Development of a relative quantification method for infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging of Arabidopsis seedlings.

RATIONALE: Mass spectrometry imaging of young seedlings is an invaluable tool in understanding how mutations affect metabolite accumulation in plant development. However, due to numerous biological considerations, established methods for the relative quantification of analytes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging are not viable options. In this study, we report a method for the quantification of auxin-related compounds using stable-isotope-labelled (SIL) indole-3-acetic acid (IAA) doped into agarose substrate. METHODS: Wild-type Arabidopsis thaliana seedlings, sur2 and wei8 tar2 loss-of-function mutants, and YUC1 gain-of-function line were grown for 3 days in the dark in standard growth medium. SIL-IAA was doped into a 1% low-melting-point agarose gel and seedlings were gently laid on top for IR-MALDESI imaging with Orbitrap mass spectrometry analysis. Relative quantification was performed post-acquisition by normalization of auxin-related compounds to SIL-IAA in the agarose. Amounts of auxin-related compounds were compared between genotypes to distinguish the effects of the mutations on the accumulation of indolic metabolites of interest. RESULTS: IAA added to agarose was found to remain stable, with repeatability and abundance features of IAA comparable with those of other compounds used in other methods for relative quantification in IR-MALDESI analyses. Indole-3-acetaldoxime was increased in sur2 mutants compared with wild-type and other mutants. Other auxin-related metabolites were either below the limits of quantification or successfully quantified but showing little difference among mutants. CONCLUSIONS: Agarose was shown to be an appropriate sampling surface for IR-MALDESI mass spectrometry imaging of Arabidopsis seedlings. SIL-IAA doping of agarose was demonstrated as a viable technique for relative quantification of metabolites in live seedlings or tissues with similar biological considerations.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging analysis of endogenous metabolites in cherry tomatoes.

We report the spatially resolved metabolic profiling of cherry tomatoes using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI), a mass spectrometry imaging (MSI) technique that operates at ambient conditions and requires no sample derivatization. Tomatoes were flash frozen, cryosectioned and imaged with adequate spatial resolution to distinguish between the major tissue structures of a tomato including the skin, mesocarp, endocarp, locular tissue, septum, placenta, seed and seed coating. Metabolites were imaged from 100-1200 m/z, enabling significant coverage of a diverse array of metabolites including amino acids and lipids along with the major secondary metabolite classes: terpenes, phenolics, glycosides, and alkaloids. During the metabolic profiling, we found endogenous carotenoid hydrocarbons, namely lycopene or its structural isomer ß-carotene, ionized as radical cations. To our knowledge, this is the first demonstration of ionizing hydrocarbons in the MSI field.

IR-MALDESI method optimization based on time-resolved measurement of ion yields.

In the field of mass spectrometry imaging, typical experiments involve ionization directly from complex samples with no pre-ionization separation, relying on high resolving power mass analyzers to separate ions of interest. When an ion trapping step is involved in the analysis, the dynamic range of the analysis may be limited by the capacity of the ion trap, which is easily exceeded. To minimize collection of undesired ambient species while maximizing collection of analyte signal, accurate timing between ion generation and collection is a requirement. Here, a method for achieving synchronicity between infrared laser ablation and ion collection on a Q Exactive Plus mass spectrometer is described and demonstrated through measurement of ion accumulation at fixed time points following a laser ablation event with electrospray post-ionization of ablated material. In a model imaging experiment using infrared matrix-assisted laser desorption electrospray ionization, fixing the injection time at the minimum duration required to capture all ions generated by the last laser pulse in a sequence is shown to maximize target ion abundances. Using optimized timing is shown to yield a doubling or better of useful signal compared to previously used parameters. Graphical abstract Illustration of the effects of signal optimization on data quality for a single lipid species (cholesterol) measured from mouse liver tissue.

Characterization of a novel miniaturized burst-mode infrared laser system for IR-MALDESI mass spectrometry imaging.

Laser systems are widely used in mass spectrometry as sample probes and ionization sources. Mid-infrared lasers are particularly suitable for analysis of high water content samples such as animal and plant tissues, using water as a resonantly excited sacrificial matrix. Commercially available mid-IR lasers have historically been bulky and expensive due to cooling requirements. This work presents a novel air-cooled miniature mid-IR laser with adjustable burst-mode output and details an evaluation of its performance for mass spectrometry imaging. The miniature laser was found capable of generating sufficient energy for complete ablation of animal tissue in the context of an IR-MALDESI experiment with exogenously added ice matrix, yielding several hundred confident metabolite identifications. Graphical abstract The use of a novel miniature 2.94 µm burst-mode laser in IR-MALDESI allows for rapid and sensitive mass spectrometry imaging of a whole mouse.

Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI.

Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations. Electrospray post-ionization can be used to circumvent many sample cleanup and desalting steps. SESI and IR-MALDESI are two examples of ionization methods that employ electrospray post-ionization at atmospheric pressure and temperature. By coupling the two techniques and doping the electrospray solvent with silver ions, olefinic terpenes of different classes and varying degrees of volatility were directly analyzed from a biological buffer system with no sample workup steps.

IR-MALDESI mass spectrometry imaging of underivatized neurotransmitters in brain tissue of rats exposed to tetrabromobisphenol A.

There is a pressing need to develop tools for assessing possible neurotoxicity, particularly for chemicals where the mode of action is poorly understood. Tetrabromobisphenol A (TBBPA), a highly abundant brominated flame retardant, has lately been targeted for neurotoxicity analysis by concerned public health entities in the EU and USA because it is a suspected thyroid disruptor and neurotoxicant. In this study, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) coupled to a Q Exactive Plus mass spectrometer was used for the analysis of neurotransmitters in the brains of rats exposed to TBBPA in gestation and lactation through their mothers. Three neurotransmitters of interest were studied in three selected regions of the brain: caudate putamen, substantia nigra (SN), and dorsal raphe. Stable isotope labeled (SIL) standards were used as internal standards and a means to achieve relative quantification. This study serves as a demonstration of a new application of IR-MALDESI, namely that neurotransmitter distributions can be confidently and rapidly imaged without derivatization.

Direct screening of enzyme activity using infrared matrix-assisted laser desorption electrospray ionization.

RATIONALE: High-throughput screening (HTS) is a critical step in the drug discovery process. However, most mass spectrometry (MS)-based HTS methods require sample cleanup steps prior to analysis. In this work we present the utility of infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for monitoring an enzymatic reaction directly from a biological buffer system with no sample cleanup and at high throughput. METHODS: IR-MALDESI was used to directly analyze reaction mixtures from a well plate at different time points after reaction initiation. The percent conversion of precursors to products was used to screen the enzyme activity. The reaction was performed with two different concentrations of precursors and enzyme in order to assess the dynamic range of the assay. Eventually, a pseudo-HTS study was designed to investigate the utility of IR-MALDESI screening enzyme activity in a high-throughput manner. RESULTS: IR-MALDESI was able to readily monitor the activity of IDH1 over time at two different concentrations of precursors and enzyme. The calculated Z-factors of 0.65 and 0.41 confirmed the suitability of the developed method for screening enzyme activity in HTS manner. Finally, in a single-blind pseudo-HTS analysis IR-MALDESI was able to correctly predict the identity of all samples, where 8/10 samples were identified with high confidence and the other two samples with lower confidence. CONCLUSIONS: The enzymatic activity of IDH1 was screened by directly analyzing the reaction content from the buffer in well plates with no sample cleanup steps. This proof-of-concept study demonstrates the robustness of IR-MALDESI for direct analysis of enzymatic reactions from biological buffers with no sample cleanup and its immense potential for HTS applications.

Rapid response of fly populations to gene dosage across development and generations.

Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the network's regulatory outputs and a decrease in fitness. We subjected these flies to EMS mutagenesis, followed by experimental evolution. After only 8-15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level.

13C-SpaceM: Spatial single-cell isotope tracing reveals heterogeneity of de novo fatty acid synthesis in cancer.

Metabolism has emerged as a key factor in homeostasis and disease including cancer. Yet, little is known about the heterogeneity of metabolic activity of cancer cells due to the lack of tools to directly probe it. Here, we present a novel method, 13C-SpaceM for spatial single-cell isotope tracing of glucose-dependent de novo lipogenesis. The method combines imaging mass spectrometry for spatially-resolved detection of 13C6-glucose-derived 13C label incorporated into esterified fatty acids with microscopy and computational methods for data integration and analysis. We validated 13C-SpaceM on a spatially-heterogeneous normoxia-hypoxia model of liver cancer cells. Investigating cultured cells, we revealed single-cell heterogeneity of lipogenic acetyl-CoA pool labelling degree upon ACLY knockdown that is hidden in the bulk analysis and its effect on synthesis of individual fatty acids. Next, we adapted 13C-SpaceM to analyze tissue sections of mice harboring isocitrate dehydrogenase (IDH)-mutant gliomas. We found a strong induction of de novo fatty acid synthesis in the tumor tissue compared to the surrounding brain. Comparison of fatty acid isotopologue patterns revealed elevated uptake of mono-unsaturated and essential fatty acids in the tumor. Furthermore, our analysis uncovered substantial spatial heterogeneity in the labelling of the lipogenic acetyl-CoA pool indicative of metabolic reprogramming during microenvironmental adaptation. Overall, 13C-SpaceM enables novel ways for spatial probing of metabolic activity at the single cell level. Additionally, this methodology provides unprecedented insight into fatty acid uptake, synthesis and modification in normal and cancerous tissues.

Developmental phenomics suggests that H3K4 monomethylation confers multi-level phenotypic robustness.

How histone modifications affect animal development remains difficult to ascertain. Despite the prevalence of histone 3 lysine 4 monomethylation (H3K4me1) on enhancers, hypomethylation appears to have minor effects on phenotype and viability. Here, we genetically reduce H3K4me1 deposition in Drosophila melanogaster and find that hypomethylation reduces transcription factor enrichment in nuclear microenvironments, disrupts gene expression, and reduces phenotypic robustness. Using a developmental phenomics approach, we find changes in morphology, metabolism, behavior, and offspring production. However, many phenotypic changes are only detected when hypomethylated flies develop outside of standard laboratory environments or with specific genetic backgrounds. Therefore, quantitative phenomics measurements can unravel how pleiotropic modulators of gene expression affect developmental robustness under conditions resembling the natural environments of a species.

Increasing quantitation in spatial single-cell metabolomics by using fluorescence as ground truth.

Imaging mass spectrometry (MS) is becoming increasingly applied for single-cell analyses. Multiple methods for imaging MS-based single-cell metabolomics were proposed, including our recent method SpaceM. An important step in imaging MS-based single-cell metabolomics is the assignment of MS intensities from individual pixels to single cells. In this process, referred to as pixel-cell deconvolution, the MS intensities of regions sampled by the imaging MS laser are assigned to the segmented single cells. The complexity of the contributions from multiple cells and the background, as well as lack of full understanding of how input from molecularly-heterogeneous areas translates into mass spectrometry intensities make the cell-pixel deconvolution a challenging problem. Here, we propose a novel approach to evaluate pixel-cell deconvolution methods by using a molecule detectable both by mass spectrometry and fluorescent microscopy, namely fluorescein diacetate (FDA). FDA is a cell-permeable small molecule that becomes fluorescent after internalisation in the cell and subsequent cleavage of the acetate groups. Intracellular fluorescein can be easily imaged using fluorescence microscopy. Additionally, it is detectable by matrix-assisted laser desorption/ionisation (MALDI) imaging MS. The key idea of our approach is to use the fluorescent levels of fluorescein as the ground truth to evaluate the impact of using various pixel-cell deconvolution methods onto single-cell fluorescein intensities obtained by the SpaceM method. Following this approach, we evaluated multiple pixel-cell deconvolution methods, the 'weighted average' method originally proposed in the SpaceM method as well as the novel 'linear inverse modelling' method. Despite the potential of the latter method in resolving contributions from individual cells, this method was outperformed by the weighted average approach. Using the ground truth approach, we demonstrate the extent of the ion suppression effect which considerably worsens the pixel-cell deconvolution quality. For compensating the ion suppression individually for each analyte, we propose a novel data-driven approach. We show that compensating the ion suppression effect in a single-cell metabolomics dataset of co-cultured HeLa and NIH3T3 cells considerably improved the separation between both cell types. Finally, using the same ground truth, we evaluate the impact of drop-outs in the measurements and discuss the optimal filtering parameters of SpaceM processing steps before pixel-cell deconvolution.

Determination of Optimal Electrospray Parameters for Lipidomics in Infrared-Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging.

Infrared matrix-assisted laser desorption ionization (IR-MALDESI) is an ambient mass spectrometry imaging (MSI) technique that relies on electrospray ionization (ESI) for ion generation of desorbed neutrals. Although many mechanisms in IR-MALDESI have been studied in depth, there has not yet been a comprehensive study of how the ESI parameters change the profiles of tissue specific lipids. Acetonitrile (ACN)/water and methanol (MeOH)/water solvent systems and compositions were varied across a series of applied ESI voltages during IR-MALDESI analysis of rat liver tissue. Gradients of 12 min were run from 5 to 95% organic solvent in both positive and negative polarities across 11 voltages between 2.25 and 4.5 kV. These experiments informed longer gradients (25-30 min) across shorter solvent gradient ranges with fewer voltages. Optimal ESI parameters for lipidomics were determined by the number and abundance of detected lipids and the relative proportion of background ions. In positive polarity, the best solvent composition was 60-75% ACN/40-25% H2O with 0.2% formic acid at 3.2 kV applied voltage. The best parameters for negative polarity analysis are 45-55% ACN/55-45% H2O with 1 mM of acetic acid for voltages between 2.25 and 3.2 kV. Using these defined parameters, IR-MALDESI positive polarity lipidomics studies can increase lipid abundances 3-fold, with 15% greater coverage, while an abundance increase of 1.5-fold and 10% more coverage can be achieved relative to commonly used parameters in negative polarity.

A Versatile Platform for Mass Spectrometry Imaging of Arbitrary Spatial Patterns.

A vision-system driven platform, RastirX, has been constructed for mass spectrometry imaging (MSI) of arbitrary two-dimensional patterns. The user identifies a region of interest (ROI) by drawing on a live video image of the sample with the computer mouse. Motion commands are automatically generated to move the sample to acquire scan data for the pixels in the ROI. Synchronization of sample stage motion with laser firing and mass spectrometer (MS) scan acquisition is fully automated. RastirX saves a co-registered optical image and the scan location information needed to convert raw MS data into imzML format. Imaging an arbitrarily shaped ROI instead of the minimal enclosing rectangle reduces contamination from off-sample material and significantly reduces acquisition time.

Methods for Cryosectioning and Mass Spectrometry Imaging of Whole-Body Zebrafish.

The zebrafish (Danio rerio) is an ideal model for whole animal studies of lipid metabolism and lipid-related disease. In this work, infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) was applied for direct visualization of lipid and metabolite distributions across various organs in whole-body zebrafish tissue sections. Detailed methods for overcoming the challenges of cryosectioning adult male zebrafish for MSI and complementary histological imaging are described. Representative two-dimensional ion maps demonstrated organ specific localization of lipid analytes allowing for visualization of areas of interest including the brain, liver, intestines, and skeletal muscle. A high resolving power mass spectrometer was utilized for accurate mass measurements, which permitted the use of open-source, web-based tools for MS1 annotations including METASPACE and METLIN. Whole-body MSI with IR-MALDESI allowed for broad lipid coverage with high spatial resolution, illustrating the potential of this technique for studying lipid-related diseases using zebrafish as a model organism.

Coupling IR-MALDESI with Drift Tube Ion Mobility-Mass Spectrometry for High-Throughput Screening and Imaging Applications.

Because of its high degree of selectivity and chemical resolution, mass spectrometry (MS) is rapidly becoming the analytical method of choice for high-throughput evaluations and clinical diagnostics. While advances in MS resolving power have increased by an order of magnitude over the past decade, advances in sample introduction are still needed for high-throughput screening applications where the time frame of chromatographic separation would limit the duty cycle. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization source that has been shown to be applicable for direct analyses and mass spectrometry imaging (MSI) of complex biological samples in a high-throughput manner. To increase a range of detectable features in IR-MALDESI experiments, we integrated the home-built ion source with a commercially available drift tube ion mobility spectrometer-mass spectrometer (IMS-MS) and analyzed small polar molecules, lipids, carbohydrates, and intact proteins. We also describe in detail how the pulsed ionization source was synchronized with IMS-MS.

3D Imaging and metabolomic profiling reveal higher neuroactive kavalactone contents in lateral roots and crown root peels of Piper methysticum (kava).

BACKGROUND: Kava is an important neuroactive medicinal plant. While kava has a large global consumer footprint for its clinical and recreational use, factors related to its use lack standardization and the tissue-specific metabolite profile of its neuroactive constituents is not well understood. RESULTS: Here we characterized the metabolomic profile and spatio-temporal characteristics of tissues from the roots and stems using cross-platform metabolomics and a 3D imaging approach. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry revealed the highest content of kavalactones in crown root peels and lateral roots. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging revealed a unique tissue-specific presence of each target kavalactone. X-ray micro-computed tomography analysis demonstrated that lateral roots have morphological characteristics suitable for synthesis of the highest content of kavalactones. CONCLUSIONS: These results provide mechanistic insights into the social and clinical practice of the use of only peeled roots by linking specific tissue characteristics to concentrations of neuroactive compounds.

Discovery and quantification of bioactive peptides in fermented cucumber by direct analysis IR-MALDESI mass spectrometry and LC-QQQ-MS.

Bioactive peptides have been identified in lactic acid bacteria fermented foods including cultured milk, sourdough, and cured meats; however, their presence has not been investigated in fermented vegetables. In this study, infrared, matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry (MS) was employed to identify bioactive peptides in fermented cucumber. Natural and starter culture fermented cucumbers were prepared in triplicate in sodium chloride brines and compared to acidified cucumbers. Putative matches of known food-derived bioactive peptides were identified by direct analysis using IR-MALDESI-MS. Peptides were confirmed by IR-MALDESI MS/MS and quantified by LC-MS/MS. Three angiotensin converting enzyme (ACE) inhibitory peptides, IPP (0.42-0.49 mg/kg), LPP (0.30-0.33 mg/kg), and VPP (0.32-0.35 mg/kg) were formed in fermented cucumbers. A fourth ACE inhibitory peptide, KP (0.93-1.5 mg/kg), was enhanced 3-5 fold in fermented cucumbers compared with acidified cucumbers. This work demonstrates that lactic acid bacteria fermentation can enhance bioactive peptide content in vegetables.

Artemisinin Biosynthesis in Non-glandular Trichome Cells of Artemisia annua.

Artemisinin-based combination therapy (ACT) forms the first line of malaria treatment. However, the yield fluctuation of artemisinin has remained an unsolved problem in meeting the global demand for ACT. This problem is mainly caused by the glandular trichome (GT)-specific biosynthesis of artemisinin in all currently used Artemisia annua cultivars. Here, we report that non-GT cells of self-pollinated inbred A. annua plants can express the artemisinin biosynthetic pathway. Gene expression analysis demonstrated the transcription of six known pathway genes in GT-free leaves and calli of inbred A. annua plants. LC-qTOF-MS/MS analysis showed that these two types of GT-free materials produce artemisinin, artemisinic acid, and arteannuin B. Detailed IR-MALDESI image profiling revealed that these three metabolites and dihydroartemisinin are localized in non-GT cells of leaves of inbred A. annua plants. Moreover, we employed all the above approaches to examine artemisinin biosynthesis in the reported A. annua glandless (gl) mutant. The resulting data demonstrated that leaves of regenerated gl plantlets biosynthesize artemisinin. Collectively, these findings not only add new knowledge leading to a revision of the current dogma of artemisinin biosynthesis in A. annua but also may expedite innovation of novel metabolic engineering approaches for high and stable production of artemisinin in the future.

Evaluation of Digital Image Recognition Methods for Mass Spectrometry Imaging Data Analysis.

Analyzing mass spectrometry imaging data can be laborious and time consuming, and as the size and complexity of datasets grow, so does the need for robust automated processing methods. We here present a method for comprehensive, semi-targeted discovery of molecular distributions of interest from mass spectrometry imaging data, using widely available image similarity scoring algorithms to rank images by spatial correlation. A fast and powerful batch search method using a MATLAB implementation of structural similarity (SSIM) index scoring with a pre-selected reference distribution is demonstrated for two sample imaging datasets, a plant metabolite study using Artemisia annua leaf, and a drug distribution study using maraviroc-dosed macaque tissue. Graphical Abstract ᅟ.

IR-MALDESI Mass Spectrometry Imaging at 50 Micron Spatial Resolution.

High spatial resolution in mass spectrometry imaging (MSI) is crucial to understanding the biology dictated by molecular distributions in complex tissue systems. Here, we present MSI using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) at 50 µm resolution. An adjustable iris, beam expander, and an aspherical focusing lens were used to reduce tissue ablation diameters for MSI at high resolution. The laser beam caustic was modeled using laser ablation paper to calculate relevant laser beam characteristics. The minimum laser spot diameter on the tissue was determined using tissue staining and microscopy. Finally, the newly constructed optical system was used to image hen ovarian tissue with and without oversampling, detailing tissue features at 50 µm resolution. Graphical Abstract ᅟ.