Dataset Information

Direct 3D sampling of the embryonic mouse head: Layer-wise nanosecond infrared laser (NIRL) ablation from scalp to cortex for spatially resolved proteomics

ABSTRACT: Common workflows in bottom-up proteomics require homogenization of tissue samples giving to get access to the biomole-cules within the cells. The homogenized tissue samples often contain many different cell types, thereby representing an average of the natural proteome composition and rare cell types are not sufficiently represented. To overcome this problem, small vol-ume sampling and spatial resolution is needed to maintain a better representation of the sample composition and their proteo-me signatures. Using nanosecond infrared laser ablation, the region of interest can be targeted in a three dimensional (3D) fash-ion whereby the spatial information is maintained during the simultaneous process of sampling and homogenization. In this study, we ablated 40 µm thick consecutive layers directly from the scalp through the cortex of the embryonic mouse heads and analyzed them by subsequent bottom-up proteomics. Extra- and intracranial ablated layers showed distinct proteome profiles comprising expected cell specific proteins. Additionally, known cortex markers like SOX2, KI67, NESTIN and MAP2 showed a layer specific spatial protein abundance distribution. We propose potential new marker proteins for cortex layers such as MTA1 and NMRAL1. The obtained data confirm that the new 3D -tissue sampling and homogenization method is well suited for investigating the spatial proteome signature of tissue samples in a layer-wise manner. Characterization of the proteome composition of embryonic skin and bone structures, meninges, and cortex lamination in situ, enables to a better understanding of molecular mechanisms of development during embryogenesis and disease pathogenesis.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Manuela Moritz

LAB HEAD: Hartmut Schlueter

PROVIDER: PXD043011 | Pride | 2024-01-26

REPOSITORIES: Pride

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Direct 3D Sampling of the Embryonic Mouse Head: Layer-wise Nanosecond Infrared Laser (NIRL) Ablation from Scalp to Cortex for Spatially Resolved Proteomics.

Navolić Jelena J Moritz Manuela M Voß Hannah H Schlumbohm Simon S Schumann Yannis Y Schlüter Hartmut H Neumann Julia E JE Hahn Jan J

Analytical chemistry 20231113 47

Common workflows in bottom-up proteomics require homogenization of tissue samples to gain access to the biomolecules within the cells. The homogenized tissue samples often contain many different cell types, thereby representing an average of the natural proteome composition, and rare cell types are not sufficiently represented. To overcome this problem, small-volume sampling and spatial resolution are needed to maintain a better representation of the sample composition and their proteome signatu ...[more]

PMID: 37956982

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Project description:Genome-wide transcriptional profiling allows characterization of the molecular underpinnings of neocortical organization, including cortical areal specialization, laminar cell type diversity and functional anatomy. Microarray analysis of individual cortical layers across sensorimotor and association cortices in rhesus macaque demonstrated robust and specific laminar and areal molecular signatures driven by differential expression of genes associated with specialized neuronal function. Gene expression corresponding with laminar architecture was generally similar across cortical areas, although genes with robust areal patterning were often highly laminar as well, and these patterns were more highly conserved between macaque and human as compared to mouse. Layer 4 of primate primary visual cortex displayed a distinct molecular signature compared to other cortical regions, a specialization not observed in mouse. Overall, transcriptome-based relationships were strongest between proximal layers in a cortical area, and between neighboring areas along the rostrocaudal axis, reflecting in vivo cortical spatial topography and therefore a developmental imprint. Tissue from male and female monkeys (n = 2-3 animals/gender) was collected from anterior cingulate gyrus (ACG), layers 2, 3, 5, 6; orbitofrontal cortex (OFC), layers 2, 3, 5, 6; dorsolateral prefrontal cortex (DLPFC), layers 2-6; primary motor cortex (M1), layers 2, 3, 5, 6; primary somatosensory cortex (S1), layers 2-6; primary auditory cortex (A1), layers 2-6; primary visual cortex (V1), layers 2-6 including 4A, 4B, 4Calpha (4Ca), 4Cbeta (4Cb); secondary visual cortex (V2), layers 2-6; middle temporal area (MT), layers 2-6; temporal area (TE), layers 2-6; hippocampus, CA1, CA2, CA3, dentate gyrus (DG); and dorsal lateral geniculate nucleus (LGN), magnocellular, parvocellular and koniocellular divisions. Due to the limited number of samples that could be amplified concurrently, amplifications were performed in 3 batches (batches A-C, containing 102, 119 and 10 experimental samples, respectively). To control for batch effects, common RNA pool control samples were amplified and hybridized in each batch. These included LCM extracted hippocampus CA3 samples from the current study and a whole rhesus brain RNA pool (Biochain). These control samples were hybridized in 6 replicates in batch A and B and in 3 replicates in batch C (3 replicates per 96 well plate).

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