Publications by authors named "Ljiljana Paša-Tolić"

167 Publications

Enhancing Top-Down Proteomics of Brain Tissue with FAIMS.

J Proteome Res 2021 May 15;20(5):2780-2795. Epub 2021 Apr 15.

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

Proteomic investigations of Alzheimer's and Parkinson's disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein's "intact" state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer's disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at -50, -40, and -30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aβ) proteoforms, including the aggregation-prone Aβ variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.
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http://dx.doi.org/10.1021/acs.jproteome.1c00049DOI Listing
May 2021

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers.

J Vis Exp 2021 03 4(169). Epub 2021 Mar 4.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory;

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.
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http://dx.doi.org/10.3791/61707DOI Listing
March 2021

Mechanisms of Manganese(II) Oxidation by Filamentous Ascomycete Fungi Vary With Species and Time as a Function of Secretome Composition.

Front Microbiol 2021 10;12:610497. Epub 2021 Feb 10.

Department of Marine Chemistry & Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA, United States.

Manganese (Mn) oxides are among the strongest oxidants and sorbents in the environment, and Mn(II) oxidation to Mn(III/IV) (hydr)oxides includes both abiotic and microbially-mediated processes. While white-rot Basidiomycete fungi oxidize Mn(II) using laccases and manganese peroxidases in association with lignocellulose degradation, the mechanisms by which filamentous Ascomycete fungi oxidize Mn(II) and a physiological role for Mn(II) oxidation in these organisms remain poorly understood. Here we use a combination of chemical and in-gel assays and bulk mass spectrometry to demonstrate secretome-based Mn(II) oxidation in three phylogenetically diverse Ascomycetes that is mechanistically distinct from hyphal-associated Mn(II) oxidation on solid substrates. We show that Mn(II) oxidative capacity of these fungi is dictated by species-specific secreted enzymes and varies with secretome age, and we reveal the presence of both Cu-based and FAD-based Mn(II) oxidation mechanisms in all 3 species, demonstrating mechanistic redundancy. Specifically, we identify candidate Mn(II)-oxidizing enzymes as tyrosinase and glyoxal oxidase in sp. SRC1lsM3a, bilirubin oxidase in sp. and AP3s5-JAC2a, and GMC oxidoreductase in all 3 species, including sp. DS3sAY3a. The diversity of the candidate Mn(II)-oxidizing enzymes identified in this study suggests that the ability of fungal secretomes to oxidize Mn(II) may be more widespread than previously thought.
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http://dx.doi.org/10.3389/fmicb.2021.610497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902709PMC
February 2021

Metabolic Interactions between and Pseudomonas fluorescens under Controlled Iron-Limited Conditions.

mSystems 2021 Jan 5;6(1). Epub 2021 Jan 5.

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

Iron (Fe) availability has well-known effects on plant and microbial metabolism, but its effects on interspecies interactions are poorly understood. The purpose of this study was to investigate metabolite exchange between the grass strain Bd21 and the soil bacterium SBW25::gfp/lux (SBW25) during Fe limitation under axenic conditions. We compared the transcriptional profiles and root exudate metabolites of plants grown semihydroponically with and without SBW25 inoculation and Fe amendment. Liquid chromatography-mass spectrometry analysis of the hydroponic solution revealed an increase in the abundance of the phytosiderophores mugineic acid and deoxymugineic acid under Fe-limited conditions compared to Fe-replete conditions, indicating greater secretion by roots presumably to facilitate Fe uptake. In SBW25-inoculated roots, expression of genes encoding phytosiderophore biosynthesis and uptake proteins increased compared to that in sterile roots, but external phytosiderophore abundances decreased. siderophores were not detected in treatments without Fe. Rather, expression of SBW25 genes encoding a porin, a transporter, and a monooxygenase was significantly upregulated in response to Fe deprivation. Collectively, these results suggest that SBW25 consumed root-exuded phytosiderophores in response to Fe deficiency, and we propose target genes that may be involved. SBW25 also altered the expression of root genes encoding defense-related enzymes and regulators, including thionin and cyanogenic glycoside production, chitinase, and peroxidase activity, and transcription factors. Our findings provide insights into the molecular bases for the stress response and metabolite exchange of interacting plants and bacteria under Fe-deficient conditions. Rhizosphere bacteria influence the growth of their host plant by consuming and producing metabolites, nutrients, and antibiotic compounds within the root system that affect plant metabolism. Under Fe-limited growth conditions, different plant and microbial species have distinct Fe acquisition strategies, often involving the secretion of strong Fe-binding chelators that scavenge Fe and facilitate uptake. Here, we studied interactions between SBW25, a plant-colonizing bacterium that produces siderophores with antifungal properties, and , a genetic model for cereal grain and biofuel grasses. Under controlled growth conditions, bacterial siderophore production was inhibited in the root system of Fe-deficient plants, bacterial inoculation altered transcription of genes involved in defense and stress response in the roots of , and SBW25 degraded phytosiderophores secreted by the host plant. These findings provide mechanistic insight into interactions that may play a role in rhizosphere dynamics and plant health in soils with low Fe solubility.
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http://dx.doi.org/10.1128/mSystems.00580-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7786132PMC
January 2021

Mapping Influenza-Induced Posttranslational Modifications on Histones from CD8+ T Cells.

Viruses 2020 12 8;12(12). Epub 2020 Dec 8.

Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38163, USA.

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.
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http://dx.doi.org/10.3390/v12121409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762524PMC
December 2020

Dynamic metabolic reprogramming in dendritic cells: An early response to influenza infection that is essential for effector function.

PLoS Pathog 2020 10 26;16(10):e1008957. Epub 2020 Oct 26.

Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.

Infection with the influenza virus triggers an innate immune response that initiates the adaptive response to halt viral replication and spread. However, the metabolic response fueling the molecular mechanisms underlying changes in innate immune cell homeostasis remain undefined. Although influenza increases parasitized cell metabolism, it does not productively replicate in dendritic cells. To dissect these mechanisms, we compared the metabolism of dendritic cells to that of those infected with active and inactive influenza A virus and those treated with toll-like receptor agonists. Using quantitative mass spectrometry, pulse chase substrate utilization assays and metabolic flux measurements, we found global metabolic changes in dendritic cells 17 hours post infection, including significant changes in carbon commitment via glycolysis and glutaminolysis, as well as mitochondrial respiration. Influenza infection of dendritic cells led to a metabolic phenotype distinct from that induced by TLR agonists, with significant resilience in terms of metabolic plasticity. We identified c-Myc as one transcription factor modulating this response. Restriction of c-Myc activity or mitochondrial substrates significantly changed the immune functions of dendritic cells, such as reducing motility and T cell activation. Transcriptome analysis of inflammatory dendritic cells isolated following influenza infection showed similar metabolic reprogramming occurs in vivo. Thus, early in the infection process, dendritic cells respond with global metabolic restructuring, that is present in inflammatory lung dendritic cells after infection, and this is important for effector function. These findings suggest metabolic switching in dendritic cells plays a vital role in initiating the immune response to influenza infection.
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http://dx.doi.org/10.1371/journal.ppat.1008957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707590PMC
October 2020

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber.

ISME J 2021 02 14;15(2):421-434. Epub 2020 Sep 14.

University of California, Davis, CA, USA.

The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa, and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 h in nylon bags within the rumen of cannulated dairy cows. Across a gene catalog covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of hemicelluloses, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8, and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks of in situ recalcitrant fiber deconstruction, and importantly, suggest that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.
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http://dx.doi.org/10.1038/s41396-020-00769-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026616PMC
February 2021

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

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry.

J Am Soc Mass Spectrom 2020 Sep 19;31(9):1783-1802. Epub 2020 Aug 19.

The University of Edinburgh, EH9 3FJ Edinburgh, United Kingdom.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.
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http://dx.doi.org/10.1021/jasms.0c00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7539639PMC
September 2020

Automated Coupling of Nanodroplet Sample Preparation with Liquid Chromatography-Mass Spectrometry for High-Throughput Single-Cell Proteomics.

Anal Chem 2020 08 22;92(15):10588-10596. Epub 2020 Jul 22.

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

Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ∼77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.
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http://dx.doi.org/10.1021/acs.analchem.0c01551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793572PMC
August 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

Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform.

Anal Chem 2020 05 6;92(10):7087-7095. Epub 2020 May 6.

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

Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. However, one significant technical gap of top-down proteomics is the inability to analyze a low amount of biological samples, which limits its access to isolated rare cells, fine needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (nanodroplet processing in one pot for trace samples), into a top-down proteomic workflow. A unique combination of a nonionic detergent dodecyl-β-d-maltopyranoside (DDM) with urea as protein extraction buffer significantly improved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing nonspecific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream analysis. The nanoPOTS-based top-down platform reproducibly and quantitatively identified ∼170 to ∼620 proteoforms from ∼70 to ∼770 HeLa cells containing ∼10 to ∼115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical research, particularly where clinical specimens are not available in amounts amenable to standard workflows.
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http://dx.doi.org/10.1021/acs.analchem.0c00467DOI Listing
May 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

An Improved Boosting to Amplify Signal with Isobaric Labeling (iBASIL) Strategy for Precise Quantitative Single-cell Proteomics.

Mol Cell Proteomics 2020 05 3;19(5):828-838. Epub 2020 Mar 3.

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99354. Electronic address:

Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio ( in general <100×) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis ( 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ∼2500 proteins and precise quantification of ∼1500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.
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http://dx.doi.org/10.1074/mcp.RA119.001857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196584PMC
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

Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.

Methods 2020 12 23;184:29-39. Epub 2019 Oct 23.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354, USA. Electronic address:

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop noted for its ability to survive water-limiting conditions. Herein, we present an analytical workflow to explore the changes in histone modifications through plant developmental stages and two drought stresses in two sorghum genotypes that differ in their response to drought. Top-down mass spectrometry (MS) is an ideal method to profile histone modifications and distinguish closely related histone proteoforms. We analyzed leaves of 48 plants and identified 26 unique histone proteins and 677 unique histone proteoforms (124 full-length and 553 truncated proteoforms). We detected trimethylation on nearly all H2B N-termini where acetylation is commonly expected. In addition, an unexpected modification on H2A histones was assigned to N-pyruvic acid 2-iminylation based on its unique neutral loss of CO. Interestingly, some of the truncated histones, in particular H4 and H3.2, showed significant changes that correlated with the growth and water conditions. The histone proteoforms could serve as targets in search of chromatin modifiers and ultimately have important ramifications in future attempts of studying plant epigenetic reprogramming under stress.
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http://dx.doi.org/10.1016/j.ymeth.2019.10.007DOI Listing
December 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

High-Throughput Single Cell Proteomics Enabled by Multiplex Isobaric Labeling in a Nanodroplet Sample Preparation Platform.

Anal Chem 2019 10 25;91(20):13119-13127. Epub 2019 Sep 25.

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

Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ∼1 600 proteins with a median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2 300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.
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http://dx.doi.org/10.1021/acs.analchem.9b03349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192326PMC
October 2019

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry.

Nat Methods 2019 07 27;16(7):587-594. Epub 2019 Jun 27.

Barnett Institute of Chemical and Biological Analysis and Departments of Chemistry & Chemical Biology and Pharmaceutical Sciences, Northeastern University, Boston, MA, USA.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.
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http://dx.doi.org/10.1038/s41592-019-0457-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719561PMC
July 2019

Online supercritical fluid extraction mass spectrometry (SFE-LC-FTMS) for sensitive characterization of soil organic matter.

Faraday Discuss 2019 08;218(0):157-171

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.

We report a novel technical approach for subcritical fluid extraction (SFE) for organic matter characterization in complex matrices such as soil. The custom platform combines on-line SFE with micro-solid phase extraction, nano liquid chromatography (LC), electrospray ionization and Fourier transform mass spectrometry (SFE-LC-FTMS). We demonstrated the utility of SFE-LC-FTMS, including results from both Orbitrap and FTICR MS, for analysis of complex mixtures of organic compounds in a solid matrix by characterizing soil organic matter in peat, a high-carbon soil. For example, in a single experiment, >6000 molecular formulas can be assigned based upon FTICR MS data from 1-50 μL of soil samples (roughly 1-50 mg of soil, dependent on soil density), nearly twice that typically obtained from direct infusion liquid solvent extraction (LSE) from an order of magnitude larger volume of the same soil. The detected species consisted predominately of lipid-like, lignin-like and protein-like compounds, based on their O/C and H/C ratios, with predominantly CHO and CHONP molecular compositions. These results clearly demonstrate that SFE has the potential to effectively extract a variety of molecular species and could become an important member of a suite of extraction methods for studying SOM and other natural organic matter. This is especially true when comprehensive coverage, minimal sample volumes, and high sensitivity are required, or when the presence of organic solvent residue in residual soil is problematic. The SFE based extraction protocol could potentially enable spatially resolved characterization of organic matter in soil with a resolution of ∼1 mm3 to facilitate studies probing the spatial heterogeneity of soil.
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http://dx.doi.org/10.1039/c9fd00011aDOI Listing
August 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

Atmo-ecometabolomics: a novel atmospheric particle chemical characterization methodology for ecological research.

Environ Monit Assess 2019 Jan 16;191(2):78. Epub 2019 Jan 16.

Department of Earth System Science, University of California, Irvine, Irvine, CA, 92697, USA.

Aerosol particles play important roles in processes controlling the composition of the atmosphere and function of ecosystems. A better understanding of the composition of aerosol particles is beginning to be recognized as critical for ecological research to further comprehend the link between aerosols and ecosystems. While chemical characterization of aerosols has been practiced in the atmospheric science community, detailed methodology tailored to the needs of ecological research does not exist yet. In this study, we describe an efficient methodology (atmo-ecometabolomics), in step-by-step details, from the sampling to the data analyses, to characterize the chemical composition of aerosol particles, namely atmo-metabolome. This method employs mass spectrometry platforms such as liquid and gas chromatography mass spectrometries (MS) and Fourier transform ion cyclotron resonance MS (FT-ICR-MS). For methodology evaluation, we analyzed aerosol particles collected during two different seasons (spring and summer) in a low-biological-activity ecosystem. Additionally, to further validate our methodology, we analyzed aerosol particles collected in a more biologically active ecosystem during the pollination peaks of three different representative tree species. Our statistical results showed that our sampling and extraction methods are suitable for characterizing the atmo-ecometabolomes in these two distinct ecosystems with any of the analytical platforms. Datasets obtained from each mass spectrometry instrument showed overall significant differences of the atmo-ecometabolomes between spring and summer as well as between the three pollination peak periods. Furthermore, we have identified several metabolites that can be attributed to pollen and other plant-related aerosol particles. We additionally provide a basic guide of the potential use ecometabolomic techniques on different mass spectrometry platforms to accurately analyze the atmo-ecometabolomes for ecological studies. Our method represents an advanced novel approach for future studies in the impact of aerosol particle chemical compositions on ecosystem structure and function and biogeochemistry.
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http://dx.doi.org/10.1007/s10661-019-7205-xDOI Listing
January 2019

We Are What We Eat: A Stoichiometric and Ecometabolomic Study of Caterpillars Feeding on Two Pine Subspecies of .

Int J Mol Sci 2018 Dec 24;20(1). Epub 2018 Dec 24.

CREAF, Center for Ecological and Forestry Applications, Cerdanyola del Vallès, 08913 Catalonia, Spain.

Many studies have addressed several plant-insect interaction topics at nutritional, molecular, physiological, and evolutionary levels. However, it is still unknown how flexible the metabolism and the nutritional content of specialist insect herbivores feeding on different closely related plants can be. We performed elemental, stoichiometric, and metabolomics analyses on leaves of two coexisting subspecies and on their main insect herbivore; the caterpillar of the processionary moth (). Caterpillars feeding on different pine subspecies had distinct overall metabolome structure, accounting for over 10% of the total variability. Although plants and insects have very divergent metabolomes, caterpillars showed certain resemblance to their plant-host metabolome. In addition, few plant-related secondary metabolites were found accumulated in caterpillar tissues which could potentially be used for self-defense. Caterpillars feeding on N and P richer needles had lower N and P tissue concentration and higher C:N and C:P ratios, suggesting that nutrient transfer is not necessarily linear through trophic levels and other plant-metabolic factors could be interfering. This exploratory study showed that little chemical differences between plant food sources can impact the overall metabolome of specialist insect herbivores. Significant nutritional shifts in herbivore tissues could lead to larger changes of the trophic web structure.
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http://dx.doi.org/10.3390/ijms20010059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337320PMC
December 2018

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

Siderophore profiling of co-habitating soil bacteria by ultra-high resolution mass spectrometry.

Metallomics 2019 01;11(1):166-175

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99354, USA.

The chemical structure of organic molecules profoundly impacts their interactions with metal ions and mineral phases in soils. Understanding the sources and cycling of metal-chelating compounds is therefore essential for predicting the bioavailability and transport of metals throughout terrestrial environments. Here we investigate the molecular speciation of organic molecules that solubilize trace metals in calcareous soils from Eastern Washington. Ultra-high performance Fourier transform ion cyclotron resonance mass spectrometry at 21 Tesla enabled fast and confident detection and identification of metal chelators that are produced by microbes that inhabit these soils based on screening for features that match diagnostic metal isotope patterns. We compared two approaches, one based on direct infusion using the incorporation of a rare isotope to validate true iron-binding features, and another based on separation with liquid chromatography and detection of isotopologues with coherent elution profiles. While the isotopic exchange method requires significantly shorter analysis time, nearly twice as many features were observed with liquid chromatography mass spectrometry (LCMS), mostly due to the reduction in ion suppression where major features limit the sensitivity of minor features. In addition, LCMS enabled the collection of higher quality fragmentation spectra and facilitated feature identification. Siderophores belonging to four major classes were identified, including ferrioxamines, pseudobactins, enterobactins, and arthrobactins. Each of these siderophores likely derives from a unique member of the microbial community, and each possesses different chemical characteristics and uptake pathways, likely contributing to fierce competition for iron within these soils. Our results provide insight into the metabolic pathways by which microbes that co-inhabit calcareous soils compete for this essential micronutrient.
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http://dx.doi.org/10.1039/c8mt00252eDOI Listing
January 2019

Increasing the Separation Capacity of Intact Histone Proteoforms Chromatography Coupling Online Weak Cation Exchange-HILIC to Reversed Phase LC UVPD-HRMS.

J Proteome Res 2018 11 8;17(11):3791-3800. Epub 2018 Oct 8.

Environmental Molecular Sciences Laboratory , Pacific Northwest National Laboratory , P.O. Box 999, Richland , Washington 99352 , United States.

Top-down proteomics is an emerging analytical strategy to characterize combinatorial protein post-translational modifications (PTMs). However, sample complexity and small mass differences between chemically closely related proteoforms often limit the resolution attainable by separations employing a single liquid chromatographic (LC) principle. In particular, for ultramodified proteins like histones, extensive and time-consuming fractionation is needed to achieve deep proteoform coverage. Herein, we present the first online nanoflow comprehensive two-dimensional liquid chromatography (nLC×LC) platform top-down mass spectrometry analysis of histone proteoforms. The described two-dimensional LC system combines weak cation exchange chromatography under hydrophilic interaction LC conditions (i.e., charge- and hydrophilicity-based separation) with reversed phase liquid chromatography (i.e., hydrophobicity-based separation). The two independent chemical selectivities were run at nanoflows (300 nL/min) and coupled online with high-resolution mass spectrometry employing ultraviolet photodissociation (UVPD-HRMS). The nLC×LC workflow increased the number of intact protein masses observable relative to one-dimensional approaches and allowed characterization of hundreds of proteoforms starting from limited sample quantities (∼1.5 μg).
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http://dx.doi.org/10.1021/acs.jproteome.8b00458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220366PMC
November 2018