MassIVE MSV000100234

Description

Domestication appears to alter the morphology and physiology of the central nervous system. Due to the limited availability of proteomic and cytoarchitectural data for comparing wild and domesticated animals, we performed a comparative analysis of the cytoarchitecture and protein profile of the primary motor cortex (M1) the key structure controlling motor activity in pigs and wild boars to evaluate the effects of domestication. Protein extraction from FFPE stored tissue sections was performed as follows: ten 10 micrometeres thick sections from each cortex area (R and L) both of pig (n 3) and wild boar (n 3) were pooled and deparaffinized in 2-5 changes of xylene for 10 min each. Then the tissues were rehydrated through a series of graded ethanol (absolute two times, 85, 70) for 10 min each. After rehydration, the tissues were resuspended in an extraction buffer of 20 mM Tris-HCl at pH 6 containing 2 SDS and 0.2 M glycine and incubated at 4 C for 1 hour under agitation. The homogenates were sonicated 3 times for 30 sec each, heated at 100 C for 20 min and successively at 60 C for 2 hours under agitation. The crude extracts were clarified by centrifugation at 14000 g for 20 min at 4 C the supernatants transferred to a new tube while the pellet resuspended in extraction buffer at pH 8.8 and incubated at 50 C for 3 hours under agitation. At the end of incubation time the sample was centrifugated at 14000 g for 20 min. The supernatant were pooled and the protein extracts were treated to reduce the content of SDS using the SDSOut precipitation kit following the manufacturer instructions ThermoFisher Scientific. Briefly, 1 volume of SDS Out precipitation reagent was added to 20 volumes of sample. Then the mixture was incubated in ice for 20 min and centrifuged at 10000 g for 10 minutes at room temperature. The supernatant was transferred to a spin cup column and centrifuged for 1 minute at 10000 g to clarify the supernatant. The samples were stored at -80 C until used. The protein content of the extracts was determined by RCDC of Biorad using serum albumin as standard. 50 micrograms of proteins were loaded onto a Nanosep 10-kDa-cutoff filter and trypsin digested according to our routine protocol. Each digested protein sample was analyzed in technical triplicate. Raw data were processed using PEAKS studio Xpro using the correct precursor only option. Spectra were matched against UniProt sus scrofa reviewed and unreviewed database, to which a list of common contaminants was appended. [doi:10.25345/C58S4K30T] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: domestication ; FFPE ; primary motor cortex ; parvalbumin ; pigs ; proteomic ; wild boars ; DatasetType:Proteomics

Contact