power and mass accuracy, ToF-SIMS can generate chemical maps with an order of magnitude better lateral resolution than the FTICR-SIMS, and the NanoSIMS instrument offers sub-100 nm spatial resolution in chemical imaging. Many commercial ToF-SIMS instruments are also capable of depth profiling that allows three-dimensional reconstructions of cell and tissue structure.
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http://dx.doi.org/10.1017/S1551929516000018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028133PMC
March 2016

Cellular Delivery of Nanoparticles Revealed with Combined Optical and Isotopic Nanoscopy.

ACS Nano 2016 04 29;10(4):4046-54. Epub 2016 Mar 29.

Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory , Richland, Washington 99354, United States.

Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.
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http://dx.doi.org/10.1021/acsnano.5b06477DOI Listing
April 2016