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1. Mass Spectrometry Imaging of Diclofenac and Its Metabolites in Tissues Using Nanospray Desorption Electrospray Ionization

   https://doi.org/10.26434/chemrxiv.13194422  

   Brown, H. ; Mesa Sanchez, D. ; Yin, R. ; Chen, B. ; Vavrek, M. ; Cancilla, M. ; Zhong, W. ; Shyong, B. ; Zhang, R. ; Li, F. ; et al ( May 2020 , ChemRxiv)

   null (Ed.)

   Glucuronidation is a common phase II metabolic process for drugs and xenobiotics which increases their solubility for excretion. Acyl glucuronides (glucuronides of carboxylic acids) present concerns of toxicity as they have been implicated in gastrointestinal toxicity and hepatic failure. Despite the substantial success in the bulk analysis of these species, little is known about their localization in tissues. Herein, we used nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI-MSI) to examine the localization of diclofenac, a widely used nonsteroidal anti-inflammatory drug, and its metabolites in mouse kidney and liver tissues. Nano-DESI allows for label-free imaging with high spatial resolution and sensitivity without special sample pretreatment. Using nano-DESI-MSI, ion images for diclofenac and its major metabolites were produced. MSI data acquired over a broad m/z range showed fairly low signals of the drug and its metabolites. At least an order of magnitude improvement in the signals was obtained using selected ion monitoring (SIM), with m/z windows centered around the low-abundance ions of interest. Using nano-DESI MSI in SIM mode, we observed that diclofenac acyl glucuronide is localized to the inner medulla and hydroxydiclofenac to the cortex of the kidney. The distributions observed for both metabolites closely match the previously reported localization of enzymes that process diclofenac into its respective metabolites. The localization of diclofenac acyl glucuronide to medulla likely indicates that the toxic metabolite is being excreted from the tissue. In contrast, a uniform distribution of diclofenac, hydroxydiclofenac and the diclofenac acyl glucuronide metabolite was observed in the liver tissue. Semiquantitative analysis found the metabolite to diclofenac ratios calculated from nano-DESI in agreement to those calculated from liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments. Collectively, our results demonstrate nano-DESI-MSI can be successfully used to image diclofenac and its primary metabolites in dosed liver and kidney tissues from mice and derive semi-quantitative data from localized tissue regions. 

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2. Microscopy-Directed Imaging Mass Spectrometry for Rapid High Spatial Resolution Molecular Imaging of Glomeruli

   https://doi.org/10.1021/jasms.3c00033  

   Esselman, Allison B. ; Patterson, Nathan Heath ; Migas, Lukasz G. ; Dufresne, Martin ; Djambazova, Katerina V. ; Colley, Madeline E. ; Van de Plas, Raf ; Spraggins, Jeffrey M. ( July 2023 , Journal of the American Society for Mass Spectrometry)

   The glomerulus is a multicellular functional tissue unit (FTU) of the nephron that is responsible for blood filtration. Each glomerulus contains multiple substructures and cell types that are crucial for their function. To understand normal aging and disease in kidneys, methods for high spatial resolution molecular imaging within these FTUs across whole slide images is required. Here we demonstrate a workflow using microscopy-driven selected sampling to enable 5 μm pixel size matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) of all glomeruli within whole slide human kidney tissues. Such high spatial resolution imaging entails large numbers of pixels, increasing the data acquisition times. Automating FTU-specific tissue sampling enables high-resolution analysis of critical tissue structures, while concurrently maintaining throughput. Glomeruli were automatically segmented using coregistered autofluorescence microscopy data, and these segmentations were translated into MALDI IMS measurement regions. This allowed high-throughput acquisition of 268 glomeruli from a single whole slide human kidney tissue section. Unsupervised machine learning methods were used to discover molecular profiles of glomerular subregions and differentiate between healthy and diseased glomeruli. Average spectra for each glomerulus were analyzed using Uniform Manifold Approximation and Projection (UMAP) and k-means clustering, yielding 7 distinct groups of differentiated healthy and diseased glomeruli. Pixel-wise k-means clustering was applied to all glomeruli, showing unique molecular profiles localized to subregions within each glomerulus. Automated microscopy-driven, FTU-targeted acquisition for high spatial resolution molecular imaging maintains high-throughput and enables rapid assessment of whole slide images at cellular resolution and identification of tissue features associated with normal aging and disease. 

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3. Enhancing Metabolite Coverage for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Through Multiple On-Tissue Chemical Derivatizations

   https://doi.org/10.1007/978-1-0716-2030-4_14  

   O’Neill, K.C. ; Dueñas, M.E. ; Larson, E. ; Forsman, T.T. ; Lee, YJ. ( January 2022 , Methods in molecular biology)

   Lee, YJ (Ed.)

   The ability to study and visualize metabolites on a cellular and sub-cellular level is important for gaining insights into biological pathways and metabolism of multicellular organisms. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool for metabolomics experiments due to its high sensitivity and small sampling size. The spatial resolution in MALDI-MSI is mainly limited by the number of molecules available in a small sampling size. When the sampling size is low enough to achieve cellular or subcellular spatial resolution, signal intensity is sacrificed making poorly ionized metabolites difficult to detect. To overcome this limitation, on-tissue chemical derivatization reactions have been used to enhance the desorption/ionization efficiency of selected classes of compounds by adding a functional group with a permanent positive charge or one that can be easily ionized. By utilizing several chemical derivatizations in parallel, metabolite coverage can be drastically improved. This chapter outlines methodology for sample preparation and data analysis for on-tissue chemical derivatization using various derivatization reagents. 

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4. Imaging mass spectrometry reveals complex lipid distributions across Staphylococcus aureus biofilm layers

   https://doi.org/10.1016/j.jmsacl.2022.09.003  

   Rivera, Emilio S. ; Weiss, Andy ; Migas, Lukasz G. ; Freiberg, Jeffrey A. ; Djambazova, Katerina V. ; Neumann, Elizabeth K. ; Van de Plas, Raf ; Spraggins, Jeffrey M. ; Skaar, Eric P. ; Caprioli, Richard M. ( November 2022 , Journal of Mass Spectrometry and Advances in the Clinical Lab)

   Introduction Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results Implementation of TIMS led to a ∼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) – CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies. 

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5. Comparison of gaseous ubiquitin ion structures obtained from a solid and solution matrix using ion mobility spectrometry/mass spectrometry

   https://doi.org/10.1002/rcm.8793  

   Inutan, Ellen D. ; Jarois, Dean R. ; Lietz, Christopher B. ; El‐Baba, Tarick J. ; Elia, Efstathios A. ; Karki, Santosh ; Sampat, Andjoe A. S. ; Foley, Casey D. ; Clemmer, David E. ; Trimpin, Sarah ( August 2020 , Rapid Communications in Mass Spectrometry)

   Examining surface protein conformations, and especially achieving this with spatial resolution, is an important goal. The recently discovered ionization processes offer spatial‐resolution measurements similar to matrix‐assisted laser desorption/ionization (MALDI) and produce charge states similar to electrospray ionization (ESI) extending higher‐mass protein applications directly from surfaces on high‐performance mass spectrometers. Studying a well‐interrogated protein by ion mobility spectrometry‐mass spectrometry (IMS‐MS) to access effects on structures using a solidvs.solvent matrix may provide insights.

   Ubiquitin was studied by IMS‐MS using new ionization processes with commercial and homebuilt ion sources and instruments (Waters SYNAPT G2(S)) and homebuilt 2 m drift‐tube instrument; MS™ sources). Mass‐to‐charge and drift‐time (t_d)‐measurements are compared for ubiquitin ions obtained byinletandvacuumionization using laserspray ionization (LSI), matrix‐ (MAI) and solvent‐assisted ionization (SAI), respectively, and compared with those from ESI under conditions that are most comparable.

   Using the same solution conditions with SYNAPT G2(S) instruments, t_d‐distributions of various ubiquitin charge states from MAI, LSI, and SAI are similar to those from ESI using a variety of solvents, matrices, extraction voltages, a laser, and temperature only, showing subtle differences in more compact features within the elongated distribution of structures. However, on a homebuilt drift‐tube instrument, within the elongated distribution of structures, both similar and different t_d‐distributions are observed for ubiquitin ions obtained by MAI and ESI. MAI‐generated ions are frequently narrower in their t_d‐distributions.

   Direct comparisons between ESI and the new ionization methods operational directly from surfaces suggest that the protein in its solution structure prior to exposure to the ionization event is either captured (frozen out) at the time of crystallization, or that the protein in the solid matrix is associated with sufficient solvent to maintain the solution structure, or, alternatively, that the observed structures are those related to what occurs in the gas phase with ESI‐ or MAI‐generated ions and not with the solution structures.

    

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