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Enhanced RNA Interactome capture (eRIC) and total proteome of mouse brain, kidney and liver

ABSTRACT: Male mice on a homogenous C57BL6/J genetic background were sacrificed at 11 to 13 weeks of age by cervical dislocation. Brain, kidneys and liver were immediately harvested, flash frozen in liquid nitrogen and stored at -80Ã‚Â°C until use. Animal handling was in accordance with guidelines approved by the European Molecular Biology Laboratory (EMBL). Organ sections (thickness 30 Ã‚Âµm) were prepared in a Cryostat (Leica Biosystems, Leica CM3050 S) set to -20 Ã‚Â°C and deposited onto SuperFrost glass slides (Carl Roth, H880); the glass slides were pre-cooled to -20Ã‚Â°C in the cryostat. ~10 sections were placed on a glass slide, and a total of around 150 sections were prepared for each sample per mouse. Tissue sections on glass slides were then transferred onto metal plates placed on dry ice. The tissue sections were exposed or not to 1 J/cm2 of 254 nm UV light in a XL 1500 UV Spectrolinker (Spectronics Corporation) and subjected to eRIC to determine the RNA-bound proteome. eRIC eluates obtained from the respective organs of two mice were combined per independent experiment. The analyses were conducted in duplicate for each organ studied. For the no crosslink controls, organ sections from four mice were pooled to generate one unique no crosslink eRIC sample per organ studied.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Liver, Kidney

SUBMITTER: Frank Stein

LAB HEAD: Matthias W. Hentze

PROVIDER: PXD032113 | Pride | 2023-03-02

REPOSITORIES: Pride

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Project description:Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10 % additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.

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Enhanced RNA Interactome capture (eRIC) and total proteome of mouse brain, kidney and liver

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