Detection and Imaging of Exposure-Related Metabolites and Xenobiotics in Hard Tissues by Laser Sampling and Mass Spectrometry.

The utility of two novel laser-based methods, laser ablation electrospray ionization (LAESI) and laser desorption ionization (LDI) from silicon nanopost array (NAPA), is explored via local analysis and mass spectrometry imaging (MSI) of hard tissues (tooth and hair) for the detection and mapping of organic components. Complex mass spectra are recorded in local analysis mode from tooth dentin and scalp hair samples. Nicotine and its metabolites (cotinine, hydroxycotinine, norcotinine, and nicotine) are detected by LAESI-MS in the teeth of rats exposed to tobacco smoke. The intensities of the detected metabolite peaks are proportional to the degree of exposure. Incorporating ion mobility separation in the LAESI-MS analysis of scalp hair enables the detection of cotinine in smoker hair along with other common molecular species, including endogenous steroid hormones and some lipids. Single hair strands are imaged by MALDI-MSI and NAPA-LDI-MSI to explore longitudinal variations in the level of small molecules. Comparing spectra integrated from NAPA-LDI-MSI and MALDI-MSI images reveals that the two techniques provide complementary information. There were 105 and 82 sample-related peaks for MALDI and NAPA, respectively, with an overlap of only 16 peaks, indicating a high degree of complementarity. Enhanced molecular coverage and spatial resolution offered by LAESI-MS and NAPA-LDI-MSI can reveal the distributions of known and potential biomarkers in hard tissues, facilitating exposome research.

Nutrient supplementation-induced metabolic profile changes and early appearance of free N-glycans in nutrient deficient tomato plants revealed by mass spectrometry.

Inorganic fertilizers are routinely used in large scale crop production for the supplementation of nitrogen, phosphorus, and potassium in nutrient poor soil. To explore metabolic changes in tomato plants grown on humic sand under different nutritional conditions, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was utilized for the analysis of xylem sap. Variations in the abundances of metabolites and oligosaccharides, including free N-glycans (FNGs), were determined. Statistical analysis of the sample-related peaks revealed significant differences in the abundance ratios of multiple metabolites, including oligosaccharides, between the control plants, grown with no fertilizers, and plants raised under "ideal" and "nitrogen deficient" nutritional conditions, i.e., under the three treatment types. Among the 36 spectral features tentatively identified as oligosaccharides, the potential molecular structures for 18 species were predicted based on their accurate masses and isotope distribution patterns. To find the spectral features that account for most of the differences between the spectra corresponding to the three different treatments, multivariate statistical analysis was carried out by orthogonal partial least squares-discriminant analysis (OPLS-DA). They included both FNGs and non-FNG compounds that can be considered as early indicators of nutrient deficiency. Our results reveal that the potential nutrient deficiency indicators can be expanded to other metabolites beyond FNGs. The m/z values for 20 spectral features with the highest variable influence on projection (VIP) scores were ranked in the order of their influence on the statistical model.

High-Throughput f-LAESI-IMS-MS for Mapping Biological Nitrogen Fixation One Cell at a Time.

For the characterization of the metabolic heterogeneity of cell populations, high-throughput single-cell analysis platforms are needed. In this study, we utilized mass spectrometry (MS) enhanced with ion mobility separation (IMS) and coupled with an automated sampling platform, fiber-based laser ablation electrospray ionization (f-LAESI), for in situ high-throughput single-cell metabolomics in soybean (Glycine max) root nodules. By fully automating the in situ sampling platform, an overall sampling rate of 804 cells/h was achieved for high numbers (>500) of tissue-embedded plant cells. This is an improvement by a factor of 13 compared to the previous f-LAESI-MS configuration. By introducing IMS, the molecular coverage improved, and structural isomers were separated on a millisecond time scale. The enhanced f-LAESI-IMS-MS platform produced 259 sample-related peaks/cell, almost twice as much as the 131 sample-related peaks/cell produced by f-LAESI-MS without IMS. Using the upgraded system, two types of metabolic heterogeneity characterization methods became possible. For unimodal metabolite abundance distributions, the metabolic noise reported on the metabolite level variations within the cell population. For bimodal distributions, the presence of metabolically distinct subpopulations was established. Discovering these latent cellular phenotypes could be linked to the presence of different cell states, e.g., proliferating bacteria in partially occupied plant cells and quiescent bacteroids in fully occupied cells in biological nitrogen fixation, or spatial heterogeneity due to altered local environments.

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.

Mass Spectrometry Imaging of Biological Tissues by Laser Desorption Ionization from Silicon Nanopost Arrays.

Mass spectrometry imaging (MSI) plays an expanding role in the label-free spatial mapping of hundreds of molecules simultaneously. Currently, matrix-assisted laser desorption ionization (MALDI) is among the most widely adopted MSI techniques. However, matrix application can impact the fidelity of spatial distributions, and matrix selection and related spectral interferences in the low mass range can lead to biased molecular coverage. Nanophotonic ionization from silicon nanopost arrays (NAPA) is an emerging matrix-free MSI platform with enhanced sensitivity for several molecular classes, for example, neutral lipids and biooligomers. Here, we describe a protocol with minimal sample preparation for NAPA-MSI of metabolites, lipids, and biooligomers from biological tissues.

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.

High-Throughput Analysis of Tissue-Embedded Single Cells by Mass Spectrometry with Bimodal Imaging and Object Recognition.

In biological tissues, cell-to-cell variations stem from the stochastic and modulated expression of genes and the varying abundances of corresponding proteins. These variations are then propagated to downstream metabolite products and result in cellular heterogeneity. Mass spectrometry imaging (MSI) is a promising tool to simultaneously provide spatial distributions for hundreds of biomolecules without the need for labels or stains. Technological advances in MSI instrumentation for the direct analysis of tissue-embedded single cells are dominated by improvements in sensitivity, sample pretreatment, and increased spatial resolution but are limited by low throughput. Herein, we introduce a bimodal microscopy imaging system combined with fiber-based laser ablation electrospray ionization (f-LAESI) MSI with improved throughput ambient analysis of tissue-embedded single cells (n > 1000) to provide insight into cellular heterogeneity. Based on automated image analysis, accurate single-cell sampling is achieved by f-LAESI leading to the discovery of cellular phenotypes characterized by differing metabolite levels.

Neuropeptide Localization in Lymnaea stagnalis: From the Central Nervous System to Subcellular Compartments.

Due to the relatively small number of neurons (few tens of thousands), the well-established multipurpose model organism Lymnaea stagnalis, great pond snail, has been extensively used to study the functioning of the nervous system. Unlike the more complex brains of higher organisms, L. stagnalis has a relatively simple central nervous system (CNS) with well-defined circuits (e.g., feeding, locomotion, learning, and memory) and identified individual neurons (e.g., cerebral giant cell, CGC), which generate behavioral patterns. Accumulating information from electrophysiological experiments maps the network of neuronal connections and the neuronal circuits responsible for basic life functions. Chemical signaling between synaptic-coupled neurons is underpinned by neurotransmitters and neuropeptides. This review looks at the rapidly expanding contributions of mass spectrometry (MS) to neuropeptide discovery and identification at different granularity of CNS organization. Abundances and distributions of neuropeptides in the whole CNS, eleven interconnected ganglia, neuronal clusters, single neurons, and subcellular compartments are captured by MS imaging and single cell analysis techniques. Combining neuropeptide expression and electrophysiological data, and aided by genomic and transcriptomic information, the molecular basis of CNS-controlled biological functions is increasingly revealed.

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

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

Mass Spectrometry Imaging of Bio-oligomer Polydispersity in Plant Tissues by Laser Desorption Ionization from Silicon Nanopost Arrays.

Mass spectrometry imaging (MSI) enables simultaneous spatial mapping for diverse molecules in biological tissues. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has been a mainstream MSI method for a wide range of biomolecules. However, MALDI-MSI of biological homopolymers used for energy storage and molecular feedstock is limited by, e.g., preferential ionization for certain molecular classes. Matrix-free nanophotonic ionization from silicon nanopost arrays (NAPAs) is an emerging laser desorption ionization (LDI) platform with ultra-trace sensitivity and molecular imaging capabilities. Here, we show complementary analysis and MSI of polyhydroxybutyric acid (PHB), polyglutamic acid (PGA), and polysaccharide oligomers in soybean root nodule sections by NAPA-LDI and MALDI. For PHB, number and weight average molar mass, polydispersity, and oligomer size distributions across the tissue section and in regions of interest were characterized by NAPA-LDI-MSI.

Multimodal imaging of biological tissues using combined MALDI and NAPA-LDI mass spectrometry for enhanced molecular coverage.

Mass spectrometry imaging (MSI) is a powerful analytical technique that enables detection, discovery, and identification of multiple classes of biomolecules, while simultaneously mapping their spatial distributions within a sample (e.g., a section of biological tissue). The limitation in molecular coverage afforded by any single MSI platform has led to the development of multimodal approaches that incorporate two or more techniques to obtain greater chemical information. Matrix-assisted laser desorption ionization (MALDI) is a preeminent ionization technique for MSI applications because the wide range of available matrices allows some degree of enhancement with respect to the detection of particular molecular classes. Nonetheless, MALDI has a limited ability to detect and image several classes of molecules, e.g., neutral lipids, in complex samples. Laser desorption ionization from silicon nanopost arrays (NAPA-LDI or NAPA) has been shown to offer complementary coverage with respect to MALDI by providing improved detection of neutral lipids and some small metabolites. Here, we present a multimodal imaging method in which a single tissue section is consecutively imaged at low and high laser fluences, generating spectra that are characteristic of MALDI and NAPA ionization, respectively. The method is demonstrated to map the distributions of species amenable to detection by MALDI (e.g., phospholipids and intermediate-mass metabolites) and NAPA (e.g., neutral lipids such as triglycerides and hexosylceramides, and small metabolites) in mouse brain and lung tissue sections.

Remote ablation chamber for high efficiency particle transfer in laser ablation electrospray ionization mass spectrometry.

Laser ablation electrospray ionization (LAESI) driven by mid-infrared laser pulses allows the direct analysis of biological tissues with minimal sample preparation. Dedicated remote ablation chambers have been developed to eliminate the need for close proximity between the sample and the mass spectrometer inlet. This also allows for the analysis of large or irregularly shaped objects, and incorporation of additional optics for microscopic imaging. Here we report on the characterization of a newly designed conical inner volume ablation chamber working in transmission geometry, where a reduced zone of stagnation was achieved by tapering the sample platform and the chamber outlet. As a result, the transmission efficiency of both large (>7.5 µm) and smaller particulates (<6.5 µm) has increased significantly. Improved analytical figures of merit, including 300 fmol limit of detection, and three orders of magnitude in dynamic range, were established. Particle residence time, measured by the FWHM of the analyte signal, was reduced from 2.0 s to 0.5 s enabling higher ablation rates and shorter analysis time. A total of six glucosinolates (sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, and glucohirsutin) were detected in plant samples with ion abundances higher by a factor of 2 to 8 for the redesigned ablation chamber.

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.

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

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 in situ 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.

In Vivo Chemical Analysis of Plant Sap from the Xylem and Single Parenchymal Cells by Capillary Microsampling Electrospray Ionization Mass Spectrometry.

In plants, long-distance transport of chemicals from source to sink takes place through the transfer of sap inside complex trafficking systems. Access to this information provides insight into the physiological responses that result from the interactions between the organism and its environment. In vivo analysis offers minimal perturbation to the physiology of the organism, thus providing information that represents the native physiological state more accurately. Here we describe capillary microsampling with electrospray ionization mass spectrometry (ESI-MS) for the in vivo analysis of xylem sap directly from plants. Initially, fast MS profiling was performed by ESI from the whole sap exuding from wounds of living plants in their native environment. This sap, however, originated from the xylem and phloem and included the cytosol of damaged cells. Combining capillary microsampling with ESI-MS enabled targeted sampling of the xylem sap and single parenchymal cells in the pith, thereby differentiating their chemical compositions. With this method we analyzed soybean plants infected by nitrogen-fixing bacteria and uninfected plants to investigate the effects of symbiosis on chemical transport through the sap. Infected plants exhibited higher abundances for certain nitrogen-containing metabolites in their sap, namely allantoin, allantoic acid, hydroxymethylglutamate, and methylene glutamate, compared to uninfected plants. Using capillary microsampling, we localized these compounds to the xylem, which indicated their transport from the roots to the upper parts of the plant. Differences between metabolite levels in sap from the infected and uninfected plants indicated that the transport of nitrogen-containing and other metabolites is regulated depending on the source of nitrogen supply.

The Molecular Composition of Soot.

Soot (sometimes referred to as black carbon) is produced when hydrocarbon fuels are burned. Our hypothesis is that polynuclear aromatic hydrocarbon (PAH) molecules are the dominant component of soot, with individual PAH molecules forming ordered stacks that agglomerate into primary particles (PP). Here we show that the PAH composition of soot can be exactly determined and spatially resolved by low-fluence laser desorption ionization, coupled with high-resolution mass spectrometry imaging. This analysis revealed that PAHs of 239-838 Da, containing few oxygenated species, comprise the soot observed in an ethylene diffusion flame. As informed by chemical graph theory (CGT), the vast majority of species observed in the sampled particulate matter may be described as benzenoids, consisting of only fused 6-membered rings. Within that limit, there is clear evidence for the presence of radical PAH in the particulate samples. Further, for benzenoid structures the observed empirical formulae limit the observed isomers to those which are nearly circular with high aromatic conjugation lengths for a given aromatic ring count. These results stand in contrast to recent reports that suggest higher aliphatic composition of primary particles.

Mass spectrometry imaging of triglycerides in biological tissues by laser desorption ionization from silicon nanopost arrays.

Mass spectrometry imaging (MSI) is used increasingly to simultaneously detect a broad range of biomolecules while mapping their spatial distributions within biological tissue sections. Matrix-assisted laser desorption ionization (MALDI) is recognized as the method-of-choice for MSI applications due in part to its broad molecular coverage. In spite of the remarkable advantages offered by MALDI, imaging of neutral lipids, such as triglycerides (TGs), from tissue has remained a significant challenge due to ion suppression of TGs by phospholipids, e.g. phosphatidylcholines (PCs). To help overcome this limitation, silicon nanopost array (NAPA) substrates were introduced to selectively ionize TGs from biological tissue sections. This matrix-free laser desorption ionization (LDI) platform was previously shown to provide enhanced ionization of certain lipid classes, such as hexosylceramides (HexCers) and phosphatidylethanolamines (PEs) from mouse brain tissue. In this work, we present NAPA as an MSI platform offering enhanced ionization efficiency for TGs from biological tissues relative to MALDI, allowing it to serve as a complement to MALDI-MSI. Analysis of a standard lipid mixture containing PC(18:1/18:1) and TG(16:0/16:0/16:0) by LDI from NAPA provided an ~49 and ~227-fold higher signal for TG(16:0/16:0/16:0) relative to MALDI, when analyzed without and with the addition of a sodium acetate, respectively. In contrast, MALDI provided an ~757 and ~295-fold higher signal for PC(18:1/18:1) compared with NAPA, without and with additional Na+ . Averaged signal intensities for TGs from MSI of mouse lung and human skin tissues exhibited an ~105 and ~49-fold increase, respectively, with LDI from NAPA compared with MALDI. With respect to PCs, MALDI provided an ~2 and ~19-fold increase in signal intensity for mouse lung and human skin tissues, respectively, when compared with NAPA. The complementary coverage obtained by the two platforms demonstrates the utility of using both techniques to maximize the information obtained from lipid MS or MSI experiments.

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

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 Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae 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.

Identification of Metabolites in Single Cells by Ion Mobility Separation and Mass Spectrometry.

Non-targeted metabolic analysis of single cells by mass spectrometry (MS) is important for understanding individual cell functions and characterizing cell-to-cell heterogeneity. However, identifying biomolecules in single cells presents significant challenges due to the low picoliter volume samples and the structural diversity of metabolites. Capillary microsampling electrospray ionization (ESI) MS with ion mobility separation (IMS) enables the analysis of single cells under ambient conditions with minimum sample pretreatment and improved specificity. Here, we describe a protocol for the analysis of the metabolic makeup, and the identification of ions produced from single cells by capillary microsampling ESI-IMS-MS.

Toward Single Cell Molecular Imaging by Matrix-Free Nanophotonic Laser Desorption Ionization Mass Spectrometry.

In recent years, innovations in mass spectrometry imaging (MSI) have enabled simultaneous detection and mapping of biomolecules and xenobiotics directly from biological tissues and single cells. Matrix-assisted laser desorption ionization (MALDI) has been the most widely embraced MSI technique. However, this technique can exhibit ion suppression effects hindering metabolite coverage and possesses a narrow dynamic range. Nanophotonic platforms, e.g., silicon nanopost array (NAPA) structures, can be used as an alternative for matrix-free imaging of biological tissues. Here, we present a protocol for MSI of large and small adherent cell clusters by laser desorption ionization from NAPA with minimal sample preparation.