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.

Global and Spatial Metabolomics of Individual Cells Using a Tapered Pneumatically Assisted nano-DESI Probe.

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 µm 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.

Identification and Imaging of Prostaglandin Isomers Utilizing MS3 Product Ions and Silver Cationization.

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.

Matrix Effects Free Imaging of Thin Tissue Sections Using Pneumatically Assisted Nano-DESI MSI.

Mass spectrometry imaging has the potential to reveal important molecular interaction in morphological regions in tissue. However, the simultaneous ionization of the continuously altered and complex chemistry in each pixel can introduce artifacts that result in skewed molecular distributions in the compiled ion images. These artifacts are known as matrix effects. Mass spectrometry imaging using nanospray desorption electrospray ionization (nano-DESI MSI) enables the elimination of matrix effects by doping the nano-DESI solvent with internal standards. Carefully selected internal standards ionize similarly and simultaneously with the extracted analytes from thin tissue sections, and the matrix effects are eliminated through a robust data normalization method. Herein we describe the setup and use of pneumatically assisted (PA) nano-DESI MSI with standards doped in the solvent for elimination of matrix effects in ion images.

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.

Screening autism-associated environmental factors in differentiating human neural progenitors with fractional factorial design-based transcriptomics.

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.

Ischemic Stroke Causes Disruptions in the Carnitine Shuttle System.

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 ß-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 ß-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.

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.

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.

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.

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.

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.

Single-cell metabolomics: where are we and where are we going?

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.

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.

Silver-Doped Nano-DESI MSI for Increased Specificity and Sensitivity of Alkenes.

Nanospray desorption electrospray ionization (nano-DESI) is a technique that can be used for mass spectrometry imaging (MSI) experiments. Due to the separation of sampling and ionization events, the nano-DESI solvent can be doped with additives to alter the specificity and selectivity of the experiment. In this chapter, we describe the addition of silver ions to the nano-DESI solvent for increased specificity and sensitivity of double bond containing analyte molecules for nano-DESI MSI.

In situ imaging reveals disparity between prostaglandin localization and abundance of prostaglandin synthases.

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 D2, 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 D2 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.

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.

Determination of Monounsaturated Fatty Acid Isomers in Biological Systems by Modeling MS3 Product Ion Patterns.

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 µM, 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.