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GNPS - High mass resolution lipid MALDI imaging of rat brain

ABSTRACT: 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.

INSTRUMENT(S): 7T solariX FT-ICR

ORGANISM(S): Rattus Rattus

SUBMITTER: Boone M. Prentice

PROVIDER: MSV000095124 | GNPS | Sun Jun 23 17:56:00 BST 2024

REPOSITORIES: GNPS

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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.

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GNPS - High mass resolution lipid MALDI imaging of rat brain

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