ABSTRACT: Maldi imaging with NEDC matrix of a rat brain tissue section. Image was acquired with 50 um resolution. Ion mobility seperation enabled. Negative ion mode.
Project description:Maldi imaging with NEDC matrix of a rat brain tissue section. Image was acquired with 50 um resolution. Ion mobility seperation enabled. Negative ion mode.
Project description:Rat brain tissues for imaging mass spectrometry were removed from animal organs, frozen on dry ice, and then stored at -80-degree C until analysis. 10-micrometer rat brain tissues were sectioned using a Leica CM 3050S Research Cryostat (Leica Biosystems, Wetzlar, Germany), prior to thaw mounting onto indium tin oxide-coated microscope slides (Delta Technologies, Loveland, CO, USA). 1,5-diaminonaphthalene (DAN) MALDI matrix layer was sublimated to the microscope slide using an in-house sublimation apparatus. MALDI imaging mass spectrometry was performed on a 7T solariX Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a dynamically harmonized ParaCell XR (Bruker Daltonics, Bremen, Germany). Analysis was performed in negative ion mode from m/z 400 to 2000 with ~0.5 s time-domain transient length, resulting in a resolution of ~35,000 FWHM at m/z ~760. The MALDI source is equipped with a Smartbeam II Nd:YAG MALDI laser and was used to sample at a pixel spacing of 100 micrometers in the x and y dimensions using 200 laser shots per pixel (large laser focus, 2 kHz frequency) with Smart Walk enabled.
Project description:We developed and validated ‘HIT-MAP’ (High-resolution Informatics Toolbox in MALDI-MSI Proteomics), an open-source bioinformatics workflow using peptide mass fingerprint analysis and a dual scoring system to computationally assign peptide and protein annotations to high mass resolution MSI datasets, and generate customisable spatial distribution maps. The uploaded files are an example dataset for the HiTMaP proteomics search engine, designed for MALDI-imaging proteomics annotation. The example data files contain one bovine lens tissue section and one mouse brain tissue section. The ID folder contains the protein/peptide identification result for each tissue segment, and the summary folder contains the protein cluster images.
Project description:Autosomal recessive polycystic kidney disease is a severe, monogenetically inherited kidney and liver disease and PCK rats carrying the orthologous mutant gene serve as a model of human disease. We combined selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis of imaging data sets for identification of candidate lipid markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. Beside increased levels of serum-cholesterol these sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases suggesting that they be molecular markers of the disease. Genome-scale gene expression profiling of PCK and SD livers as control was performed to attempt elucidation of some of the underlying mechanisms leading to increases of cholesterol and taurine-conjugated bile acids in the PCK rat. Several pathways were found to be changed in cystic livers with up regulation or down regulation of important gene sets. Enhanced expression of steroid biosynthesis genes might result in the observed increased levels of cholesterol. In contrast, primary bile acid biosynthesis was found to be down regulated in diseased livers. These findings might be explained by compensatory mechanisms of liver metabolism to reduce toxic levels of accumulated bile acids. Snap-frozen liver tissue of 10 week old rats were subjected for RNA extraction and hybridization on Affymetrix microarrays to perform genome-scale gene expression profiling of n = 6 diseased PCK and n = 6 Sprague Dawley rat livers as control.
Project description:Autosomal recessive polycystic kidney disease is a severe, monogenetically inherited kidney and liver disease and PCK rats carrying the orthologous mutant gene serve as a model of human disease. We combined selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis of imaging data sets for identification of candidate lipid markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. Beside increased levels of serum-cholesterol these sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases suggesting that they be molecular markers of the disease. Genome-scale gene expression profiling of PCK and SD livers as control was performed to attempt elucidation of some of the underlying mechanisms leading to increases of cholesterol and taurine-conjugated bile acids in the PCK rat. Several pathways were found to be changed in cystic livers with up regulation or down regulation of important gene sets. Enhanced expression of steroid biosynthesis genes might result in the observed increased levels of cholesterol. In contrast, primary bile acid biosynthesis was found to be down regulated in diseased livers. These findings might be explained by compensatory mechanisms of liver metabolism to reduce toxic levels of accumulated bile acids.
Project description:Rat brain tissues for imaging mass spectrometry were removed from animal organs, frozen on dry ice, and then stored at -80-degree C until analysis. 10-micrometer rat brain tissues were sectioned using a Leica CM 3050S Research Cryostat (Leica Biosystems, Wetzlar, Germany), prior to thaw mounting onto indium tin oxide-coated microscope slides (Delta Technologies, Loveland, CO, USA). 1,5-diaminonaphthalene (DAN) MALDI matrix layer was sublimated to the microscope slide using an in-house sublimation apparatus. MALDI imaging mass spectrometry was performed on a 7T solariX Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer equipped with a dynamically harmonized ParaCell XR (Bruker Daltonics, Bremen, Germany). Analysis was performed in negative ion mode from m/z 400 to 2000 with ~0.5 s time-domain transient length, resulting in a resolution of ~35,000 FWHM at m/z ~760. The MALDI source is equipped with a Smartbeam II Nd:YAG MALDI laser and was used to sample at a pixel spacing of 100 micrometers in the x and y dimensions using 200 laser shots per pixel (large laser focus, 2 kHz frequency) with Smart Walk enabled.
Project description:INTRODUCTION: Mass Spectrometry Imaging (MSI) is a hybrid mass spectrometry technique that integrates aspects of traditional microscopy and mass spectrometry-based omics analysis. The traditional MALDI TOF/TOF instrument still remains the dominant platform for this type of anal-ysis. However, with reduced mass resolution compared to other platforms it is insufficient to rely on mass resolution alone for peptide identification. Here we propose a hybrid method of data analysis that integrates both image-based analysis and a parallel protein identification workflow using peptide mass fingerprinting, and its successful application to the detection and validation of viral biomarkers. METHODS: FFPE samples were imaged as described previously in an UltrafleXtreme MALDI TOF/TOF. Total mass spectra were exported and searched against the mouse FASTA da-tabase, while companion images were exported and processed with image J. RESULTS: Peptide mass fingerprinting (PMF) revealed 14 target peptides that were successfully assigned to viral proteins while a pixel based correlational analysis revealed a very high R2 correlation (>0.81) be-tween those same peptides assigned to the NS1 and VP1 viral proteins. CONCLUSIONS: We successfully identified and validated the presence of viral biomarkers to a high degree of confidence using MALDI MSI.
Project description:Spatial metabolomics describes the location and chemistry of small molecules involved in metabolic phenotypes, defense molecules and chemical interactions in natural communities. Most current techniques are unable to spatially link the genotype and metabolic phenotype of microorganisms in situ at a scale relevant to microbial interactions. Here, we present a spatial metabolomics pipeline (metaFISH) that combines fluorescence in situ hybridization (FISH) microscopy and high-resolution atmospheric pressure mass spectrometry imaging (AP-MALDI-MSI) to image host-microbe symbioses and their metabolic interactions. metaFISH aligns and integrates metabolite and fluorescent images at the micrometer-scale for a spatial assignment of host and symbiont metabolites on the same tissue section. To illustrate the advantages of metaFISH, we mapped the spatial metabolome of a deep-sea mussel and its intracellular symbiotic bacteria at the scale of individual epithelial host cells. Our analytical pipeline revealed metabolic adaptations of the epithelial cells to the intracellular symbionts, a variation in metabolic phenotypes in one symbiont type, and novel symbiosis metabolites. metaFISH provides a culture-independent approach to link metabolic phenotypes to community members in situ - a powerful tool for microbiologists across fields. <br><br> <b>MALDI imaging MS assay</b> protocols and data are reported in the current study <b>MTBLS744</b>. <br><br> <b>UPLC-MS/MS assay</b> protocols and data associated to this study are reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS746><b>MTBLS746</b></a>.<br> <b>On-tissue MALDI imaging MS/MS assay</b> protocols and data associated to this study are reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS805><b>MTBLS805</b></a>.<br> <b>MRMS assay</b> protocols and data associated to this study are reported in <a href=https://www.ebi.ac.uk/metabolights/MTBLS811><b>MTBLS811</b></a>.<br>
Project description:Highly specialized cells are fundamental for proper functioning of complex organs. Variations in cell-type specific gene expression and protein composition have been linked to a variety of diseases. Although single cell technologies have emerged as valuable tools to address this cellular heterogeneity, a majority of these workflows lack sufficient in situ resolution for functional classification of cells and are associated with extremely long analysis time, especially when it comes to in situ proteomics. In addition, lack of understanding of single cell dynamics within their native environment limits our ability to explore the altered physiology in disease development. This limitation is particularly relevant in the mammalian brain, where different cell types perform unique functions and exhibit varying sensitivities to insults. The hippocampus, a brain region crucial for learning and memory, is of particular interest due to its obvious involvement in various neurological disorders. Here, we present a combination of experimental and data integration approaches for investigation of cellular heterogeneity and functional disposition within the mouse brain hippocampus using MALDI Imaging mass spectrometry (MALDI-IMS) and shotgun proteomics (LC-MS/MS) coupled with laser-capture microdissection (LCM) along with spatial transcriptomics. Within the dentate gyrus granule cells we identified two proteomically distinct cellular subpopulations that are characterized by a substantial number of discriminative proteins. These cellular clusters contribute to the overall functionality of the dentate gyrus by regulating redox homeostasis, mitochondrial organization, RNA processing, and microtubule organization. Importantly, most of the identified proteins matched their transcripts, verifying the in situ protein identification and supporting their functional analyses. By combining high-throughput spatial proteomics with transcriptomics, our approach enables reliable near-single-cell scale identification of proteins and profiling of inter-cellular heterogeneity within similar cell-types in tissues. This methodology has the potential to be applied to different biological conditions and tissues, providing a deeper understanding of cellular subpopulations in situ.