The rumen is a major the site of digestion and absorption of energy predominantly in the form of short chain fatty acids as well as amino acids and simple carbohydrates. The value of crops such as perennial wheat (PW) for grain and grazing compared to conventional wheat (W), or the addition of lucerne to PW (PWL) is still being determined. The research sought to determine if these diets representing high potassium (K): low sodium (Na) ratio (W, PW and PWL) was associated with changes in the membrane bound proteins that transport nutrients in the rumen epithelium (RE). Information about these proteins and the influence of different diets is scarce due to difficulties developing a procedure to examine them using quantitative proteomics. The research focuses on proteins extracted using an enzymatic procedure (Bond et al. 2019) from the rumen epithelium layer of the sheep’s’ forestomach. A membrane protein enriched fraction was then extracted and prepared for TMT-MS. The comprehensive assignment of proteins (>4000) to cellular location and quantification of differences in protein abundance will progress the annotation and understanding of protein functions in the ruminant genomes.