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The importance of metabolites and their isomeric structures in biological function and dysfunction is increasingly recognized. However, achieving quantitative mapping of metabolites within tissue regions, particularly with isomeric specificity, remains an analytical challenge. This work presents the development of a quantitative surface sampling capillary electrophoresis method for spatial metabolomics with isomeric resolution. Five quantitation strategies were evaluated, with the optimal approach identified as sequential injection of metabolites directly from tissue alongside standards. This methodology was applied to a rat brain tissue section in a proof-of-principle study, enabling quantitative spatial analysis of metabolites, neurotransmitters, and isomeric species. Among the findings, the aromatic amino acids tyrosine, phenylalanine, and tryptophan exhibited the most dynamic distributions across four brain regions, while leucine and isoleucine demonstrated distinct spatial profiles, with leucine consistently being the more abundant isomer. This method offers a promising tool for advancing the understanding of spatially resolved biochemical processes underlying biological function and dysfunction.

Background: Mass spectrometry imaging (MSI) and Raman spectroscopy imaging are two chemical imaging techniques that can reveal and visualize the chemical distributions in thin tissue sections. The two techniques are complementary with Raman spectroscopy detecting the Raman shift of molecular bonds while MSI detects mass to-charge of ionized molecules. Furthermore, MSI requires molecules to be desorbed, ablated or sputtered from the surface, making Raman spectroscopy less destructive in comparison. With a multimodal analysis workflow, the advantages of both techniques can be combined to yield enhanced chemical information of surfaces, such as thin tissue sections. Here, we combine the two imaging modalities and map the chemical signature in the exact same location on thin tissue section to uncover MSI derived chemical alterations in tissue.

Results: In this study, we present a multimodal workflow integrating pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) MSI and Raman spectroscopy on the same tissue section. This requires some adaptation of MSI to the sample restrictions of Raman. We demonstrate successful integration and the power of combined analysis in characterization of tissue chemistry. Raman spectroscopy proved highly effective for identifying solvent-induced chemical alterations in tissue caused by PA nano-DESI MSI with methanol or acetonitrile-based solvents. First, we describe chemical alterations connected to different sampling modes, such as different probe speeds and oversampling. Following, we characterize chemical changes in the resulting tissue exposed to up to five repeated imaging experiments. The results from the two modalities were in excellent agreement and showed that methanol desorbs more material from the tissue compared to acetonitrile, and that both solvents cause protein denaturation in the tissue.

Significance: This study presents the establishment of a multimodal workflow combining imaging with PA nano-DESI MSI and Raman spectroscopy from the exact same tissue sections and locations. The combination enabled chemical characterization of the tissue after MSI and revealed previously unknown alterations in tissue chemistry upon MSI. Overall, our findings offer new insights into how exposure to solvents impact tissue chemistry and highlight the potential of combining MSI with Raman spectroscopy for in depth chemical characterization of tissue sections.

Bioactive lipid mediators (LMs) derived from polyunsaturated fatty acids (PUFAs) are key molecules in both the initiation and resolution of inflammatory responses. Previous findings suggest that a dysregulated LM balance, especially within the arachidonic acid (AA) pathway, may contribute to an impaired resolution response and subsequent chronic neuroinflammation in multiple sclerosis (MS). However, to date, the local biosynthesis and signaling of LMs within the brain of people with MS (PwMS) remains unexplored. In this study, we, therefore, mapped the distribution of AA and its key downstream LM prostaglandin E₂ (PGE₂) in white matter MS brain tissue and of non-neurological controls (NNCs) for the first time using mass spectrometry imaging. We found that AA levels are lower in MS cases compared to NNCs and reduced in MS lesions compared to peri-lesional tissue. Furthermore, the PGE₂/AA ratio, indicating the PGE₂ synthesis from the AA substrate, was increased in lesion areas compared to fully myelinated regions in MS. In line with that, the expression of prostaglandin synthesizing enzymes as measured by RT-qPCR was partially increased in MS tissue compared to NNCs. In addition, the expression of prostaglandin E2 receptor 4 (EP4) decreased, while prostaglandin E2 receptor 2 (EP2) showed increased expression levels in MS lesions compared to NNCs and localized specifically to microglia. We also found that PGE₂ addition to pro-inflammatory human-induced pluripotent stem cell (iPSC)-derived microglia resulted in enhanced cytokine signaling pathways, but also the upregulation of its synthase PTGES and homeostatic/resolving signaling, the latter of which might mainly occur through EP2 signaling. Collectively, our results provide detailed information about the region-specific levels of AA and PGE₂ in MS lesions and we propose enhanced PGE₂-EP2 signaling in inflamed microglia in MS.

Spatial metabolomics offers the combination of molecular identification and localization. As a tool for spatial metabolomics, mass spectrometry imaging (MSI) can provide detailed information on localization. However, molecular annotation with MSI is challenging due to the lack of separation prior to mass spectrometric analysis. Contrarily, surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS) provides detailed molecular information, although the size of the sampling sites is modest. Here, we describe a platform for spatial metabolomics where MSI using pneumatically assisted nanospray desorption electrospray ionization (PA-nano-DESI) is combined with SS-CE-MS to gain both in-depth chemical information and spatial localization from thin tissue sections. We present the workflow, including the user-friendly setup and switching between the techniques, compare the obtained data, and demonstrate a quantitative approach when using the platform for spatial metabolomics of ischemic stroke.

In mass spectrometry imaging (MSI), analytes are desorbed and ionized directly from a complex and unique chemical microenvironment in each pixel, which makes their quantification challenging. Matrix effects have been addressed by the use of isotopically labeled internal standards (IS), either included in the solvent or sprayed over the tissue section, for pixel-by-pixel relative quantification. However, in addition to requiring preselection, isotopically labeled IS may be costly or unavailable. Here, we introduce a novel approach for quantification in MSI, based on the standard addition method. We report a workflow for both acquiring and processing quantitative data. Furthermore, we compare the detected concentrations obtained by standard addition to the detected concentrations obtained using both IS quantification and external calibration. Finally, we show the applicability of using molecules extracted from tissue as an easily accessible standard mixture for standard addition quantification in MSI. The possibility of using analytical standards and readily available endogenous analytes as a source of calibration standards makes our standard addition-based quantitative approach cost-effective, accessible, and versatile.

Background

Identification and characterization of steroids from complex mixtures with isomeric precision is key to studying endocrine-related metabolism and disorders. Whereas the golden standard chromatography, including liquid chromatography and gas chromatography, can be coupled with mass spectrometry to separate steroids prior to ionization, this separation is time-consuming. Contrarily, direct infusion techniques can offer increased throughput; however, these are often hampered by limited structural specificity. Thus, it is important to develop new analytical tools for direct infusion mass spectrometry that will provide isomeric specificity.

Results

Herein, we show that direct infusion with electrospray ionization in combination with silver adduction and cyclic ion mobility mass spectrometry (cIMS) enables mobility separation and improves the detectability of steroid isomers. Specifically, silver ion adduction of steroids increases instrumental response up to 14 times and enables almost baseline mobility separation of closely related structural steroid isomers even at low cIMS resolution. By combining experimental and theoretical data, we show that the silver interacts with the steroid isomer at single or multiple sites, which introduces conformational changes that enable mobility separation. Moreover, we show that the combination of cIMS and silver adduct fragmentation in tandem mass spectrometry provides an additional dimension for annotation of steroid isomers. Thus, the simple introduction of silver ions into the electrospray solvent provides a great gain in the analytical discernment of steroid isomers.

Significance

For the first time, we show that the use of silver adduction introduces a conformational change in steroids that allows for them to be separated with low-resolution ion mobility spectrometry without any prior derivatization, chromatographic separation, or instrumental modification. This is a new and important tool for analyzing steroid isomers that can unravel their importance in biological systems.

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.

Background/Objectives: Profiling of metabolites and lipids in biological samples can provide invaluable insights into life-sustaining chemical processes. The ability to detect both metabolites and lipids in the same sample can enhance these understandings and connect cellular dynamics. However, simultaneous detection of metabolites and lipids is generally hampered by chromatographic systems tailored to one molecular type. This void can be filled by direct infusion mass spectrometry (MS), where all ionizable molecules can be detected simultaneously. However, in direct infusion MS, the high chemical complexity of biological samples can introduce limitations in detectability due to matrix effects causing ionization suppression.

Methods: Decreased sample complexity and increased detectability and molecular coverage was provided by combining our direct infusion probe (DIP) with liquid-liquid extraction (LLE) and directly sampling the different phases for direct infusion. Three commonly used LLE methods for separating lipids and metabolites were evaluated.

Results: The butanol-methanol (BUME) method was found to be preferred since it provides high molecular coverage and have low solvent toxicity. The established BUME DIP-MS method was used as a fast and sensitive analysis tool to study chemical changes in insulin-secreting cells upon glucose stimulation. By analyzing the metabolome at distinct time points, down to 1-min apart, we found high dynamics of the intracellular metabolome.

Conclusions: The rapid workflow with LLE DIP-MS enables higher sensitivity of phase separated metabolites and lipids. The application of BUME DIP-MS provides novel information on the dynamics of the intracellular metabolome of INS-1 during the two phases of insulin release for both metabolite and lipid classes.

Single-cell metabolomics has the potential to reveal unique insights into intracellular mechanisms and biological processes. However, the detection of metabolites from individual cells is challenging due to their versatile chemical properties and concentrations. Here, we demonstrate a tapered probe for pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) mass spectrometry that enables both chemical imaging of larger cells and global metabolomics of smaller 15 mu m cells. Additionally, by depositing cells in predefined arrays, we show successful metabolomics from three individual INS-1 cells per minute, which enabled the acquisition of data from 479 individual cells. Several cells were used to optimize analytical conditions, and 93 or 97 cells were used to monitor metabolome alterations in INS-1 cells after exposure to a low or high glucose concentration, respectively. Our analytical approach offers insights into cellular heterogeneity and provides valuable information about cellular processes and responses in individual cells.

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.