Spatial and Depth Profiling of Agricultural Formulations in Leaf Tissue Using LAESI Mass Spectrometry.

Formulating agrochemical products involves combining several chemical components, including the active ingredient(s), to obtain a final product with desirable efficacy. A formulated product incorporates additional components to modulate properties that enhance the efficacy of the active(s) by modifying physical properties such as viscosity, hydrophobicity, miscibility, and others. In plants, understanding the formulation's ability to spread on tissues and penetrate through the outer layer is critical in evaluating the efficacy of the final product. We have previously demonstrated the use of mass spectrometry imaging to determine spreadability. In this study, we show that laser ablation electrospray mass spectrometry (LAESI-MS) can be a valuable tool to assess the penetrability of formulations into the leaf tissues by selectively sampling various layers of leaf tissue by manipulating the laser intensity and analyzing the ablated material using a mass spectrometer. Using this technique, we were able to identify a formulation composition that can improve the penetration and uptake of active ingredients.

CPP impairs contextual learning at concentrations below those that block pyramidal neuron NMDARs and LTP in the CA1 region of the hippocampus.

Drugs that block N-methyl-d-aspartate receptors (NMDARs) suppress hippocampus-dependent memory formation; they also block long-term potentiation (LTP), a cellular model of learning and memory. However, the fractional block that is required to achieve these effects is unknown. Here, we measured the dose-dependent suppression of contextual memory in vivo by systemic administration of the competitive antagonist (R,S)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP); in parallel, we measured the concentration-dependent block by CPP of NMDAR-mediated synapses and LTP of excitatory synapses in hippocampal brain slices in vitro. We found that the dose of CPP that suppresses contextual memory in vivo (EC50 = 2.3 mg/kg) corresponds to a free concentration of 53 nM. Surprisingly, applying this concentration of CPP to hippocampal brain slices had no effect on the NMDAR component of evoked field excitatory postsynaptic potentials (fEPSPNMDA), or on LTP. Rather, the IC50 for blocking the fEPSPNMDA was 434 nM, and for blocking LTP was 361 nM - both nearly an order of magnitude higher. We conclude that memory impairment produced by systemically administered CPP is not due primarily to its blockade of NMDARs on hippocampal pyramidal neurons. Rather, systemic CPP suppresses memory formation by actions elsewhere in the memory-encoding circuitry.

Symbiont-Mediated Protection of Acromyrmex Leaf-Cutter Ants from the Entomopathogenic Fungus Metarhizium anisopliae.

Many fungus-growing ants engage in a defensive symbiosis with antibiotic-producing ectosymbiotic bacteria in the genus Pseudonocardia, which help protect the ants' fungal mutualist from a specialized mycoparasite, Escovopsis. Here, using germfree ant rearing and experimental pathogen infection treatments, we evaluate if Acromyrmex ants derive higher immunity to the entomopathogenic fungus Metarhizium anisopliae from their Pseudonocardia symbionts. We further examine the ecological dynamics and defensive capacities of Pseudonocardia against M. anisopliae across seven different Acromyrmex species by controlling Pseudonocardia acquisition using ant-nonnative Pseudonocardia switches, in vitro challenges, and in situ mass spectrometry imaging (MSI). We show that Pseudonocardia protects the ants against M. anisopliae across different Acromyrmex species and appears to afford higher protection than metapleural gland (MG) secretions. Although Acromyrmex echinatior ants with nonnative Pseudonocardia symbionts receive protection from M. anisopliae regardless of the strain acquired compared with Pseudonocardia-free conditions, we find significant variation in the degree of protection conferred by different Pseudonocardia strains. Additionally, when ants were reared in Pseudonocardia-free conditions, some species exhibit more susceptibility to M. anisopliae than others, indicating that some ant species depend more on defensive symbionts than others. In vitro challenge experiments indicate that Pseudonocardia reduces Metarhizium conidiospore germination area. Our chemometric analysis using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) reveals that Pseudonocardia-carrying ants produce more chemical signals than Pseudonocardia-free treatments, indicating that Pseudonocardia produces bioactive metabolites on the Acromyrmex cuticle. Our results indicate that Pseudonocardia can serve as a dual-purpose defensive symbiont, conferring increased immunity for both the obligate fungal mutualist and the ants themselves. IMPORTANCE In some plants and animals, beneficial microbes mediate host immune response against pathogens, including by serving as defensive symbionts that produce antimicrobial compounds. Defensive symbionts are known in several insects, including some leaf-cutter ants where antifungal-producing Actinobacteria help protect the fungal mutualist of the ants from specialized mycoparasites. In many defensive symbioses, the extent and specificity of defensive benefits received by the host are poorly understood. Here, using "aposymbiotic" rearing, symbiont switching experiments, and imaging mass spectrometry, we explore the ecological and chemical dynamics of the model defensive symbiosis between Acromyrmex ants and their defensive symbiotic bacterium Pseudonocardia. We show that the defensive symbiont not only protects the fungal crop of Acromyrmex but also provides protection from fungal pathogens that infect the ant workers themselves. Furthermore, we reveal that the increased immunity to pathogen infection differs among strains of defensive symbionts and that the degree of reliance on a defensive symbiont for protection varies across congeneric ant species. Taken together, our results suggest that Acromyrmex-associated Pseudonocardia have evolved broad antimicrobial defenses that promote strong immunity to diverse fungal pathogens within the ancient fungus-growing ant-microbe symbiosis.

MALDI Mass Spectrometry Imaging of Peptides in Medicago truncatula Root Nodules.

Mass spectrometry imaging is routinely used to visualize the distributions of biomolecules in tissue sections. In plants, mass spectrometry imaging of metabolites is more often conducted, but the imaging of larger molecules is less frequently performed despite the importance of proteins and endogenous peptides to the plant. Here, we describe a matrix-assisted laser desorption/ionization mass spectrometry imaging method for the imaging of peptides in Medicago truncatula root nodules. Sample preparation steps including embedding in gelatin, sectioning, and matrix application are described. The method described is employed to determine the spatial distribution of hundreds of peptide peaks.

Gut Microbial and Metabolic Responses to Salmonella enterica Serovar Typhimurium and Candida albicans.

The gut microbiota confers resistance to pathogens of the intestinal ecosystem, yet the dynamics of pathogen-microbiome interactions and the metabolites involved in this process remain largely unknown. Here, we use gnotobiotic mice infected with the virulent pathogen Salmonella enterica serovar Typhimurium or the opportunistic pathogen Candida albicans in combination with metagenomics and discovery metabolomics to identify changes in the community and metabolome during infection. To isolate the role of the microbiota in response to pathogens, we compared mice monocolonized with the pathogen, uninfected mice "humanized" with a synthetic human microbiome, or infected humanized mice. In Salmonella-infected mice, by 3 days into infection, microbiome community structure and function changed substantially, with a rise in Enterobacteriaceae strains and a reduction in biosynthetic gene cluster potential. In contrast, Candida-infected mice had few microbiome changes. The LC-MS metabolomic fingerprint of the cecum differed between mice monocolonized with either pathogen and humanized infected mice. Specifically, we identified an increase in glutathione disulfide, glutathione cysteine disulfide, inosine 5'-monophosphate, and hydroxybutyrylcarnitine in mice infected with Salmonella in contrast to uninfected mice and mice monocolonized with Salmonella These metabolites potentially play a role in pathogen-induced oxidative stress. These results provide insight into how the microbiota community members interact with each other and with pathogens on a metabolic level.IMPORTANCE The gut microbiota is increasingly recognized for playing a critical role in human health and disease, especially in conferring resistance to both virulent pathogens such as Salmonella, which infects 1.2 million people in the United States every year (E. Scallan, R. M. Hoekstra, F. J. Angulo, R. V. Tauxe, et al., Emerg Infect Dis 17:7-15, 2011, https://doi.org/10.3201/eid1701.P11101), and opportunistic pathogens like Candida, which causes an estimated 46,000 cases of invasive candidiasis each year in the United States (Centers for Disease Control and Prevention, Antibiotic Resistance Threats in the United States, 2013, 2013). Using a gnotobiotic mouse model, we investigate potential changes in gut microbial community structure and function during infection using metagenomics and metabolomics. We observe that changes in the community and in biosynthetic gene cluster potential occur within 3 days for the virulent Salmonella enterica serovar Typhimurium, but there are minimal changes with a poorly colonizing Candida albicans In addition, the metabolome shifts depending on infection status, including changes in glutathione metabolites in response to Salmonella infection, potentially in response to host oxidative stress.

Imaging with Mass Spectrometry of Bacteria on the Exoskeleton of Fungus-Growing Ants.

Mass spectrometry imaging is a powerful analytical technique for detecting and determining spatial distributions of molecules within a sample. Typically, mass spectrometry imaging is limited to the analysis of thin tissue sections taken from the middle of a sample. In this work, we present a mass spectrometry imaging method for the detection of compounds produced by bacteria on the outside surface of ant exoskeletons in response to pathogen exposure. Fungus-growing ants have a specialized mutualism with Pseudonocardia, a bacterium that lives on the ants' exoskeletons and helps protect their fungal garden food source from harmful pathogens. The developed method allows for visualization of bacterial-derived compounds on the ant exoskeleton. This method demonstrates the capability to detect compounds that are specifically localized to the bacterial patch on ant exoskeletons, shows good reproducibility across individual ants, and achieves accurate mass measurements within 5 ppm error when using a high-resolution, accurate-mass mass spectrometer.

Examination of Endogenous Peptides in Medicago truncatula Using Mass Spectrometry Imaging.

Plant science is an important, rapidly developing area of study. Within plant science, one area of study that has grown tremendously with recent technological advances, such as mass spectrometry, is the field of plant-omics; however, plant peptidomics is relatively underdeveloped in comparison with proteomics and metabolomics. Endogenous plant peptides can act as signaling molecules and have been shown to affect cell division, development, nodulation, reproduction, symbiotic associations, and defense reactions. There is a growing need to uncover the role of endogenous peptides on a molecular level. Mass spectrometric imaging (MSI) is a valuable tool for biological analyses as it allows for the detection of thousands of analytes in a single experiment and also displays spatial information for the detected analytes. Despite the prediction of a large number of plant peptides, their detection and imaging with spatial localization and chemical specificity is currently lacking. Here we analyzed the endogenous peptides and proteins in Medicago truncatula using matrix-assisted laser desorption/ionization (MALDI)-MSI. Hundreds of endogenous peptides and protein fragments were imaged, with interesting peptide spatial distribution changes observed between plants in different developmental stages.

Mass Spectrometry in Plant-omics.

Plant-omics is rapidly becoming an important field of study in the scientific community due to the urgent need to address many of the most important questions facing humanity today with regard to agriculture, medicine, biofuels, environmental decontamination, ecological sustainability, etc. High-performance mass spectrometry is a dominant tool for interrogating the metabolomes, peptidomes, and proteomes of a diversity of plant species under various conditions, revealing key insights into the functions and mechanisms of plant biochemistry.

Multifaceted investigation of metabolites during nitrogen fixation in Medicago via high resolution MALDI-MS imaging and ESI-MS.

Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Owing to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula-Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula-Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional versus nonfunctional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology.

MALDI-MS-assisted molecular imaging of metabolites in legume plants.

Mass spectrometric imaging (MSI) is a powerful analytical tool that provides spatial information of several compounds in a single experiment. This technique has been used extensively to study proteins, peptides, and lipids, and is becoming more common for studying small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected within a biological tissue section. Herein, we present a method developed specifically for imaging metabolites in legume plant roots and root nodules which can be adapted for studying metabolites in other legume organs and even other biological tissue samples. We focus on essential steps such as sample preparation and matrix application, comparing several useful techniques, and present a standard workflow that can be easily modified for different tissue types and instrumentation.

Optimization and comparison of multiple MALDI matrix application methods for small molecule mass spectrometric imaging.

The matrix application technique is critical to the success of a matrix-assisted laser desorption/ionization (MALDI) experiment. This work presents a systematic study aiming to evaluate three different matrix application techniques for MALDI mass spectrometric imaging (MSI) of endogenous metabolites from legume plant, Medicago truncatula, root nodules. Airbrush, automatic sprayer, and sublimation matrix application methods were optimized individually for detection of metabolites in the positive ionization mode exploiting the two most widely used MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA). Analytical reproducibility and analyte diffusion were examined and compared side-by-side for each method. When using DHB, the optimized method developed for the automatic matrix sprayer system resulted in approximately double the number of metabolites detected when compared to sublimation and airbrush. The automatic sprayer method also showed more reproducible results and less analyte diffusion than the airbrush method. Sublimation matrix deposition yielded high spatial resolution and reproducibility but fewer analytes in the higher m/z range (500-1000 m/z). When the samples were placed in a humidity chamber after sublimation, there was enhanced detection of higher mass metabolites but increased analyte diffusion in the lower mass range. When using CHCA, the optimized automatic sprayer method and humidified sublimation method resulted in double the number of metabolites detected compared to standard airbrush method.

MALDI-mass spectrometric imaging for the investigation of metabolites in Medicago truncatula root nodules.

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied(1). Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples(1-5). This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

Challenges and recent advances in mass spectrometric imaging of neurotransmitters.

Mass spectrometric imaging (MSI) is a powerful tool that grants the ability to investigate a broad mass range of molecules, from small molecules to large proteins, by creating detailed distribution maps of selected compounds. To date, MSI has demonstrated its versatility in the study of neurotransmitters and neuropeptides of different classes toward investigation of neurobiological functions and diseases. These studies have provided significant insight in neurobiology over the years and current technical advances are facilitating further improvements in this field. Herein, we briefly review new MSI studies of neurotransmitters, focusing specifically on the challenges and recent advances of MSI of neurotransmitters.

Measurement of NMDA Receptor Antagonist, CPP, in Mouse Plasma and Brain Tissue Following Systematic Administration Using Ion-Pair LCMS/MS.

(RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) is a competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor and is routinely used with rodent models to investigate the role of NMDA receptors in brain function. This highly polar compound is difficult to separate from biological matrices. A reliable and sensitive assay was developed for the determination of CPP in plasma and tissue. In order to overcome the challenges relating to the physicochemical properties of CPP we employed an initial separation using solid phase extraction harnessing mixed-mode anion exchange. Then an ion-pair UPLC C18 separation was performed followed by MS/MS with a Waters Acquity UPLC interfaced to an AB Sciex QTrap 5500 mass spectrometer, which was operated in positive ion ESI mode. Multiple reaction monitoring (MRM) mode was utilized to detect the analyte and internal standard. The precursor to product ions used for quantitation for CPP and internal standard were m/z 252.958 → 207.100 and 334.955 → 136.033, respectively. This method was applied to a pharmacokinetic study and examined brain tissue and plasma concentrations following intravenous and intraperitoneal injections of CPP. The elimination half-life (t1/2) of CPP was 8.8 minutes in plasma and 14.3 minutes in brain tissue, and the plasma to brain concentration ratio was about 18:1. This pharmacokinetic data will aid the interpretation of the vast number of studies using CPP to investigate NMDA receptor function in rodents and the method itself can be used to study many other highly polar analytes of interest.

Qualitative and quantitative mass spectrometry imaging of drugs and metabolites.

Mass spectrometric imaging (MSI) has rapidly increased its presence in the pharmaceutical sciences. While quantitative whole-body autoradiography and microautoradiography are the traditional techniques for molecular imaging of drug delivery and metabolism, MSI provides advantageous specificity that can distinguish the parent drug from metabolites and modified endogenous molecules. This review begins with the fundamentals of MSI sample preparation/ionization, and then moves on to both qualitative and quantitative applications with special emphasis on drug discovery and delivery. Cutting-edge investigations on sub-cellular imaging and endogenous signaling peptides are also highlighted, followed by perspectives on emerging technology and the path for MSI to become a routine analysis technique.

MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula-Sinorhizobium meliloti symbiosis.

Symbiotic associations between leguminous plants and nitrogen-fixing rhizobia culminate in the formation of specialized organs called root nodules, in which the rhizobia fix atmospheric nitrogen and transfer it to the plant. Efficient biological nitrogen fixation depends on metabolites produced by and exchanged between both partners. The Medicago truncatula-Sinorhizobium meliloti association is an excellent model for dissecting this nitrogen-fixing symbiosis because of the availability of genetic information for both symbiotic partners. Here, we employed a powerful imaging technique - matrix-assisted laser desorption/ionization (MALDI)/mass spectrometric imaging (MSI) - to study metabolite distribution in roots and root nodules of M. truncatula during nitrogen fixation. The combination of an efficient, novel MALDI matrix [1,8-bis(dimethyl-amino) naphthalene, DMAN] with a conventional matrix 2,5-dihydroxybenzoic acid (DHB) allowed detection of a large array of organic acids, amino acids, sugars, lipids, flavonoids and their conjugates with improved coverage. Ion density maps of representative metabolites are presented and correlated with the nitrogen fixation process. We demonstrate differences in metabolite distribution between roots and nodules, and also between fixing and non-fixing nodules produced by plant and bacterial mutants. Our study highlights the benefits of using MSI for detecting differences in metabolite distributions in plant biology.

A vision for better health: mass spectrometry imaging for clinical diagnostics.

BACKGROUND: Mass spectrometry imaging (MSI) is a powerful tool that grants the ability to investigate a broad mass range of molecules from small molecules to large proteins by creating detailed distribution maps of selected compounds. Its usefulness in biomarker discovery towards clinical applications has obtained success by correlating the molecular expression of tissues acquired from MSI with well-established histology. RESULTS: To date, MSI has demonstrated its versatility in clinical applications, such as biomarker diagnostics of different diseases, prognostics of disease severities and metabolic response to drug treatment, etc. These studies have provided significant insight in clinical studies over the years and current technical advances are further facilitating the improvement of this field. Although the underlying concept is simple, factors such as choice of ionization method, sample preparation, instrumentation and data analysis must be taken into account for successful applications of MSI. Herein, we briefly reviewed these key elements yet focused on the clinical applications of MSI that cannot be addressed by other means. CONCLUSIONS: Challenges and future perspectives in this field are also discussed to conclude that the ever-growing applications with continuous development of this powerful analytical tool will lead to a better understanding of the biology of diseases and improvements in clinical diagnostics.

Mass spectrometric evaluation of neuropeptidomic profiles upon heat stabilization treatment of neuroendocrine tissues in crustaceans.

Tissue heat stabilization is a vital component in successful mammalian neuropeptidomic studies. Heat stabilization using focused microwave irradiation, conventional microwave irradiation, boiling, and treatment with the Denator Stabilizor T1 have all proven effective in arresting post-mortem protein degradation. Although research has reported the presence of protein fragments in crustacean hemolymph when protease inhibitors were not added to the sample, the degree to which post-mortem protease activity affects neuropeptidomic tissue studies in crustacean species has not been investigated in depth. This work examines the need for Stabilizor T1 or boiling tissue stabilization methods for neuropeptide studies of Callinectes sapidus (blue crab) pericardial organ tissue. Neuropeptides in stabilized and nonstabilized tissue were extracted using acidified methanol or N,N-dimethylformamide (DMF) and analyzed by MALDI-TOF and nanoLC-ESI-MS/MS platforms. Post-mortem fragments did not dramatically affect MALDI analysis in the range m/z 650-1600, but observations in ESI MS/MS experiments suggest that putative post-mortem fragments can mask neuropeptide signal and add spectral complexity to crustacean neuropeptidomic studies. The impact of the added spectral complexity did not dramatically affect the number of detected neuropeptides between stabilized and nonstabilized tissues. However, it is prudent that neuropeptidomic studies of crustacean species include a preliminary experiment using the heat stabilization method to assess the extent of neuropeptide masking by larger, highly charged molecular species.