A proposed sample handling of ovine cotyledon for proteomic studies

Abstract

Ovine trophoblast is a suitable material for placental studies. However, a universal protocol for handling ruminant cotyledon samples has still not been reported. Considering the villous structure of ovine cotyledon, we suggest procedures to prepare cotyledons with limited inherent contamination, using semi-dry conditions to avoid freezing damage and sample errors. The cytosolic water-soluble proteins were physically extracted from the frozen cotyledons. High homogeneity was demonstrated between the replicates of both tissue and extract samples. Importantly, the chemical lysis of placental crude extracts was necessary for protein separation and immunoreaction. The integrity of stored tissues was histologically validated using a formalin-fixed paraffin-embedded technique. Using label-free proteomics, we detected 388 Ovis protein-groups in at least two of three biological replicates of either the tissue or extract. Although the water-soluble proteins were dominated by hemoglobin subunits, ten proteins were identified exclusively in all extract replicates. The physical extraction selectively reduced the membrane, extracellular matrix, and cytoskeleton proteins. The hydrolase enzymes, in the extract, hindered the identification of some specific proteins, such as histone H2A. In summary, the proposed workflow may guide further proteomic investigations of ovine cotyledon biology. Furthermore, our proteomic data have inferred some potential mechanisms of ovine trophoblast at parturition.

Keywords: Ovine cotyledon proteome; Proteomic assays; Sample handling; Water-soluble proteins.