Background: The sheep (Ovis aries) is a powerful, translational large animal model in neuroscience. The sheep brain has several characteristics in common with the human brain that rodent models lack including comparable brain size, the presence of gyri and sulci and white matter proportion that more closely resemble those of the human brain. Despite these benefits, the proteomic profile of the disease-free ovine brain remains under-characterized, limiting the use of this model in characterising the on- and off-target effects of trial therapies.

Aims:  characterise the proteome of disease-free sheep brain using cutting-edge mass spectrometry.

Methods: Naïve Sheep brain tissue from each hemisphere was collected and digested into peptides. Unbiased, unlabelled mass spectrometry analysis was performed using an Orbitrap Exploris 480 coupled with high-field asymmetric waveform ion mobility spectrometry (FAIMS) at 40, 50, 60 and 70 compensation voltages. Peptide matches were be adjusted for 1% false discovery rate, and bioinformatic analysis was conducted using Perseus and Ingenuity Pathway Analysis (IPA, QIAGEN) to identify activity of canonical molecular pathways. Comparative analysis between the two hemispheres was conducted to explore differences in their proteomes.

Results: Biological replicates were highly correlated (Pearson correlation >0.89). There is no significant difference in protein abundance between biological replicates (p = 0.902), and no significant differences in protein abundance between brain hemispheres within an individual. IPA analysis of proteins in the top quartile of abundance showed significant enrichment in the synaptogenesis, EIF2 and Rho GTPase cycle pathways.

Conclusion: We present, to date, the most in-depth characterisation of the sheep brain proteome. The results of this study are a valuable resource for future research in translational neuroscience and will assist in the testing of new diagnostic and therapeutic strategies for brain-related disorders.