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.

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.

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.

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.

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.

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.

Delicate polydimethylsiloxane hollow fibre membrane interfaces for condensed phase membrane introduction mass spectrometry (CP-MIMS).

RATIONALE: On-line analytical techniques such as condensed phase membrane introduction mass spectrometry (CP-MIMS) permit direct and rapid analyte measurements in complex samples. Direct, rapid analytical methods are desirable because they eliminate potential contamination and/or dilution from sample workup steps, facilitate rapid sample screening and allow 'real-time' monitoring applications. METHODS: PDMS hollow fibre membrane (HFM) flow cell interfaces (215 µm, 35 µm, and 0.5 µm thick composite) were coupled with an electrospray ionization (ESI) triple quadrupole mass spectrometer. A simultaneous push/pull methanol acceptor phase delivery system and membrane mounting via epoxy potting ensured that the delicate membranes were not ruptured during construction or sample measurements. Both flow cell and direct insertion 'J-Probe' interfaces using the 0.5 µm thick composite PDMS HFM were utilized for direct naphthenic acid measurements. RESULTS: Delicate HFM CP-MIMS interfaces were used for the rapid screening and continuous, on-line monitoring of carboxylic acids and hydroxylated compounds directly in complex sample matrices under ambient conditions at pptr - ppb detection limits. Push/pull acceptor phase (methanol) delivery maintained ambient hydrostatic pressures within the HFMs, improving ESI stability and analytical sensitivity, especially with stopped acceptor flow operation. Signal response times less than 2 min were achieved for thin, composite PDMS HFMs at 30°C. The continuous monitoring of naphthenic acid degradation was demonstrated. CONCLUSIONS: Delicate PDMS HFM CP-MIMS interfaces were developed and used for the direct, on-line detection of low volatility, polar analytes in complex aqueous samples. Composite PDMS HFM interfaces yielded the best overall analytical performance improvements, and were used to demonstrate the direct measurement of naphthenic acids in complex aqueous samples.

Mass spectrometry imaging methods for visualizing tumor heterogeneity.

Profiling spatial distributions of lipids, metabolites, and proteins in tumors can reveal unique cellular microenvironments and provide molecular evidence for cancer cell dysfunction and proliferation. Mass spectrometry imaging (MSI) is a label-free technique that can be used to map biomolecules in tumors in situ. Here, we discuss current progress in applying MSI to uncover molecular heterogeneity in tumors. First, the analytical strategies to profile small molecules and proteins are outlined, and current methods for multimodal imaging to maximize biological information are highlighted. Second, we present and summarize biological insights obtained by MSI of tumor tissue. Finally, we discuss important considerations for designing MSI experiments and several current analytical challenges.

A miniature condensed-phase membrane introduction mass spectrometry (CP-MIMS) probe for direct and on-line measurements of pharmaceuticals and contaminants in small, complex samples.

RATIONALE: High-throughput, automated analytical measurements are desirable in many analytical scenarios, as are rapid sample pre-screening techniques to identify 'positive' samples for subsequent measurements using more time-consuming conventional methodologies (e.g., liquid chromatography/mass spectrometry (LC/MS)). A miniature condensed-phase membrane introduction mass spectrometry (CP-MIMS) probe for the direct and continuous, on-line measurement of pharmaceuticals and environmental contaminants in small, complex samples is presented. METHODS: A miniature polydimethylsiloxane hollow fibre membrane (PDMS-HFM) probe is coupled with an electrospray ionization (ESI) triple quadrupole mass spectrometer. Analytes are transported from the probe to the ESI source by a methanol acceptor phase. The probe can be autosampler mounted and directly inserted in small samples (≥400 µL) allowing continuous and simultaneous pptr-ppb level detection of target analytes (chlorophenols, triclosan, gemfibrozil, nonylphenol) in complex samples (artificial urine, beer, natural water, waste water, plant tissue). RESULTS: The probe has been characterized and optimized for acceptor phase flow rate, sample mixing and probe washing. Signal response times, detection limits and calibration data are given for selected ion monitoring (SIM) and tandem mass spectrometry (MS/MS) measurements of target analytes at trace levels. Comparisons with flow cell type CP-MIMS systems are given. Analyte depletion effects are evaluated for small samples (≥400 µL). On-line measurements in small volumes of complex samples, temporally resolved reaction monitoring and in situ/in vivo demonstrations are presented. CONCLUSIONS: The miniature CP-MIMS probe developed was successfully used for the direct, on-line detection of target analytes in small volumes (40 mL to 400 µL) of complex samples at pptr to low ppb levels. The probe can be readily automated as well as deployed for in situ/in vivo monitoring, including reaction monitoring, small sample measurements and direct insertion in living plant tissue.

Strategies for uncovering stable isotope tracing patterns between cell populations.

Despite practical complexities, isotope tracing studies in humans are becoming increasingly feasible. However, several technological challenges need to be addressed in order to take full advantage of human tracing studies. First, absolute metabolic flux measurements in mice are not so easily applied to human models, given that tissue resection is restricted to a single surgical time point. Second, isotope tracing has yet to be employed to detect metabolic differences between cells types in vivo. Here, we discuss the current models and propose an alternative, liquid tumor environment, that could overcome these limitations. Furthermore, we highlight current strategies used to maintain isotopolog enrichment following cell isolation techniques to facilitate cell-type-specific analysis.

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.

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.

Direct analysis of naphthenic acids in constructed wetland samples by condensed phase membrane introduction mass spectrometry.

The application of direct mass spectrometry techniques to the analysis of complex samples has a number of advantages including reduced sample handling, higher sample throughput, in situ process monitoring, and the potential for adaptation to on-site analysis. We report the application of a semi-permeable capillary hollow fibre membrane probe (immersed directly into an aqueous sample) coupled to a triple quadrupole mass spectrometer by a continuously flowing methanol acceptor phase for the rapid analysis of naphthenic acids with unit mass resolution. The intensity of the naphthenic acid-associated peaks in the mass spectrum are normalized to an internal standard in the acceptor phase for quantitation and the relative abundance of the peaks in the mass spectrum are employed to monitor compositional changes in the naphthenic acid mixture using principle component analysis. We demonstrate the direct analysis of a synthetic oil sands process-affected water for classical naphthenic acids (CnH2n+zO2) as they are attenuated through constructed wetlands containing sedge (Carex aquatilis), cattail (Typha latifolia), or bulrush (Schoenoplectus acutus). Quantitative results for on-line membrane sampling compare favourably to those obtained by solid-phase extraction high-resolution mass spectrometry. Additionally, chemometric analysis of the mass spectra indicates a clear discrimination between naphthenic acid-influenced and natural background waters. Furthermore, the compositional changes within complex naphthenic acid mixtures track closely with the degree of attenuation. Overall, the technique is successful in following changes in both the concentration and composition of naphthenic acids from synthetic oil sands process-affected waters, with the potential for high throughput screening and environmental forensics.

Rapid Screening of Carboxylic Acids from Waste and Surface Waters by ESI-MS/MS Using Barium Ion Chemistry and On-Line Membrane Sampling.

Negative ion tandem mass spectrometric analysis of aliphatic carboxylic acids often yields only non-diagnostic ([M - H](-)) ions with limited selective fragmentation. However, carboxylates cationized with Ba(2+) have demonstrated efficient dissociation in positive ion mode, providing structurally diagnostic product ions. We report the application of barium adducts followed by collision induced dissociation (CID), to improve selectivity for rapid screening of carboxylic acids in complex aqueous samples. The quantitative MS/MS method presented utilizes common product ions of [M - H + Ba](+) precursor ions. The mechanism of product ion formation is investigated using isotopically labeled standards and a series of structurally related carboxylic acids. The results suggest that hydrogen atoms in the ß and γ positions yield common product ions ([BaH](+) and [BaOH](+)). Furthermore, the diagnostic product ion at m/z 196 serves as a qualifying ion for carboxylate species. This methodology has been successfully used in conjunction with condensed phase membrane introduction mass spectrometry (CP-MIMS), with barium acetate added directly to the methanol acceptor phase. The combination enables rapid screening of carboxylic acids directly from acidified water samples (wastewater effluent, spiked natural waters) using a capillary hollow fiber PDMS membrane immersion probe. We have applied this technique for the direct analysis of complex naphthenic acid mixtures spiked into natural surface waters using CP-MIMS. Selectivity at the ionization and tandem mass spectrometry level eliminate isobaric interferences from hydroxylated species present within the samples, which have been observed in negative electrospray ionization.

A semi-quantitative approach for the rapid screening and mass profiling of naphthenic acids directly in contaminated aqueous samples.

We report the use of a direct sampling, online analytical approach for the determination of acid extractable naphthenic acids in complex aqueous samples, known as condensed phase membrane introduction mass spectrometry (CP-MIMS). The technique employs a capillary hollow fibre semi-permeable membrane probe configured for immersion into a pH adjusted sample. A continuously flowing methanol acceptor phase transfers naphthenic acids to an electrospray ionization source, operated in negative ion mode, whereupon they are analysed by mass spectrometry as [M-H](-) ions. High-resolution mass spectrometry is used to characterize the influence of sample pH on membrane transport of multiple components of complex naphthenic acid mixtures. We demonstrate the use of CP-MIMS for semi-quantitative analysis of real-world samples using selected ion monitoring and full scan mass spectra at unit mass resolution. The technique has also been employed to continuously monitor the temporal evolution in the mass profile and concentrations of individual naphthenic acid isomer classes in heterogeneous solutions during adsorption processes. Copyright © 2015 John Wiley & Sons, Ltd.