Mapping microhabitats of lignocellulose decomposition by a microbial consortium.

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Regulation of ß-cell death by ADP-ribosylhydrolase ARH3 via lipid signaling in insulitis.

BACKGROUND: Lipids are regulators of insulitis and ß-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate ß-cell death. METHODS: We performed lipidomics using three models of insulitis: human islets and EndoC-ßH1 ß cells treated with the pro-inflammatory cytokines interlukine-1ß and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. RESULTS: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced ß-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. CONCLUSIONS: Our data provide insights into the change of lipidomics landscape in ß cells during insulitis and identify a protective mechanism by omega-3 fatty acids. Video Abstract.

Soil metabolome response to whole-ecosystem warming at the Spruce and Peatland Responses under Changing Environments experiment.

In this study, a suite of complementary environmental geochemical analyses, including NMR and gas chromatography-mass spectrometry (GC-MS) analyses of central metabolites, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of secondary metabolites, and lipidomics, was used to investigate the influence of organic matter (OM) quality on the heterotrophic microbial mechanisms controlling peatland CO2, CH4, and CO2:CH4 porewater production ratios in response to climate warming. Our investigations leverage the Spruce and Peatland Responses under Changing Environments (SPRUCE) experiment, where air and peat warming were combined in a whole-ecosystem warming treatment. We hypothesized that warming would enhance the production of plant-derived metabolites, resulting in increased labile OM inputs to the surface peat, thereby enhancing microbial activity and greenhouse gas production. Because shallow peat is most susceptible to enhanced warming, increases in labile OM inputs to the surface, in particular, are likely to result in significant changes to CO2 and CH4 dynamics and methanogenic pathways. In support of this hypothesis, significant correlations were observed between metabolites and temperature consistent with increased availability of labile substrates, which may stimulate more rapid turnover of microbial proteins. An increase in the abundance of methanogenic genes in response to the increase in the abundance of labile substrates was accompanied by a shift toward acetoclastic and methylotrophic methanogenesis. Our results suggest that as peatland vegetation trends toward increasing vascular plant cover with warming, we can expect a concomitant shift toward increasingly methanogenic conditions and amplified climate-peatland feedbacks.

A Preprocessing Tool for Enhanced Ion Mobility-Mass Spectrometry-Based Omics Workflows.

The ability to improve the data quality of ion mobility-mass spectrometry (IM-MS) measurements is of great importance for enabling modular and efficient computational workflows and gaining better qualitative and quantitative insights from complex biological and environmental samples. We developed the PNNL PreProcessor, a standalone and user-friendly software housing various algorithmic implementations to generate new MS-files with enhanced signal quality and in the same instrument format. Different experimental approaches are supported for IM-MS based on Drift-Tube (DT) and Structures for Lossless Ion Manipulations (SLIM), including liquid chromatography (LC) and infusion analyses. The algorithms extend the dynamic range of the detection system, while reducing file sizes for faster and memory-efficient downstream processing. Specifically, multidimensional smoothing improves peak shapes of poorly defined low-abundance signals, and saturation repair reconstructs the intensity profile of high-abundance peaks from various analyte types. Other functionalities are data compression and interpolation, IM demultiplexing, noise filtering by low intensity threshold and spike removal, and exporting of acquisition metadata. Several advantages of the tool are illustrated, including an increase of 19.4% in lipid annotations and a two-times faster processing of LC-DT IM-MS data-independent acquisition spectra from a complex lipid extract of a standard human plasma sample. The software is freely available at https://omics.pnl.gov/software/pnnl-preprocessor.

Ceramide synthase 2 deletion decreases the infectivity of HIV-1.

The lipid composition of HIV-1 virions is enriched in sphingomyelin (SM), but the roles that SM or other sphingolipids (SLs) might play in the HIV-1 replication pathway have not been elucidated. In human cells, SL levels are regulated by ceramide synthase (CerS) enzymes that produce ceramides, which can be converted to SMs, hexosylceramides, and other SLs. In many cell types, CerS2, which catalyzes the synthesis of very long chain ceramides, is the major CerS. We have examined how CerS2 deficiency affects the assembly and infectivity of HIV-1. As expected, we observed that very long chain ceramide, hexosylceramide, and SM were reduced in CerS2 knockout cells. CerS2 deficiency did not affect HIV-1 assembly or the incorporation of the HIV-1 envelope (Env) protein into virus particles, but it reduced the infectivites of viruses produced in the CerS2-deficient cells. The reduced viral infection levels were dependent on HIV-1 Env, since HIV-1 particles that were pseudotyped with the vesicular stomatitis virus glycoprotein did not exhibit reductions in infectivity. Moreover, cell-cell fusion assays demonstrated that the functional defect of HIV-1 Env in CerS2-deficient cells was independent of other viral proteins. Overall, our results indicate that the altered lipid composition of CerS2-deficient cells specifically inhibit the HIV-1 Env receptor binding and/or fusion processes.

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F1FO ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.

Interpreting the lipidome: bioinformatic approaches to embrace the complexity.

BACKGROUND: Improvements in mass spectrometry (MS) technologies coupled with bioinformatics developments have allowed considerable advancement in the measurement and interpretation of lipidomics data in recent years. Since research areas employing lipidomics are rapidly increasing, there is a great need for bioinformatic tools that capture and utilize the complexity of the data. Currently, the diversity and complexity within the lipidome is often concealed by summing over or averaging individual lipids up to (sub)class-based descriptors, losing valuable information about biological function and interactions with other distinct lipids molecules, proteins and/or metabolites. AIM OF REVIEW: To address this gap in knowledge, novel bioinformatics methods are needed to improve identification, quantification, integration and interpretation of lipidomics data. The purpose of this mini-review is to summarize exemplary methods to explore the complexity of the lipidome. KEY SCIENTIFIC CONCEPTS OF REVIEW: Here we describe six approaches that capture three core focus areas for lipidomics: (1) lipidome annotation including a resolvable database identifier, (2) interpretation via pathway- and enrichment-based methods, and (3) understanding complex interactions to emphasize specific steps in the analytical process and highlight challenges in analyses associated with the complexity of lipidome data.

Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Statistically Driven Metabolite and Lipid Profiling of Patients from the Undiagnosed Diseases Network.

Advancements in molecular separations coupled with mass spectrometry have enabled metabolome analyses for clinical cohorts. A population of interest for metabolome profiling is patients with rare disease for which abnormal metabolic signatures may yield clues into the genetic basis, as well as mechanistic drivers of the disease and possible treatment options. We undertook the metabolome profiling of a large cohort of patients with mysterious conditions characterized through the Undiagnosed Diseases Network (UDN). Due to the size and enrollment procedures, collection of the metabolomes for UDN patients took place over 2 years. We describe the study designed to adjust for measurements collected over a long time scale and how this enabled statistical analyses to summarize the metabolome of individual patients. We demonstrate the removal of time-based batch effects, overall statistical characteristics of the UDN population, and two case studies of interest that demonstrate the utility of metabolome profiling for rare diseases.

Lipid Mini-On: mining and ontology tool for enrichment analysis of lipidomic data.

SUMMARY: Here we introduce Lipid Mini-On, an open-source tool that performs lipid enrichment analyses and visualizations of lipidomics data. Lipid Mini-On uses a text-mining process to bin individual lipid names into multiple lipid ontology groups based on the classification (e.g. LipidMaps) and other characteristics, such as chain length. Lipid Mini-On provides users with the capability to conduct enrichment analysis of the lipid ontology terms using a Shiny app with options of five statistical approaches. Lipid classes can be added to customize the user's database and remain updated as new lipid classes are discovered. Visualization of results is available for all classification options (e.g. lipid subclass and individual fatty acid chains). Results are also visualized through an editable network of relationships between the individual lipids and their associated lipid ontology terms. The utility of the tool is demonstrated using biological (e.g. human lung endothelial cells) and environmental (e.g. peat soil) samples. AVAILABILITY AND IMPLEMENTATION: Rodin (R package: https://github.com/PNNL-Comp-Mass-Spec/Rodin), Lipid Mini-On Shiny app (https://github.com/PNNL-Comp-Mass-Spec/LipidMiniOn) and Lipid Mini-On online tool (https://omicstools.pnnl.gov/shiny/lipid-mini-on/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Exploiting the Semidestructive Nature of Gas Cluster Ion Beam Time-of-Flight Secondary Ion Mass Spectrometry Imaging for Simultaneous Localization and Confident Lipid Annotations.

Lipids have been recognized as key players in cell signaling and disease. Information on their location and distribution within a biological system, under varying conditions, is necessary to understand the contributions of different lipid species to an altered phenotype. Imaging mass spectrometry techniques, such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and matrix-assisted laser desorption/ionization (MALDI), are capable of revealing global lipid distributions in tissues in an untargeted fashion. However, to confidently identify the species present in a sample, orthogonal analyses like tandem MS (MS/MS) are often required. This can be accomplished by bulk sample analysis with liquid chromatography (LC)-MS/MS, which can provide confident lipid identifications, at the expense of losing location-specific information. Here, using planarian flatworms as a model system, we demonstrate that imaging gas cluster ion beam (GCIB)-ToF-SIMS has the unique capability to simultaneously detect, identify, and image lipid species with subcellular resolution in tissue sections. The parallel detection of both, intact lipids and their respective fragments, allows for unique identification of some species without the need of performing an additional orthogonal MS/MS analysis. This was accomplished by correlating intact lipid and associated fragment SIMS images. The lipid assignments, respective fragment identities, and locations gathered from ToF-SIMS data were confirmed via LC-MS/MS on lipid extracts and ultrahigh mass resolution MALDI-MS imaging. Together, these data show that the semidestructive nature of ToF-SIMS can be utilized advantageously to enable both confident molecular annotations and to determine the locations of species within a biological sample.

Lipid Coverage in Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging of Mouse Lung Tissues.

Lipids are a naturally occurring group of molecules that not only contribute to the structural integrity of the lung preventing alveolar collapse but also play important roles in the anti-inflammatory responses and antiviral protection. Alteration in the type and spatial localization of lipids in the lung plays a crucial role in various diseases, such as respiratory distress syndrome (RDS) in preterm infants and oxidative stress-influenced diseases, such as pneumonia, emphysema, and lung cancer following exposure to environmental stressors. The ability to accurately measure spatial distributions of lipids and metabolites in lung tissues provides important molecular insights related to lung function, development, and disease states. Nanospray desorption electrospray ionization (nano-DESI) and other ambient ionization mass spectrometry techniques enable label-free imaging of complex samples in their native state with minimal to absolutely no sample preparation. However, lipid coverage obtained in nano-DESI mass spectrometry imaging (MSI) experiments has not been previously characterized. In this work, the depth of lipid coverage in nano-DESI MSI of mouse lung tissues was compared to liquid chromatography tandem mass spectrometry (LC-MS/MS) lipidomics analysis of tissue extracts prepared using two different procedures: standard Folch extraction method of the whole lung samples and extraction into a 90% methanol/10% water mixture used in nano-DESI MSI experiments. A combination of positive and negative ionization mode nano-DESI MSI identified 265 unique lipids across 20 lipids subclasses and 19 metabolites (284 in total) in mouse lung tissues. Except for triacylglycerols (TG) species, nano-DESI MSI provided comparable coverage to LC-MS/MS experiments performed using methanol/water tissue extracts and up to 50% coverage in comparison with the Folch extraction-based whole lung lipidomics analysis. These results demonstrate the utility of nano-DESI MSI for comprehensive spatially resolved analysis of lipids in tissue sections. A combination of nano-DESI MSI and LC-MS/MS lipidomics is particularly useful for exploring changes in lipid distributions during lung development, as well as resulting from disease or exposure to environmental toxicants.

LIQUID: an-open source software for identifying lipids in LC-MS/MS-based lipidomics data.

SUMMARY: We introduce an open-source software, LIQUID, for semi-automated processing and visualization of LC-MS/MS-based lipidomics data. LIQUID provides users with the capability to process high throughput data and contains a customizable target library and scoring model per project needs. The graphical user interface provides visualization of multiple lines of spectral evidence for each lipid identification, allowing rapid examination of data for making confident identifications of lipid molecular species. LIQUID was compared to other freely available software commonly used to identify lipids and other small molecules (e.g. CFM-ID, MetFrag, GNPS, LipidBlast and MS-DIAL), and was found to have a faster processing time to arrive at a higher number of validated lipid identifications. AVAILABILITY AND IMPLEMENTATION: LIQUID is available at http://github.com/PNNL-Comp-Mass-Spec/LIQUID . CONTACT: jennifer.kyle@pnnl.gov or thomas.metz@pnnl.gov. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Corrigendum: Saliniramus fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from Hot Lake, Washington, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

There was an error in the proposed genus name in the published article, in that the genus 'Salinivirga' was effectively published while this article was in review. Therefore, the genus 'Salinivirga' should be replaced with 'Saliniramus'. For the convenience of future readers, we have included the complete corrected article below, in which all occurrences of the incorrect genus name have been amended: A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Saliniramus fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Saliniramus and Salinarimonas (the type genus of the family).

Salinivirga fredricksonii gen. nov., sp. nov., a heterotrophic halophile isolated from a photosynthetic mat, a member of a novel lineage (Salinarimonadaceae fam. nov.) within the order Rhizobiales, and reclassification of the genus Salinarimonas Liu et al. 2010 into Salinarimonadaceae.

A halophilic bacterial strain, HL-109T, was isolated from the unicyanobacterial consortium UCC-O, which was obtained from the photosynthetic mat of Hot Lake (Washington, USA). A polyphasic approach using phenotypic, genotypic and chemotaxonomic data was used to classify the strain within the order Rhizobiales. The organism stained Gram-negative and was a moderate thermophile with a growth optimum of 45 °C. It was obligately aerobic, heterotrophic and halophilic, growing in both NaCl and MgSO4 brines. The novel isolate had a polymorphic cellular morphology of short rods with occasional branching, and cells were monotrichous. The major fatty acids detected were C18 : 1, C18 : 0, C16 : 0 and C18 : cyc. Phylogenetic analysis of the 16S rRNA gene placed the strain in the order Rhizobiales and it shared 94 % identity with the type strain of its nearest relative, Salinarimonas ramus. Morphological, chemotaxonomic and phylogenetic results did not affiliate the novel organism with any of the families in the Rhizobiales; therefore, HL-109T is representative of a new lineage, for which the name Salinivirga fredricksonii gen. nov., sp. nov. is proposed, with the type strain HL-109T (=JCM 31876T=DSM 102886T). In addition, examination of the phylogenetics of strain HL-109T and its nearest relatives, Salinarimonas ramus and Salinarimonasrosea, demonstrates that these halophiles form a clade distinct from the described families of the Rhizobiales. We further propose the establishment of a new family, Salinarimonadaceae fam. nov., to accommodate the genera Salinivirga and Salinarimonas (the type genus of the family).

Comparing identified and statistically significant lipids and polar metabolites in 15-year old serum and dried blood spot samples for longitudinal studies.

RATIONALE: The use of dried blood spots (DBS) has many advantages over traditional plasma and serum samples such as the smaller blood volume required, storage at room temperature, and ability to sample in remote locations. However, understanding the robustness of different analytes in DBS samples is essential, especially in older samples collected for longitudinal studies. METHODS: Here we analyzed the stability of polar metabolites and lipids in DBS samples collected in 2000-2001 and stored at room temperature. The identified and statistically significant molecules were then compared to matched serum samples stored at -80°C to determine if the DBS samples could be effectively used in a longitudinal study following metabolic disease. RESULTS: A total of 400 polar metabolites and lipids were identified in the serum and DBS samples using gas chromatograph/mass spectrometry (GC/MS), liquid chromatography (LC)/MS, and LC/ion mobility spectrometry-MS (LC/IMS-MS). The identified polar metabolites overlapped well between the sample types, though only one statistically significant metabolite was conserved in a case-control study of older diabetic males with low amounts of high-density lipoproteins and high body mass indices, triacylglycerides and glucose levels when compared to non-diabetic patients with normal levels, indicating that degradation in the DBS samples affects polar metabolite quantitation. Differences in the lipid identifications indicated that some oxidation occurs in the DBS samples. However, 36 statistically significant lipids correlated in both sample types. CONCLUSIONS: The difference in the number of statistically significant polar metabolites and lipids indicated that the lipids did not degrade to as great of a degree as the polar metabolites in the DBS samples and lipid quantitation was still possible. Copyright © 2016 John Wiley & Sons, Ltd.

MPLEx: a method for simultaneous pathogen inactivation and extraction of samples for multi-omics profiling.

The continued emergence and spread of infectious agents is of great concern, and systems biology approaches to infectious disease research can advance our understanding of host-pathogen relationships and facilitate the development of new therapies and vaccines. Molecular characterization of infectious samples outside of appropriate biosafety containment can take place only subsequent to pathogen inactivation. Herein, we describe a modified Folch extraction using chloroform/methanol that facilitates the molecular characterization of infectious samples by enabling simultaneous pathogen inactivation and extraction of proteins, metabolites, and lipids for subsequent mass spectrometry-based multi-omics measurements. This single-sample metabolite, protein and lipid extraction (MPLEx) method resulted in complete inactivation of clinically important bacterial and viral pathogens with exposed lipid membranes, including Yersinia pestis, Salmonella Typhimurium, and Campylobacter jejuni in pure culture, and Yersinia pestis, Campylobacter jejuni, and West Nile, MERS-CoV, Ebola, and influenza H7N9 viruses in infection studies. In addition, >99% inactivation, which increased with solvent exposure time, was also observed for pathogens without exposed lipid membranes including community-associated methicillin-resistant Staphylococcus aureus, Clostridium difficile spores and vegetative cells, and adenovirus type 5. The overall pipeline of inactivation and subsequent proteomic, metabolomic, and lipidomic analyses was evaluated using a human epithelial lung cell line infected with wild-type and mutant influenza H7N9 viruses, thereby demonstrating that MPLEx yields biomaterial of sufficient quality for subsequent multi-omics analyses. Based on these experimental results, we believe that MPLEx will facilitate systems biology studies of infectious samples by enabling simultaneous pathogen inactivation and multi-omics measurements from a single specimen with high success for pathogens with exposed lipid membranes.

Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry.

Understanding how biological molecules are generated, metabolized and eliminated in living systems is important for interpreting processes such as immune response and disease pathology. While genomic and proteomic studies have provided vast amounts of information over the last several decades, interest in lipidomics has also grown due to improved analytical technologies revealing altered lipid metabolism in type 2 diabetes, cancer, and lipid storage disease. Mass spectrometry (MS) measurements are currently the dominant approach for characterizing the lipidome by providing detailed information on the spatial and temporal composition of lipids. However, interpreting lipids' biological roles is challenging due to the existence of numerous structural and stereoisomers (i.e. distinct acyl chain and double-bond positions), which are often unresolvable using present approaches. Here we show that combining liquid chromatography (LC) and structurally-based ion mobility spectrometry (IMS) measurement with MS analyses distinguishes lipid isomers and allows insight into biological and disease processes.