Interleukin-13 Treatment of Living Lung Tissue Model Alters the Metabolome and Proteome-A Nano-DESI MS Metabolomics and Shotgun Proteomics Study.

Asthma is a chronic respiratory disease with one of the largest numbers of cases in the world; thus, constant investigation and technical development are needed to unravel the underlying biochemical mechanisms. In this study, we aimed to develop a nano-DESI MS method for the in vivo characterization of the cellular metabolome. Using air-liquid interface (ALI) cell layers, we studied the role of Interleukin-13 (IL-13) on differentiated lung epithelial cells acting as a lung tissue model. We demonstrate the feasibility of nano-DESI MS for the in vivo monitoring of basal-apical molecular transport, and the subsequent endogenous metabolic response, for the first time. Conserving the integrity of the ALI lung-cell layer enabled us to perform temporally resolved metabolomic characterization followed by "bottom-up" proteomics on the same population of cells. Metabolic remodeling was observed upon histamine and corticosteroid treatment of the IL-13-exposed lung cell monolayers, in correlation with alterations in the proteomic profile. This proof of principle study demonstrates the utility of in vivo nano-DESI MS for characterizing ALI tissue layers, and the new markers identified in our study provide a good starting point for future, larger-scale studies.

Revealing Structure and Localization of Steroid Regioisomers through Predictive Fragmentation Patterns in Mass Spectrometry Imaging.

Identifying and mapping steroids in tissues can provide opportunities for biomarker discovery, the interrogation of disease progression, and new therapeutics. Although separation coupled to mass spectrometry (MS) has emerged as a powerful tool for studying steroids, imaging and annotating steroid isomers remains challenging. Herein, we present a new method based on the fragmentation of silver-cationized steroids in tandem MS, which produces distinctive and consistent fragmentation patterns conferring confidence in steroid annotation at the regioisomeric level without using prior derivatization, separation, or instrumental modification. In addition to predicting the structure of the steroid with isomeric specificity, the method is simple, flexible, and inexpensive, suggesting that the wider community will easily adapt to it. We demonstrate the utility of our approach by visualizing steroids and steroid isomer distributions in mouse brain tissue using silver-doped pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging.

Ion-to-Image, i2i, a Mass Spectrometry Imaging Data Analysis Platform for Continuous Ionization Techniques.

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.

Surface sampling capillary electrophoresis-mass spectrometry for a direct chemical characterization of tissue and blood samples.

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.

Host-Guest Chemistry for Simultaneous Imaging of Endogenous Alkali Metals and Metabolites with Mass Spectrometry.

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 host-guest chemistry provides the added dimension of alkali metal ion imaging to provide unique insights into biological processes.

A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples.

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 µL/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 µL 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 metabolism: current and future trends.

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.

Membrane Sampling Separates Naphthenic Acids from Biogenic Dissolved Organic Matter for Direct Analysis by Mass Spectrometry.

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 Kow ∼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 O2 class of NAs and can be used prior to conventional analysis or as an on-line analytical strategy when coupled directly to mass spectrometry.

Quantitative determination of sn-positional phospholipid isomers in MSn using silver cationization.

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-µM 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.

CpG preconditioning reduces accumulation of lysophosphatidylcholine in ischemic brain tissue after middle cerebral artery occlusion.

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.

Spatially Defined Surface Sampling Capillary Electrophoresis 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.

Advances in mass spectrometry based single-cell metabolomics.

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.

Metabolite aberrations in early diabetes detected in rat kidney using mass spectrometry imaging.

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 2 weeks 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. Graphical abstract.

Oversampling To Improve Spatial Resolution for Liquid Extraction Mass Spectrometry Imaging.

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.

Quantitative Mass Spectrometry Imaging of Prostaglandins as Silver Ion Adducts with Nanospray Desorption Electrospray Ionization.

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 ∼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.

Profiling and quantifying endogenous molecules in single cells using nano-DESI 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.

A pneumatically assisted nanospray desorption electrospray ionization source for increased solvent versatility and enhanced metabolite detection from tissue.

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.

Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization.

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.

Three-dimensional imaging of lipids and metabolites in tissues by nanospray desorption electrospray ionization mass spectrometry.

Three-dimensional (3D) imaging of tissue sections is a new frontier in mass spectrometry imaging (MSI). Here, we report on fast 3D imaging of lipids and metabolites associated with mouse uterine decidual cells and embryo at the implantation site on day 6 of pregnancy. 2D imaging of 16-20 serial tissue sections deposited on the same glass slide was performed using nanospray desorption electrospray ionization (nano-DESI)-an ambient ionization technique that enables sensitive localized analysis of analytes on surfaces without special sample pretreatment. In this proof-of-principle study, nano-DESI was coupled to a high-resolution Q-Exactive instrument operated at high repetition rate of >5 Hz with moderate mass resolution of 35,000 (m/Δm at m/z 200), which enabled acquisition of the entire 3D image with a spatial resolution of ∼150 µm in less than 4.5 h. The results demonstrate localization of acetylcholine in the primary decidual zone (PDZ) of the implantation site throughout the depth of the tissue examined, indicating an important role of this signaling molecule in decidualization. Choline and phosphocholine-metabolites associated with cell growth-are enhanced in the PDZ and abundant in other cellular regions of the implantation site. Very different 3D distributions were obtained for fatty acids (FA), oleic acid and linoleic acid (FA 18:1 and FA 18:2), differing only by one double bond. Localization of FA 18:2 in the PDZ indicates its important role in decidualization while FA 18:1 is distributed more evenly throughout the tissue. In contrast, several lysophosphatidylcholines (LPC) observed in this study show donut-like distributions with localization around the PDZ. Complementary distributions with minimal overlap were observed for LPC 18:0 and FA 18:2 while the 3D image of the potential precursor phosphatidylcholine 36:2 (PC 36:2) showed a significant overlap with both LPC 18:0 and FA 18:2.

Shotgun approach for quantitative imaging of phospholipids using nanospray desorption electrospray ionization mass spectrometry.

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.