Dataset Information

Tissue sampling and homogenization with NIRL enables spatially resolved cell layer specific proteomic analysis of the murine intestine

ABSTRACT: For investigating the molecular physiology and pathophysiology in organs the most exact data will be obtained, if not organ specific cell lines are analysed or the whole organ is homogenized, followed by the analysis of its biomolecules, but if the morphological organisation of the organ can be addressed and in the best case the composition of molecules in single cells of the target organ can be analysed. Laser capture microdissection (LCM) is a technique, which is enabling the selection of specific cells of a tissue for further analysis of their molecules. However, LCM is a time-consuming 2-dimensional technique and optimal results are only obtained, if the tissue is fixed e.g. by formalin. Especially for proteome analysis formalin fixation is reducing the number of identifiable proteins, which is an additional drawback. Recently, it was demonstrated that sampling of fresh-frozen (non-fixed) tissue with an infrared-laser is giving higher yields with respect to the absolute protein amount and number of identifiable proteins than conventional mechanical homogenization of tissues. In this study the applicability of the infrared laser tissue sampling for proteome analysis of different cell layers of murine intestine was investigated, using LC-MS/MS-based differential quantitative bottom-up proteomics. By laser ablation eight consecutive layers of the colon tissue were obtained and analyzed. Beside a clear distinguishability of protein profiles between ascending, descending and transversal colon, we identified in the different intestinal cell layers proteins, which are cell-specific as confirmed with data from the human protein atlas. Thus, for the first time the sampling directly from intact fresh-frozen tissue with 3-dimensional resolution is giving access to the different proteomes of the different cell layers of colon tissue.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Colon

SUBMITTER: Hannah Voß

LAB HEAD: Prof. Dr. Hartmut Schlüter

PROVIDER: PXD033584 | Pride | 2022-06-22

REPOSITORIES: Pride

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Publications

Efficacy of military antishock trousers in compensatory and decompensatory hemorrhagic hypotension.

Johnson G G Bond R F RF Stack L B LB Class C A CA Hardebeck J R JR

Circulatory shock 19870101 3

The compensatory cardiovascular response to hemorrhage includes a baroreceptor-induced activation of the sympathetic nervous system resulting in an attempt to reestablish MAP through peripheral vasoconstriction. If the hypotension is not reversed this compensatory vasoconstriction will progress to a loss of vascular tone known as vascular decompensation. The primary purpose of the present study was to compare the effectiveness of military antishock trousers (MAST) applied during the compensatory ...[more]

PMID: 3568281

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Project description:Background: Aquaculture of the black tiger prawn Penaeus monodon remains severely constrained by an almost total dependence on wild-caught broodstock. Reliance on wild-caught broodstock stems, for the most part, from reduced reproductive potential of captive-reared females. Reproductive performance of captive-reared females is usually characterised by longer latency period, lower egg production, egg hatch rates and post-larval survivorship compared with their wild-caught counterparts. Improved understanding of the cellular and associated molecular events occurring during peneaid ovarian maturation could therefore be fundamental to improving reproductive success of captive-reared animals. Methodology/Principle Findings: In support of other studies, our histological analyses of developing oocytes revealed differences between wild-caught and captive-reared P. monodon, including reduced lipid accumulation in oocytes of captive-reared animals. We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Custom oligonucleotide microarrays were constructed and screened with transcripts derived from the ovary, cephalothorax and eyestalk from animals of all ovarian maturation stages. Ovarian maturation-related differential expression patterns were observed for 111 transcripts, with 53 transcripts displaying differential expression between wild-caught and captive-reared animals. Notably transcripts encoding vitellogenin - the major egg yolk protein precursor, and a lipid storage droplet protein (which we named pmLSD) which is involved in lipid accumulation, were found to be more highly expressed in wild-caught animals. pmLSD transcripts localise to pre-vitellogenic oocytes of wild-caught animals and the pmLSD protein is exclusively localised to the surface of lipid droplets of oocytes at vitellogenic and cortical rod stages. We have employed oligonucleotide microarray analysis to compare expression profiles of genes involved in ovarian maturation among wild-caught and captive-reared animals. Target preparation and microarray hybridisation. Ovarian RNA samples from nine wild-caught animals representing six ovarian maturation stages (P, 2, 24, V, R, E) were used in microarray hybridisations. Similarly, RNA samples from three captive-reared animals representing four maturation stages (P, 24, V, E) were used in microarray hybridisations. For wild-caught animals, samples from each ovarian maturation stage were pooled into groups of four and five, enabling two hybridisations. For captive-reared animals, samples from each ovarian maturation stage from all three animals were pooled enabling one hybridisation for each stage. Importantly, as the four stages for captive-reared animals were (1) pre-ablation pre-vitellogenic, (2) post-ablation pre-vitellogenic, (3) post-ablation vitellogenic, (4) post-ablation vitellogenic with cortical rods, this arrangement allowed for 2 samples of captive-reared pre-vitellogenic and 2 samples of captive-reared vitellogenic, thereby enabling t-tests between samples, while also allowing analysis across the whole 4 stages via cluster analysis. All hybridisations were single channel hybridisations conducted using equal amounts of RNA pooled from each individual.

Dataset Information

Tissue sampling and homogenization with NIRL enables spatially resolved cell layer specific proteomic analysis of the murine intestine

Publications

Efficacy of military antishock trousers in compensatory and decompensatory hemorrhagic hypotension.

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