High-Throughput Mining of Novel Compounds from Known Microbes: A Boost to Natural Product Screening.

Advanced techniques can accelerate the pace of natural product discovery from microbes, which has been lagging behind the drug discovery era. Therefore, the present review article discusses the various interdisciplinary and cutting-edge techniques to present a concrete strategy that enables the high-throughput screening of novel natural compounds (NCs) from known microbes. Recent bioinformatics methods revealed that the microbial genome contains a huge untapped reservoir of silent biosynthetic gene clusters (BGC). This article describes several methods to identify the microbial strains with hidden mines of silent BGCs. Moreover, antiSMASH 5.0 is a free, accurate, and highly reliable bioinformatics tool discussed in detail to identify silent BGCs in the microbial genome. Further, the latest microbial culture technique, HiTES (high-throughput elicitor screening), has been detailed for the expression of silent BGCs using 500-1000 different growth conditions at a time. Following the expression of silent BGCs, the latest mass spectrometry methods are highlighted to identify the NCs. The recently emerged LAESI-IMS (laser ablation electrospray ionization-imaging mass spectrometry) technique, which enables the rapid identification of novel NCs directly from microtiter plates, is presented in detail. Finally, various trending 'dereplication' strategies are emphasized to increase the effectiveness of NC screening.

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

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.

Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry.

Imaging mass spectrometry (IMS) is a rapidly advancing molecular imaging modality that can map the spatial distribution of molecules with high chemical specificity. IMS does not require prior tagging of molecular targets and is able to measure a large number of ions concurrently in a single experiment. While this makes it particularly suited for exploratory analysis, the large amount and high-dimensional nature of data generated by IMS techniques make automated computational analysis indispensable. Research into computational methods for IMS data has touched upon different aspects, including spectral preprocessing, data formats, dimensionality reduction, spatial registration, sample classification, differential analysis between IMS experiments, and data-driven fusion methods to extract patterns corroborated by both IMS and other imaging modalities. In this work, we review unsupervised machine learning methods for exploratory analysis of IMS data, with particular focus on (a) factorization, (b) clustering, and (c) manifold learning. To provide a view across the various IMS modalities, we have attempted to include examples from a range of approaches including matrix assisted laser desorption/ionization, desorption electrospray ionization, and secondary ion mass spectrometry-based IMS. This review aims to be an entry point for both (i) analytical chemists and mass spectrometry experts who want to explore computational techniques; and (ii) computer scientists and data mining specialists who want to enter the IMS field. © 2019 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc. Mass SpecRev 00:1-47, 2019.

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

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.

Screening of Bacillus mojavensis biofilms and biosurfactants using laser ablation electrospray ionization mass spectroscopy.

AIMS: Biofilms are composed of micro-organisms within a matrix of chemically complex polymer compounds and from these structures many unknown competitive factors are suggested that many considered are important consequences for biological control. This research was undertaken to study further the endophyte, Bacillus mojavensis and its relationships to biofilm and two classes of lipopeptides considered relevant for biocontrol of plant pathogens. METHODS AND RESULTS: Laser ablation electrospray ionization mass spectrometry and conventional MS/MS were used to study in situ biofilm production and the production of lipopeptides fengycin and surfactin in different strains of B. mojavensis in plate and test tube culture on two media. All strains were capable of producing biofilm in vitro along with the accumulation of surfactin and fengycin although no concentration-dependent relationship between lipopeptide accumulation and biofilm was observed. CONCLUSION: All strains studied produce biofilms in culture with the accumulated surfactin and fengycin, demonstrating that endophytic bacteria also produced biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that this endophytic species produced biofilms along with two biocontrol compounds of which one, surfactin, considered by others as a quorum sensor, highlighting its ecological role as a signalling mechanism in planta.

In situ ergot alkaloid detection in three Balansia epichloe-infected grass species.

AIMS: The objectives of this work were to characterize molecularly the morphologically described endophyte Balansia epichloe symbiotic on three grass species, and to determine the in situ production of ergot alkaloids on these three symbiota. METHODS AND RESULTS: Balansia epichloe symbiotic with smut grass (Sporobolus poiretii), love grass (Eragrostis hirsuta) and lace grass (Eragrostis capillaries, a new host) were characterized using DNA barcoding. Laser ablation electro spray ionization (LAESI)-mass spectrometry was used to detect ergot alkaloids in situ for each symbiotum. CONCLUSIONS: The three morphologically described symbionts on the three host grasses were indicated as belonging to the species B. epichloe, DNA barcoding suggested they were related although a cryptic species was suggested. LAESI-mass spectrometry showed that ergot alkaloids were produced in vivo in two hosts but not the third although this same symbiotum was related to one of the ergot alkaloid producing symbiota as revealed by the DNA-barcoding procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: These results established the accumulation of ergot alkaloids in pot culture by a morpho species although there were variations with each species of grass. Barcoding described divergence among species, but considering its limitation, the suggested existence of cryptic species among this morphospecies requires substantiation by studies that are more rigorous.

Laser ablation electrospray ionization mass spectrometry imaging as a new tool for accessing patulin diffusion in mold-infected fruits.

This work describes the use of laser ablation electrospray ionization mass spectrometry imaging (LAESI imaging) to investigate the diffusion of the mycotoxin patulin from rotten to healthy areas of fruits. Slices of mold-infected and uninfected (control) apples and strawberries were prepared, and this was the only sample preparation step used. An infrared laser beam (2.94 µm) was used to irradiate the slices, resulting in the ablation of sample compounds directly ionized by electrospray and analyzed by mass spectrometry. Multivariate curve resolution - alternating least squares was applied in unfolded LAESI images to obtain relative quantity information. Patulin was not detected in the control samples but was seen in all mold-infected fruits. LAESI images showed the diffusion of patulin from the rotten area to unaffected parts of the fruits. This study points out the advantage of LAESI imaging over traditional analytical methods used to study the diffusion of mycotoxins in fruits.

Single-Cell Metabolomics with Rapid Determination of Chemical Formulas from Isotopic Fine Structures.

Metabolomic measurements can provide functional readouts of cellular states and phenotypes. Here, we present a protocol for single-cell metabolomics that permits direct untargeted detection of a broad number of metabolites under ambient conditions, without the need for sample processing, and with high confidence in the discovery and identification of the molecular formulas for detected metabolites. This protocol describes combining fiber-based laser ablation electrospray ionization (f-LAESI) with a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T-FTICR-MS) to obtain high confidence molecular formula information about detected metabolites. The f-LAESI source utilizes mid-infrared laser ablation through a sharp optical fiber tip, affording direct ambient analysis of cells without the need for sample processing. Using the 21T-FTICR-MS as a mass analyzer enabled measurement of the isotopic fine structure (IFS) for numerous metabolites simultaneously from single cells, and the IFSs were in turn computationally processed to rapidly determine the corresponding elemental compositions. This metabolomics technique complements other single cell omics measurement methods, helping to resolve complex molecular interactions that take place within cells unattainable from single cell transcriptomic and proteomics methods.

Ambient Single-Cell Analysis and Native Tissue Imaging Using Laser-Ablation Electrospray Ionization Mass Spectrometry with Increased Spatial Resolution.

Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is an emerging method that has the potential to transform the field of metabolomics. This is in part due to LAESI-MS being an ambient ionization method that requires minimal sample preparation and uses (endogenous) water for in situ analysis. This application note details the employment of the "LAESI microscope" source to perform spatially resolved MS analysis of cells and MS imaging (MSI) of tissues at high spatial resolution. This source configuration utilizes a long-working-distance reflective objective that permits both visualization of the sample and a smaller LAESI laser beam profile than conventional LAESI setups. Here, we analyzed 200 single cells of Allium cepa (red onion) and imaged Fittonia argyroneura (nerve plant) in high spatial resolution using this source coupled to a Fourier transform mass spectrometer for high-mass-resolution and high-mass-accuracy metabolomics.

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

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.

Metabolomic Profiling of Adherent Mammalian Cells In Situ by LAESI-MS with Ion Mobility Separation.

Ambient ionization-based mass spectrometry (MS) methods coupled with ion mobility separation (IMS) have emerged as promising approaches for high-throughput in situ analysis for biomedical to environmental applications. These methods are capable of direct profiling and molecular imaging of metabolites, lipids, peptides, and xenobiotics from biological tissues with minimal sample preparation. Furthermore, employing IMS within the workflow improves the molecular coverage, resolves isobaric species, and improves biomolecule identifications through accurate collision cross section measurements. Laser ablation electrospray ionization (LAESI)-MS coupled with IMS has been successful in profiling and molecular imaging of small biomolecules directly from biological tissues and single cells. Herein, we describe a protocol for the direct analysis of adherent mammalian cells with limited perturbations by LAESI-IMS-MS. A benefit of IMS is that within the same LAESI acquisition, the spectral features related to the ESI background, washing buffer, and cell signal can be extracted and isolated separately.

Integrating ion mobility and imaging mass spectrometry for comprehensive analysis of biological tissues: A brief review and perspective.

Imaging mass spectrometry (IMS) technologies are capable of mapping a wide array of biomolecules in diverse cellular and tissue environments. IMS has emerged as an essential tool for providing spatially targeted molecular information due to its high sensitivity, wide molecular coverage, and chemical specificity. One of the major challenges for mapping the complex cellular milieu is the presence of many isomers and isobars in these samples. This challenge is traditionally addressed using orthogonal liquid chromatography (LC)-based analysis, though, common approaches such as chromatography and electrophoresis are not able to be performed at timescales that are compatible with most imaging applications. Ion mobility offers rapid, gas-phase separations that are readily integrated with IMS workflows in order to provide additional data dimensionality that can improve signal-to-noise, dynamic range, and specificity. Here, we highlight recent examples of ion mobility coupled to IMS and highlight their importance to the field.

Petrobactin Protects against Oxidative Stress and Enhances Sporulation Efficiency in Bacillus anthracis Sterne.

Bacillus anthracis is a Gram-positive bacillus that under conditions of environmental stress, such as low nutrients, can convert from a vegetative bacillus to a highly durable spore that enables long-term survival. The sporulation process is regulated by a sequential cascade of dedicated transcription factors but requires key nutrients to complete, one of which is iron. Iron acquisition by the iron-scavenging siderophore petrobactin is required for vegetative growth of B. anthracis under iron-depleted conditions and in the host. However, the extent to which petrobactin is involved in spore formation is unknown. This work shows that efficient in vitro sporulation of B. anthracis requires petrobactin, that the petrobactin biosynthesis operon (asbA to -F) is induced prior to sporulation, and that the siderophore itself associates with spores. Petrobactin is also required for oxidative stress protection during late-stage growth and for wild-type levels of sporulation in sporulation medium. Sporulation in bovine blood was found to be petrobactin dependent. Collectively, the in vitro contributions of petrobactin to sporulation as well as growth imply that petrobactin may be required for B. anthracis transmission via the spore during natural infections, in addition to its key known functions during active anthrax infections.IMPORTANCEBacillus anthracis causes the disease anthrax, which is transmitted via its dormant, spore phase. However, conversion from bacillus to spore is a complex, energetically costly process that requires many nutrients, including iron. B. anthracis requires the siderophore petrobactin to scavenge iron from host environments. We show that, in the Sterne strain, petrobactin is required for efficient sporulation, even when ample iron is available. The petrobactin biosynthesis operon is expressed during sporulation, and petrobactin is biosynthesized during growth in high-iron sporulation medium, but instead of being exported, the petrobactin remains intracellular to protect against oxidative stress and improve sporulation. It is also required for full growth and sporulation in blood (bovine), an essential step for anthrax transmission between mammalian hosts.

Laser Ablation Electrospray Ionization-Mass Spectrometry Imaging (LAESI-MS) for Spatially Resolved Plant Metabolomics.

There is increasing demand to bring the level of metabolomics analyses down to the tissue or cell level. Significant progress has been made involving the use of in situ metabolomics imaging techniques where no tissue collection or extraction is needed prior to analysis. In this chapter we describe a relatively new method which is simple and easy to use. No ectopic matrix or vacuum is required, and analyses are performed with living plant materials directly from (or even still attached to) the plant. Although relatively straightforward, there are still a few caveats as regards this method which are described at the end of the chapter.

High-throughput quantitative analysis of domoic acid directly from mussel tissue using Laser Ablation Electrospray Ionization - tandem mass spectrometry.

Eliminating sample extraction or liquid chromatography steps from methods for analysis of the neurotoxin Domoic Acid (DA) in shellfish could greatly increase throughput in food safety testing laboratories worldwide. To this end, we have investigated the use of Laser Ablation Electrospray Ionization (LAESI) with tandem mass spectrometry (MS/MS) detection for DA analysis directly from mussel tissue homogenates without sample extraction, cleanup or separation. DA could be selectively detected directly from mussel tissue homogenates using MS/MS in selected reaction monitoring scan mode. The quantitative capabilities of LAESI-MS/MS for DA analysis from mussel tissue were evaluated by analysis of four mussel tissue reference materials using matrix-matched calibration. Linear response was observed from 1 mg/kg to 40 mg/kg and the method limit of detection was 1 mg/kg. Results for DA analysis in tissue within the linear range were in good agreement with two established methods, LC-UV and LC-MS/MS (recoveries from 103 to 125%). Beyond the linear range, extraction and clean-up were required to achieve good quantitation. Most notable is the extremely rapid analysis time of about 10 s per sample by LAESI-MS/MS, which corresponds to a significant increase in sample throughput compared with existing methodology for routine DA analysis.

Surface analysis of lipids by mass spectrometry: more than just imaging.

Mass spectrometry is now an indispensable tool for lipid analysis and is arguably the driving force in the renaissance of lipid research. In its various forms, mass spectrometry is uniquely capable of resolving the extensive compositional and structural diversity of lipids in biological systems. Furthermore, it provides the ability to accurately quantify molecular-level changes in lipid populations associated with changes in metabolism and environment; bringing lipid science to the "omics" age. The recent explosion of mass spectrometry-based surface analysis techniques is fuelling further expansion of the lipidomics field. This is evidenced by the numerous papers published on the subject of mass spectrometric imaging of lipids in recent years. While imaging mass spectrometry provides new and exciting possibilities, it is but one of the many opportunities direct surface analysis offers the lipid researcher. In this review we describe the current state-of-the-art in the direct surface analysis of lipids with a focus on tissue sections, intact cells and thin-layer chromatography substrates. The suitability of these different approaches towards analysis of the major lipid classes along with their current and potential applications in the field of lipid analysis are evaluated.