Single cell metabolomics is an emerging and rapidly developing field that complements developments in single cell analysis by genomics and proteomics. Major goals include mapping and quantifying the metabolome in sufficient detail to provide useful information about cellular function in highly heterogeneous systems such as tissue, ultimately with spatial resolution at the individual cell level. The chemical diversity and dynamic range of metabolites poses particular challenges for detection, identification and quantification. In this review we discuss both significant technical issues of measurement and interpretation, and progress toward addressing them, with recent examples from diverse biological systems. We provide a framework for further directions aimed at improving workflow and robustness so that such analyses may become commonly applied, especially in combination with metabolic imaging and single cell transcriptomics and proteomics.
Research continues to identify genetic variation, environmental exposures, and their mixtures underlying different diseases and conditions. There is a need for screening methods to understand the molecular outcomes of such factors. Here, we investigate a highly efficient and multiplexable, fractional factorial experimental design (FFED) to study six environmental factors (lead, valproic acid, bisphenol A, ethanol, fluoxetine hydrochloride and zinc deficiency) and four human induced pluripotent stem cell line derived differentiating human neural progenitors. We showcase the FFED coupled with RNA-sequencing to identify the effects of low-grade exposures to these environmental factors and analyse the results in the context of autism spectrum disorder (ASD). We performed this after 5-day exposures on differentiating human neural progenitors accompanied by a layered analytical approach and detected several convergent and divergent, gene and pathway level responses. We revealed significant upregulation of pathways related to synaptic function and lipid metabolism following lead and fluoxetine exposure, respectively. Moreover, fluoxetine exposure elevated several fatty acids when validated using mass spectrometry-based metabolomics. Our study demonstrates that the FFED can be used for multiplexed transcriptomic analyses to detect relevant pathway-level changes in human neural development caused by low-grade environmental risk factors. Future studies will require multiple cell lines with different genetic backgrounds for characterising the effects of environmental exposures in ASD.
Mass Spectrometry Imaging (MSI) characterizes changes in chemical composition between regions of biological samples such as tissues. One goal of statistical analysis of MSI experiments is class comparison, i.e. determining analytes that change in abundance between conditions more systematically than as expected by random variation. To reach accurate and reproducible conclusions, statistical analysis must appropriately reflect the initial research question, the design of the MSI experiment, and all the associated sources of variation. This manuscript highlights the importance of following these general statistical principles. Using the example of two case studies with complex experimental designs, and with different strategies of data acquisition, we demonstrate the extent to which choices made at key points of this workflow impact the results, and provide suggestions for appropriate design and analysis of MSI experiments that aim at detecting differentially abundant analytes. (C) 2018 Elsevier B.V. All rights reserved.
Over the past few decades, the chemical characterization of single cells has improved immensely. In particular, mass spectrometry (MS) has pioneered direct analysis of metabolites, lipids, and peptides from single cells. This progress has been enabled by new and improved strategies for ionization and sampling, where a multitude of techniques for single‐cell MS has contributed unique insights to many different disciplines. Here, an overview of the main three techniques secondary ion mass spectrometry (SIMS), matrix‐assisted laser desorption ionization (MALDI), and ambient ionization for direct single‐cell MS analysis are presented, including some example studies detailing the use of single‐cell MS.
Molecular profiling of single cells has the potential to significantly advance our understanding of cell function and cellular processes of importance to health and disease. In particular, small molecules with rapid turn-over rates can reveal activated metabolic pathways resulting from an altered chemical environment or cellular events such as differentiation. Consequently, techniques for quantitative metabolite detection acquired in a higher throughput manner are needed to characterize the biological variability between seemingly homogenous cells. Here, we show that nanospray desorption electrospray ionization (nano-DESI) mass spectrometry ( MS) enables sensitive molecular profiling and quantification of endogenous species in single cells in a higher throughput manner. Specifically, we show a large number of detected amino acids and phospholipids, including plasmalogens, readily detected from single cheek cells. Further, by incorporating a phosphatidylcholine ( PC) internal standard into the nano-DESI solvent, we determined the total amount of PC in one cell to be 1.2 pmoles. Finally, we describe a higher throughput approach where molecules in single cells are automatically profiled. These developments in single cell analysis provide a basis for future studies to understand cellular processes related to drug effects, cell differentiation and altered chemical microenvironments.
Diabetic kidney disease is a serious complication of diabetes that can ultimately lead to end-stage renal disease. The pathogenesis of diabetic kidney disease is complex, and fundamental research is still required to provide a better understanding of the driving forces behind it. We report regional metabolic aberrations from an untargeted mass spectrometry imaging study of kidney tissue using an insulinopenic rat model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidneys were harvested 2weeks thereafter. Imaging was performed using nanospray desorption electrospray ionization connected to a high-mass-resolving mass spectrometer. No histopathological changes were observed in the kidney sections; however, mass spectrometry imaging revealed a significant increase in several 18-carbon unsaturated non-esterified fatty acid species and monoacylglycerols. Notably, these 18-carbon acyl chains were also constituents of several increased diacylglycerol species. In addition, a number of short- and long-chain acylcarnitines were found to be accumulated while several amino acids were depleted. This study presents unique regional metabolic data indicating a dysregulated energy metabolism in renal mitochondria as an early response to streptozotocin-induced type I diabetes.
Small molecule neurotransmitters are essential for the function of the nervous system, and neurotransmitter imbalances are often connected to neurological disorders. The ability to quantify such imbalances is important to provide insights into the biochemical mechanisms underlying the disorder. This proof-of-principle study presents online quantification of small molecule neurotransmitters, specifically acetylcholine, γ-aminobutyric acid (GABA) and glutamate, in rat brain tissue sections using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging. By incorporating deuterated internal standards in the nano-DESI solvent we show identification, accurate mapping, and quantification of these small neurotransmitters in rat brain tissue without introducing any additional sample preparation steps. We find that GABA is about twice as abundant in the medial septum-diagonal band complex (MSDB) as in the cortex, while glutamate is about twice as abundant in the cortex as compared to the MSDB. The study shows that nano-DESI is well suited for imaging of small molecule neurotransmitters in health and disease.
A simple and rapid capillary electrophoresis method with direct ultraviolet (UV) detection was set up for the determination of four organic acids in beverages. The method included dilution and filtration as simple sample preparation steps. The electrophoretic separation and detection of oxalic, malic, citric and lactic acids in wines and beers were performed in 8 min. For the method validation, linearity, detection and quantification limits, repeatability and recovery in wine and beer matrices were studied. Good linearity was observed from 25 to 500 mg/L for all acids excluding lactic acid, for which it started from 50 mg/L. The limits of quantitation of oxalic, malonic and citric acid were set 9.5 to 28.5 mg/L. Repeatability of this method was from 3.2 to 7.3%, recoveries ranged from 90.1 to 110.1%. The validated method was applied to the analysis of different wines and beers and showed great variability in their composition.
Counterfeit pharmaceutical drugs imply an increasing threat to the global public health. It is necessary to have systems to control the products that reach the market and to detect falsified medicines. In this work, molecules in several pharmaceutical tablets were directly analyzed using nanospray desorption electrospray ionization mass spectrometry (nano-DESI MS). Nano-DESI is an ambient surface sampling technique which enables sampling of molecules directly from the surface of the tablets without any sample pretreatment. Both the active pharmaceutical ingredients (APIs) and some excipients were detected in all analyzed tablets. Principal component analysis was used to analyze mass spectral features from different tablets showing strong clustering between tablets with different APIs. The obtained results suggest nano-DESI MS as future tool for forensic analysis to discern APIs present in unknown tablet samples.
Polydimethylsiloxane (PDMS) is among the most widely used materials for organ-on-chip systems. Despite itsmultiple beneficial characteristics from an engineering point of view, there is a concern about the effect of PDMSon the cells cultured in such devices. The aim of this study was to enhance the understanding of the effect of PDMSon cellular behavior in a context relevant for on-chip studies. The focus was put on an indirect effect of PDMS,namely leaching of uncrosslinked oligomers, particularly for bone regeneration applications. PDMS-based chipswere prepared and analyzed for the potential release of PDMS oligomers within the microfluidic channel whenkept at different flow rates. Leaching of uncrosslinked oligomers from PDMS was quantified as silicon concen-tration by inductively coupled plasma - optical emission spectrometry and further confirmed by mass spec-trometry. Subsequently, PDMS-leached media, with a silicon concentration matching the on-chip experiment,were prepared to study cell proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts and humanmesenchymal stem cells. The silicon concentration initially detected in the media was inversely proportional tothe tested flow rates and decreased to control levels within 52 h. In addition, by curing the material overnightinstead of 2 h, regardless of the curing temperature (65 and 80 C), a large reduction in silicon concentration wasfound, indicating the importance of the PDMS curing parameters. Furthermore, it was shown that PDMS oligo-mers enhanced the differentiation of MC3T3-E1 pre-osteoblasts, this being a cell type dependent effect as nochanges in cell differentiation were observed for human mesenchymal stem cells. Overall, this study illustrates theimportance of optimization steps when using PDMS devices for biological studies, in particular PDMS curingconditions and extensive washing steps prior to an experiment.
Liquid extraction mass spectrometry imaging (MSI) experiments provide users with direct analysis of biological surfaces with minimal sample preparation. Until now, much of the effort to increase spatial resolution for MSI with liquid extraction techniques has focused on reducing the size of the sampling area. However, this can be experimentally challenging. Here, we present oversampling as a simple alternative to increase the spatial resolution using nanospray desorption electrospray ionization (nano-DESI) MSI. By imaging partial rat spinal cord tissue sections, two major concerns with oversampling are addressed: whether endogenous molecules are significantly depleted from repeated sampling events and whether analytes are redistributed as a result of oversampling. In depth examination of ion images for representative analytes show that depletion and redistribution do not affect analyte localization in the tissue sample. Nano-DESI MSI experiments using three times oversampling provided higher spatial resolution, allowing the observation of features not visible with undersampling. Although proper care must be taken to ensure that oversampling will work in specific applications, we envision oversampling as a simple approach to increase image quality for liquid extraction MSI techniques.
Nanospray desorption electrospray ionization (nano-DESI) has been established as a powerful technique for mass spectrometry imaging (MSI) of biomolecules from tissue samples. The direct liquid extraction of analytes from a surface at ambient pressure negates the need for significant sample preparation or matrix application. Although many recent studies have applied nano-DESI to new and exciting applications, there has not been much work in the development and improvement of the nano-DESI source. Here, we incorporate a nebulizer to replace the self-aspirating secondary capillary in the conventional nano-DESI setup, and characterize the device by use of rat kidney tissue sections. We find that the pneumatically assisted nano-DESI device offers improved sensitivity for metabolite species by 1-3 orders of magnitude through more complete desolvation and reduced ionization suppression. Further, the pneumatically assisted nano-DESI device reduces the dependence on probe-to-surface distance and enables sampling and imaging using pure water as the nano-DESI solvent. This provides exclusive detection and imaging of many highly polar endogenous species. Overall, the developed pneumatically assisted nano-DESI device provides more versatile solvent selection and an increased sensitivity for metabolites, which generates ion images of higher contrast - allowing for more intricate studies of metabolite distribution.
Prostaglandins (PG) are an important class of lipid biomolecules that are essential in many biological processes, including inflammation and successful pregnancy. Despite a high bioactivity, physiological concentrations are typically low, which makes direct mass spectrometric analysis of endogenous PG species challenging. Consequently, there have not been any studies investigating PG localization to specific morphological regions in tissue sections using mass spectrometry imaging (MSI) techniques. Herein, we show that silver ions, added to the solvent used for nanospray desorption electrospray ionization (nano-DESI) MSI, enhances the ionization of PGs and enables nano-DESI MSI of several species in uterine tissue from day 4 pregnant mice. It was found that detection of [PG + Ag](+) ions increased the sensitivity by similar to 30 times, when compared to [PG - H](-) ions. Further, the addition of isotopically labeled internal standards enabled generation of quantitative ion images for the detected PG species. Increased sensitivity and quantitative MSI enabled the first proof-of-principle results detailing PG localization in mouse uterus tissue sections. These results show that PG species primarily localized to cellular regions of the luminal epithelium and glandular epithelium in uterine tissue. Further, this study provides a unique scaffold for future studies investigating the PG distribution within biological tissue samples.
Metabolomics has grown into a prominent field contributing to the molecular understanding of complex biological processes in both health and disease. Furthermore, single-cells are known to display metabolic differences between seemingly homogeneous populations of cells. Single-cell metabolomics attempts to analyze many cellular metabolites from single cells to understand phenotypic heterogeneity, which is a significant challenge due to the low analyte abundances and limited sample volumes. Label-free metabolite detection can be achieved with mass spectrometry, which is capable of simultaneously analyzing hundreds of metabolites. Herein, we review the recent advances in mass spectrometry based single-cell metabolomics, highlighting the current state-of-the-art within the last three years, and identify the challenges to move the field forward.
Oil sands process waters can release toxic naphthenic acids (NAs) into aquatic environments. Analytical techniques for NAs are challenged by sample complexity and interference from naturally occurring dissolved organic matter (DOM). Herein, we report the use of a poly(dimethylsiloxane) (PDMS) polymer membrane for the on-line separation of NAs from DOM and use direct infusion electrospray ionization mass spectrometry to yield meaningful qualitative and quantitative information with minimal sample cleanup. We compare the composition of membrane-permeable species from natural waters fortified with a commercial NA mixture to those derived from weak anion exchange solid-phase extraction (SPE) using high-resolution mass spectrometry. The results show that SPE retains a wide range of carboxylic acids, including biogenic DOM, while permeation through PDMS was selective for petrogenic classically defined NAs (CnH2n+zO2). A series of model compounds (log K-ow similar to 1-7) were used to characterize the perm-selectivity and reveal the separation is based on hydrophobicity. This convenient sample cleanup method is selective for the O-2 class of NAs and can be used prior to conventional analysis or as an on-line analytical strategy when coupled directly to mass spectrometry.
Capillary electrophoresis mass spectrometry (CE-MS) is an established technique for targeted and untargeted analysis of metabolites from complex biological samples. However, current CE-MS devices rely on liquid sample extracts, which restricts acquisition of spatially defined chemical information from tissue samples. The ability to chemically profile distinct cellular regions in tissue can contribute better understanding to molecular foundations in health and disease. Therefore, we describe the first CE-MS device capable of untargeted metabolite profiling directly from defined morphological regions of solid tissue sections. With surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS), endogenous molecules are sampled and detected from a single defined tissue location. Characterization of SS-CE MS from different locations of the outer epidermal layer of A. Cepa demonstrated reproducible relative migration times and a peak area RSD of 20% (n = 5). Further, relative migration times were conserved for endogenous metabolites in tissues with varying complexities, including brain, spinal cord, and kidney. Results from proof-of-principle experiments from distinct morphological tissue regions reveal simultaneous analysis of small and large biomolecules, confident metabolite annotation, identification of in-source fragmentation interferences, and discrete isomeric abundances related to biological function. We envision that this new tool will provide in-depth chemical profiling and annotation of molecules in distinct cellular regions of tissue for improved biological understanding.
Duncan et al. use a mass spectrometry imaging method to assess the localization and concentration of prostaglandins (PGs) in mouse tissues during pregnancy. This study brings new biological insights into the spatial evaluation of PGs in tissues, which could reveal the functional significance of each PGs during different stages of embryo development/pregnancy. Prostaglandins are important lipids involved in mediating many physiological processes, such as allergic responses, inflammation, and pregnancy. However, technical limitations of in-situ prostaglandin detection in tissue have led researchers to infer prostaglandin tissue distributions from localization of regulatory synthases, such as COX1 and COX2. Herein, we apply a novel mass spectrometry imaging method for direct in situ tissue localization of prostaglandins, and combine it with techniques for protein expression and RNA localization. We report that prostaglandin D-2, its precursors, and downstream synthases co-localize with the highest expression of COX1, and not COX2. Further, we study tissue with a conditional deletion of transformation-related protein 53 where pregnancy success is low and confirm that PG levels are altered, although localization is conserved. Our studies reveal that the abundance of COX and prostaglandin D-2 synthases in cellular regions does not mirror the regional abundance of prostaglandins. Thus, we deduce that prostaglandins tissue localization and abundance may not be inferred by COX or prostaglandin synthases in uterine tissue, and must be resolved by an in situ prostaglandin imaging.
Capillary electrophoresis (CE) is a powerful separation tool for non-targeted analysis of chemically complex samples, such as blood, urine, and tissue. However, traditionally CE requires samples in solution for analysis, which limits information on analyte distribution and heterogeneity in tissue. The recent development of surface sampling CE–mass spectrometry (SS-CE–MS) brings these advantages of CE to solid samples and enables chemical mapping directly from the tissue surface without laborious sample preparation. Here, we describe developments of SS-CE–MS to increase reproducibility and stability for metabolite, lipid, and protein extraction from tissue sections and dried blood spots. Additionally, we report the first electrokinetic sequential sample injection for high throughput analysis. We foresee that the wide molecular coverage from a distinct tissue region in combination with higher throughput will provide novel information on biological function and dysfunction.
BACKGROUND: Although apoE-/- mice are characterized by hypercholesterolemia, the bile acid enterohepatic circulation, which plays a crucial role in cholesterol homeostasis, has not been examined in these mice. The differences between apoE-/- and C57BL/6 mice in expression of the ileal ASBT and ILBP and in intestinal bile acid absorption were studied.
METHODS: The intestinal tissues of the fetal, neonatal and post-weaning mice were processed for immunohistochemistry. Body retention and fecal excretion of 75SeHCAT were measured. The bile acid pool size and its composition were analysed by HPLC.
RESULTS: In apoE-/- and C57BL/6 mice, the bile acid pool size was 75 +/- 13 and 78 +/- 13 micromol/ 100 g body weight, respectively, while the ratio of cholic acid/beta-muricholic acid was 1.8 +/- 0.3 and 1.4 +/- 0.3 (P < 0.05), respectively. The daily body retention of 75SeHCAT was 48% = 1.8% in C57 black mice and 58.4% +/- 2.7% in apoE-/- mice (P < 0.05). In both mouse strains, ASBT expression in the small intestine was found in the near-term fetal and post-weaning mice, while ILBP expression was found in all postnatal mice. In the post-weaning mice, ILBP expression was limited to the distal 25%-30% of the small intestine, while ASBT expression was limited to the distal 18%.
CONCLUSIONS: The bile acid enterohepatic circulation in apoE-/- mice probably does not differ greatly from that in C57BL/6 mice.
Nanospray desorption electrospray ionization (nano-DESI) combined with tandem mass spectrometry (MS/MS), high-resolution mass analysis of the fragment ions (m/Δm = 17 500 at m/z 200), and rapid spectral acquisition enabled simultaneous imaging and identification of a large number of metabolites and lipids from 92 selected m/z windows (±1 Da) with a spatial resolution of better than 150 μm. Mouse uterine sections of implantation sites on day 6 of pregnancy were analyzed in the ambient environment without any sample pretreatment. MS/MS imaging was performed by scanning the sample under the nano-DESI probe at 10 μm/s, while higher-energy collision-induced dissociation (HCD) spectra were acquired for a targeted inclusion list of 92 m/z values at a rate of ∼6.3 spectra/s. Molecular ions and their corresponding fragments, separated by high-resolution mass analysis, were assigned on the basis of accurate mass measurement. Using this approach, we were able to identify and image both abundant and low-abundance isobaric and isomeric species within each m/z window. MS/MS analysis enabled efficient separation and identification of isomeric and isobaric phospholipids that are difficult to separate in full-scan mode. Furthermore, we identified several metabolites associated with early pregnancy and obtained the first 2D images of these molecules.
Here we demonstrate that conditional deletion of mouse uterine Trp53 (p53(d/d)), molecularly linked to mTORC1 activation and causally linked to premature uterine senescence and preterm birth, results in aberrant lipid signatures within the heterogeneous cell types of embryo implantation sites on day 8 of pregnancy. In situ nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) was used to characterize the molecular speciation of free fatty acids, monoacylglycerol species, unmodified and oxidized phosphatidylcholine (PC/Ox-PC), and diacylglycerol (DG) species within implantation sites of p53(d/d) mice and floxed littermates. Implantation sites from p53(d/d) mice exhibited distinct spatially resolved changes demonstrating accumulation of DG species, depletion of Ox-PC species, and increase in species with more unsaturated acyl chains, including arachidonic and docosahexaenoic acid. Understanding abnormal changes in the abundance and localization of individual lipid species early in the progression to premature birth is an important step toward discovering novel targets for treatments and diagnosis.
Microorganisms release a diversity of organic compounds that couple interspecies metabolism, enable communication, or provide benefits to other microbes. Increased knowledge of microbial metabolite production will contribute to understanding of the dynamic microbial world and can potentially lead to new developments in drug discovery, biofuel production, and clinical research. Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that enables detailed chemical characterization of molecules from a specific location on a surface without special sample pretreatment. Due to its ambient nature, living bacterial colonies growing on agar plates can be rapidly analyzed without affecting the viability of the colony. In this study we demonstrate for the first time the utility of nano-DESI for spatial profiling of chemical gradients generated by microbial communities on agar plates. We found that despite the high salt content of the agar used in this study (~350 mM), nano-DESI analysis enables detailed characterization of metabolites produced by the Synechococcus sp. PCC 7002 colonies. High resolution mass spectrometry and MS/MS analysis of the living Synechococcus sp. PCC 7002 colonies allowed us to detect metabolites and lipids on the colony and on the surrounding agar, and confirm their identities. High sensitivity of nano-DESI enabled identification of several glycolipids that have not been previously reported by extracting the cells using conventional methods. Spatial profiling demonstrated that a majority of lipids and metabolites were localized on the colony while sucrose and glucosylglycerol, an osmoprotective compound produced by cyanobacteria, were secreted onto agar. Furthermore, we demonstrated that the chemical gradients of sucrose and glucosylglycerol on agar depend on the age of the colony. The methodology presented in this study will facilitate future studies focused on molecular-level characterization of interactions between bacterial colonies.
An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours by precisely controlling the distance between the sample and the nano-DESI probe. This is achieved by mounting the sample holder onto an automated XYZ stage, defining the tilt of the sample plane, and recalculating the vertical position of the stage at each point. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSI QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. Low background noise enables simultaneous detection of lipids and metabolites in the tissue section. High-resolution mass analysis combined with tandem mass spectometry (MS/MS) experiments enabled identification of the observed species. In addition, the high dynamic range (>2000) of the technique allowed us to generate ion images of low-abundance isobaric lipids. A high-spatial resolution image was acquired over a small region of the tissue section revealing the distribution of an abundant brain metabolite, creatine, on the boundary between the white and gray matter. The observed distribution is consistent with the literature data obtained using magnetic resonance spectroscopy.
Since the discovery of the anaerobic ammonium oxidizing (anammox) bacteria, many attempts have been made in order to identify these environmentally important bacteria in natural environments. Anammox bacteria contain a unique class of lipids, called ladderane lipids and here we present a novel method to detect viable anammox bacteria in sediments and waste water treatment plants based on the use of a ladderane lipid biomarker. Intact ladderane phosphatidylcholine (PC) lipids are analyzed using reversed-phase liquid chromatography-electrospray ionization-mass spectrometry. Following extraction from the complex sediment matrix, reversed-phase LC is used to separate ladderane PC lipids based on their tail group hydrophobicity as well as their ether or ester link to the glycerol backbone in the sn-2 position. We investigate the presence of intact ladderane lipids in natural sediments displaying anammox activity and illustrate the use of a specific intact membrane forming PC lipid as a biomarker for viable anammox bacterial cells. The presented method can be used to elucidate the whereabouts of viable anammox bacteria, subsequently enabling an estimation of anammox activity. This will greatly increase the knowledge of anammox bacteria and their importance in the global nitrogen cycle.
Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) imaging yields molecule-specific images showing the spatial distribution of specific lipids with submicron resolution, making it a potentially powerful method for studying a variety of biological questions in single cells. In order to make possible the analysis of hydrated cells in vacuum, we have designed a device for in situ freeze-fracture of cell samples. PC12 cells are frozen between two silicon shards in a sandwich geometry, and the fracture is conducted under vacuum inside the analysis chamber of the instrument. After freeze-fracture, ToF-SIMS analysis was carried out using a Bi cluster ion source, providing high-resolution images of single cells. We also show that when combining freeze-fracture and cluster SIMS imaging, low abundant biologically important lipids, specifically PE, PC and non-SM PC, can be imaged in single cells. Finally, K+ ion localization was used as a diagnostic to identify fracture planes through the cell for these fractured samples.
An in situ freeze fracture device featuring a spring-loaded trap system has been designed and characterized for time of flight secondary ion mass spectrometry (TOF SIMS) analysis of single cells. The device employs the sandwich assembly, which is typically used in freeze fracture TOF SIMS experiments to prepare frozen, hydrated cells for high-resolution SIMS imaging. The addition of the spring-loaded trap system to the sandwich assembly offers two advances to this sample preparation method. First, mechanizing the fracture by adding a spring standardizes each fracture by removing the need to manually remove the top of the sandwich assembly with a cryogenically cooled knife. A second advance is brought about because the top of the sandwich is not discarded after the sandwich assembly has been fractured. This results in two imaging surfaces effectively doubling the sample size and providing the unique ability to image both sections of a cell bifurcated by the fracture. Here, we report TOF SIMS analysis of freeze fractured rat pheochromocytoma (PC12) cells using a Bi cluster ion source. This work exhibits the ability to obtain single cell chemical images with subcellular lateral resolution from cells preserved in an ice matrix. In addition to preserving the cells, the signal from lipid fragment ions rarely identified in single cells are better observed in the freeze-fractured samples for these experiments. Furthermore, using the accepted argument that K(+) signal indicates a cell that has been fractured though the cytoplasm, we have also identified different fracture planes of cells over the surface. Coupling a mechanized freeze fracture device to high-resolution cluster SIMS imaging will provide the sensitivity and resolution as well as the number of trials required to carry out biologically relevant SIMS experiments.
Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that uses localized liquid extraction for mass spectrometry imaging of molecules on surfaces. Nano-DESI enables imaging of ionizable molecules from a sample in its native state without any special sample pretreatment. In this chapter we describe the protocol for nano-DESI imaging of thin tissue sections.
Time of Flight secondary ion mass spectrometry (TOF-SIMS) has been used to explore the distribution of phospholipids in the plasma membrane of Tetrahymena pyriformis during cell division. The dividing cells were freeze dried prior to analysis followed by line scan and region of interest analysis at various stages of cell division. The results showed no signs of phospholipid domain formation at the junction between the dividing cells. Instead the results showed that the sample preparation technique had a great impact on one of the examined phospholipids, namely phosphatidylcholine (PC). Phosphatidylcholine and 2-aminoethylphosphonolipid (2-AEP) have therefore been evaluated in Tetrahymena cells that have been subjected to different sample preparation techniques: freeze drying ex situ, freeze fracture, and freeze fracture with partial or total freeze drying in situ. The result suggests that freeze-drying ex situ causes the celia to collapse and cover the plasma membrane.
Single-cell metabolomics with mass spectrometry enables a large variety of metabolites to be simultaneously detected from individual cells, without any preselection or labelling, to map phenotypes on the single cell level. Although the field is relatively young, it is steadily progressing with an increasing number of active research groups, techniques for cell sampling and ionization, tools for data analysis, and applications to answer important biomedical and environmental questions. In addition, the community shows great creativity in overcoming challenges associated with low sample volumes, a wide range of metabolite species, and large datasets. Here, we briefly discuss publications since 2019 and aim to provide the unfamiliar reader with an insight into the field and the expert reader with an update on the current status of the field.
Time of flight secondary ion mass spectrometry (TOF-SIMS) imaging has been used to investigate the incorporation of phospholipids into the plasma membrane of PC12 cells after incubation with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The incubations were done at concentrations previously shown to change the rate of exocytosis in model cell lines. The use of TOF-SIMS in combination with an in situ freeze fracture device enables the acquisition of ion images from the plasma membrane in single PC12 cells. By incubating cells with deuterated phospholipids and acquiring ion images at high mass resolution, specific deuterated fragment ions were used to monitor the incorporation of lipids into the plasma membrane. The concentration of incorporated phospholipids relative to the original concentration of PC was thus determined. The observed relative amounts of phospholipid accumulation in the membrane range from 0.5 to 2% following 19 h of incubation with PC at 100-300 μM and from 1 to 9% following incubation with PE at the same concentrations. Phospholipid accumulation is therefore shown to be dependent on the concentration in the surrounding media. In combination with previous exocytosis results, the present data suggests that very small changes in the plasma membrane phospholipid concentration are sufficient to produce significant effects on important cellular processes, such as exocytosis in PC12 cells.
Matrix effects in mass spectrometry imaging (MSI) may affect the observed molecular distribution in chemical and biological systems. In this study, we use mouse brain tissue of a middle cerebral artery occlusion (MCAO) stroke model to examine matrix effects in nanospray desorption electrospray ionization MSI (nano-DESI MSI). This is achieved by normalizing the intensity of the sodium and potassium adducts of endogenous phosphatidylcholine (PC) species to the intensity of the corresponding adduct of the PC standard supplied at a constant rate with the nano-DESI solvent. The use of MCAO model with an ischemic region localized to one hemisphere of the brain enables immediate comparison of matrix effects within one ion image. Furthermore, significant differences in sodium and potassium concentrations in the ischemic region in comparison with the healthy tissue allowed us to distinguish between two types of matrix effects. Specifically, we discuss matrix effects originating from variations in alkali metal concentrations and matrix effects originating from variations in the molecular composition of the tissue. Compensation for both types of matrix effects was achieved by normalizing the signals corresponding to endogenous PC to the signals of the standards. This approach, which does not introduce any complexity in sample preparation, efficiently compensates for signal variations resulting from differences in the local concentrations of sodium and potassium in tissue sections and from the complexity of the extracted analyte mixture derived from local variations in molecular composition.
Imaging mass spectrometry offers simultaneous spatially resolved detection of drugs, drug metabolites, and endogenous substances in a single experiment. This is important when evaluating effects of a drug on a complex organ system such as the brain, where there is a need to understand how regional drug distribution impacts function. Nanospray desorption electrospray ionization, nano-DESI, is a new ambient technique that enables spatially resolved analysis of a variety of samples without special sample pretreatment. This study introduces an experimental approach for accurate spatial mapping of drugs and metabolites in tissue sections by nano-DESI imaging. In this approach, an isotopically labeled standard is added to the nano-DESI solvent to compensate for matrix effects and ion suppression. The analyte image is obtained by normalizing the analyte signal to the signal of the standard in each pixel. We demonstrate that the presence of internal standard enables online quantification of analyte molecules extracted from tissue sections. Ion images are subsequently mapped to the anatomical brain regions in the analyzed section by use of an atlas mesh deformed to match the optical image of the section. Atlas-based registration accounts for the physical variability between animals, which is important for data interpretation. The new approach was used for mapping the distribution of nicotine in rat brain tissue sections following in vivo drug administration. We demonstrate the utility of nano-DESI imaging for sensitive detection of the drug in tissue sections with subfemtomole sensitivity in each pixel of a 27 μm × 150 μm area. Such sensitivity is necessary for spatially resolved detection of low-abundance molecules in complex matrices.
Mass spectrometry imaging (MSI) has been extensively used for determining spatial distributions of molecules in biological samples, and there is increasing interest in using MSI for quantification. Nanospray desorption electrospray ionization (nano-DESI) is an ambient MSI technique where a solvent is used for localized extraction of molecules followed by nanoelectrospray ionization. Doping the nano-DESI solvent with carefully selected standards enables online quantification during MSI experiments. In this proof-of-principle study, we demonstrate that this quantification approach can be extended to provide shotgun-like quantification of phospholipids in thin brain tissue sections. Specifically, two phosphatidylcholine (PC) standards were added to the nano-DESI solvent for simultaneous imaging and quantification of 22 endogenous PC species observed in nano-DESI MSI. Furthermore, by combining the quantitative data obtained in the individual pixels, we demonstrate quantification of these PC species in seven different regions of a rat brain tissue section.
Unsaturated free fatty acids are natively present in biological samples as isomers, where double bonds can be situated on different carbons in the acyl chain. While these isomers can have different actions and impacts on biological systems, they are inherently difficult to identify and differentiate by mass spectrometry alone. To address this challenge, several techniques for derivatization of the double bond or metal cationization at the carboxylic group have yielded diagnostic product ions for the respective isomer in tandem mass spectrometry. However, diagnostic product ions do not necessarily reflect quantitative isomeric ratios since fatty acid isomers have different ionization and fragmentation efficiencies. Here, we introduce a simple and rapid approach to predict the quantitative ratio of isomeric monounsaturated fatty acids. Specifically, empirically derived MS3 product ion patterns from fatty acid silver adducts are modeled using a stepwise linear model. This model is then applied to predict the proportion oleic and vaccenic acid in chemically complex samples at individual concentrations between 0.45 and 5.25 mu M, with an average accuracy and precision below 2 and 5 mol %, respectively. We show that by simply including silver ions in the electrospray solvent, isomeric ratios are rapidly predicted in neat standards, rodent plasma, and tissue extract. Furthermore, we use the method to directly map isomeric ratios in tissue sections using nanospray desorption electrospray ionization MS3 imaging without any sample preparation or modification to the instrumental setup. Ultimately, this approach provides a simple and rapid solution to differentiate monounsaturated fatty acids using commonly available commercial mass spectrometers without any instrumental modifications.
Mass spectrometry imaging (MSI) techniques generate data that reveal spatial distributions of molecules on a surface with high sensitivity and selectivity. However, processing large volumes of mass spectrometry data into useful ion images is not trivial. Furthermore, data from MSI techniques using continuous ionization sources where data are acquired in line scans require different data handling strategies compared to data collected from pulsed ionization sources where data are acquired in grids. In addition, for continuous ionization sources, the pixel dimensions are influenced by the mass spectrometer duty cycle, which, in turn, can be controlled by the automatic gain control (AGC) for each spectrum (pixel). Currently, there is a lack of data-handling software for MSI data generated with continuous ionization sources and AGC. Here, we present ion-to-image (i2i), which is a MATLAB-based application for MSI data acquired with continuous ionization sources, AGC, high resolution, and one or several scan filters. The source code and a compiled installer are available at https://github.com/LanekoffLab/i2i. The application includes both quantitative, targeted, and nontargeted data processing strategies and enables complex data sets to be processed in minutes. The i2i application has high flexibility for generating, processing, and exporting MSI data both from simple full scans and more complex scan functions interlacing MSn and SIM scan data sets, and we anticipate that it will become a valuable addition to the existing MSI software toolbox.
Attaining specificity in mass spectrometry imaging (MSI) is challenging and often results in longer experimental runtime or lower spatial resolution. Herein we describe a novel method for acquisition of MS2 spectra in an ion trap parallel with high resolution accurate mass FTMS scans. Using the nanospray desorption electrospray ionization (nano-DESI) ion source for MSI, we were able to perform 28 MS2 events in an ion trap simultaneously to recording a transient for FTMS. This allowed for deep characterization of the targeted compounds and allowed for improved annotation by spatially correlating the FTMS and ITMS2 data. By combining the data streams from data with different specificities we were able to efficiently data mine the complex data set and could annotate several lipid species.
Glycerophospholipids are one of the fundamental building blocks for life. The acyl chain connectivity to the glycerol backbone constitutes different sn-positional isomers, which have great diversity and importance for biological function. However, to fully realize their impact on function, analytical techniques that can identify and quantify sn-positional isomers in chemically complex biological samples are needed. Here, we utilize silver ion cationization in combination with tandem mass spectrometry (MSn) to identify sn-positional isomers of phosphatidylcholine (PC) species. In particular, a labile carbocation is generated through a neutral loss (NL) of AgH, the dissociation of which provides diagnostic product ions that correspond to acyl chains at the sn-1 or sn-2 position. The method is comparable to currently available methods, has a sensitivity in the nM-mu M range, and is compatible with quantitative imaging using mass spectrometry in MS4. The results reveal a large difference in isomer concentrations and the ion images show that the sn-positional isomers PC 18:1_18:0 are homogeneously distributed, whereas PC 18:1_16:0 and PC 20:1_16:0 show distinct localizations to sub-hippocampal structures.
The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool for studying mechanisms of redox reactions, identification of products and intermediates, and online derivatization/recognition of analytes. This work reports a new coupling interface for EC/MS by employing nanospray desorption electrospray ionization, a recently developed ambient ionization method. We demonstrate online coupling of nanospray desorption electrospray ionization MS with a traditional electrochemical flow cell, in which the electrolyzed solution emanating from the cell is ionized by nanospray desorption electrospray ionization for MS analysis. Furthermore, we show first coupling of nanospray desorption electrospray ionization MS with an interdigitated array (IDA) electrode enabling chemical analysis of electrolyzed samples directly from electrode surfaces. Because of its inherent sensitivity, nanospray desorption electrospray ionization enables chemical analysis of small volumes and concentrations of sample solution. Specifically, good-quality signal of dopamine and its oxidized form, dopamine o-quinone, was obtained using 10 μL of 1 μM solution of dopamine on the IDA. Oxidation of dopamine, reduction of benzodiazepines, and electrochemical derivatization of thiol groups were used to demonstrate the performance of the technique. Our results show the potential of nanospray desorption electrospray ionization as a novel interface for electrochemical mass spectrometry research.
Targeted and nontargeted metabolomics has the potential to evaluate and detect global metabolite changes in biological systems. Direct infusion mass spectrometric analysis enables detection of all ionizable small molecules, thus simultaneously providing information on both metabolites and lipids in chemically complex samples. However, to unravel the heterogeneity of the metabolic status of cells in culture and tissue a low number of cells per sample should be analyzed with high sensitivity, which requires low sample volumes. Here, we present the design and characterization of the direct infusion probe, DIP. The DIP is simple to build and position directly in front of a mass spectrometer for rapid metabolomics of chemically complex biological samples using pneumatically assisted electrospray ionization at 1 mu L/min flow rate. The resulting data is acquired in a square wave profile with minimal carryover between samples that enhances throughput and enables several minutes of uniform MS signal from 5 mu L sample volumes. The DIP was applied to study the intracellular metabolism of insulin secreting INS-1 cells and the results show that exposure to 20 mM glucose for 15 min significantly alters the abundance of several small metabolites, amino acids, and lipids.
Single-cell metabolomics has the potential to reveal unique insights into intracellular mechanisms and biological processes. However, the detection of metabolites from individual cells is challenging due to their versatile chemical properties and concentrations. Here, we demonstrate a tapered probe for pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) mass spectrometry that enables both chemical imaging of larger cells and global metabolomics of smaller 15 mu m cells. Additionally, by depositing cells in predefined arrays, we show successful metabolomics from three individual INS-1 cells per minute, which enabled the acquisition of data from 479 individual cells. Several cells were used to optimize analytical conditions, and 93 or 97 cells were used to monitor metabolome alterations in INS-1 cells after exposure to a low or high glucose concentration, respectively. Our analytical approach offers insights into cellular heterogeneity and provides valuable information about cellular processes and responses in individual cells.
Sodium and potassium are biological alkali metal ions that are essential for the physiological processes of cells and organisms. In combination with small-molecule metabolite information, disturbances in sodium and potassium tissue distributions can provide a further understanding of the biological processes in diseases. However, methods using mass spectrometry are generally tailored toward either elemental or molecular detection, which limits simultaneous quantitative mass spectrometry imaging of alkali metal ions and molecular ions. Here, we provide a new method by including crown ether molecules in the solvent for nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) that combines host-guest chemistry targeting sodium and potassium ions and quantitative imaging of endogenous lipids and metabolites. After evaluation and optimization, the method was applied to an ischemic stroke model, which has highly dynamic tissue sodium and potassium concentrations, and we report 2 times relative increase in the detected sodium concentration in the ischemic region compared to healthy tissue. Further, in the same experiment, we showed the accumulation and depletion of lipids, neurotransmitters, and amino acids using relative quantitation with internal standards spiked in the nano-DESI solvent. Overall, we demonstrate a new method that with a simple modification in liquid extraction MSI techniques using hostguest chemistry provides the added dimension of alkali metal ion imaging to provide unique insights into biological processes.
Prostaglandins (PGs) are important lipid mediators involved in physiological processes, such as inflammation and pregnancy. The pleiotropic effects of the PG isomers and their differential expression from cell types impose the necessity for studying individual isomers locally in tissue to understand the molecular mechanisms. Currently, mass spectrometry (MS)-based analytical workflows for determining the PG isomers typically require homogenization of the sample and a separation method, which results in a loss of spatial information. Here, we describe a method exploiting the cationization of PGs with silver ions for enhanced sensitivity and tandem MS to distinguish the biologically relevant PG isomers PGE2, PGD2, and Δ12-PGD2. The developed method utilizes characteristic product ions in MS3 for training prediction models and is compatible with direct infusion approaches. We discuss insights into the fragmentation pathways of Ag+ cationized PGs during collision-induced dissociation and demonstrate the high accuracy and robustness of the model to predict isomeric compositions of PGs. The developed method is applied to mass spectrometry imaging (MSI) of mouse uterus implantation sites using silver-doped pneumatically assisted nanospray desorption electrospray ionization and indicates localization to the antimesometrial pole and the luminal epithelium of all isomers with different abundances. Overall, we demonstrate, for the first time, isomeric imaging of major PG isomers with a simple method that is compatible with liquid-based extraction MSI methods.
Gaining a deep understanding of the molecular mechanisms underlying ischemic stroke is necessary to develop treatment alternatives. Ischemic stroke is known to cause a cellular energy imbalance when glucose supply is deprived, enhancing the role for energy production via β-oxidation where acylcarnitines are essential for the transportation of fatty acids into the mitochondria. Although traditional bulk analysis methods enable sensitive detection of acylcarnitines, they do not provide information on their abundances in various tissue regions. However, with quantitative mass spectrometry imaging the detected concentrations and spatial distributions of endogenous molecules can be readily obtained in an unbiased way. Here, we use pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging (PA nano-DESI MSI) doped with internal standards to study the distributions of acylcarnitines in mouse brain affected by stroke. The internal standards enable quantitative imaging and annotation of endogenous acylcarnitines is achieved by studying fragmentation patterns. We report a significant accumulation of long-chain acylcarnitines due to ischemia in brain tissue of the middle cerebral artery occlusion (MCAO) stroke model. Further, we estimate activities of carnitine transporting enzymes and demonstrate disruptions in the carnitine shuttle system that affects the β-oxidation in the mitochondria. Our results show the importance for quantitative monitoring of metabolite distributions in distinct tissue regions to understand cell compensation mechanisms involved in handling damage caused by stroke.
Ischemic stroke is one of the major causes of death and permanent disability in the world. However, the molecular mechanisms surrounding tissue damage are complex and further studies are needed to gain insights necessary for development of treatment. Prophylactic treatment by administration of cytosine-guanine (CpG) oligodeoxynucleotides has been shown to provide neuroprotection against anticipated ischemic injury. CpG binds to Toll-like receptor 9 (TLR9) causing initialization of an inflammatory response that limits visible ischemic damages upon subsequent stroke. Here, we use nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) to characterize molecular effects of CpG preconditioning prior to middle cerebral artery occlusion (MCAO) and reperfusion. By doping the nano-DESI solvent with appropriate internal standards, we can study and compare distributions of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the ischemic hemisphere of the brain despite the large changes in alkali metal abundances. Our results show that CpG preconditioning not only reduces the infarct size but it also decreases the degradation of PC and accumulation of LPC species, which indicates reduced cell membrane breakdown and overall ischemic damage. Our findings show that molecular mechanisms of PC degradation are intact despite CpG preconditioning but that these are limited due to the initialized inflammatory response.