| Summary: | <i>Opuntia</i> spp. is an economically important vegetable crop with high stress-tolerance and health benefits. However, proteomic analysis of the plant has been difficult due to the composition of its succulent cladodes; the abundant polysaccharides interfere with protein extraction. To facilitate proteomic analysis of this plant, we present a rapid and simple protein extraction method for <i>Opuntia ficus-indica</i> (L.) Miller. The optimized method produced highly reproducible protein patterns and was compatible with a gel-free quantitative workflow without the need for additional purification. We successfully analyzed the cladode mesocarp and exocarp tissues, resulting in the identification of 319 proteins. In addition, we used this method to examine the relative changes in the <i>Opuntia</i> proteome in response to salt stress to determine whether physiological changes could be captured. Qualified observations were obtained, revealing that salt stress increased phosphoenolpyruvate carboxylase abundance and decreased ribulose-bisphosphate carboxylase in young <i>O. ficus-indica</i> plants. These findings suggest that Crassulacean acid metabolism is promoted under salinity. This study highlights the efficacy of our optimized protein extraction method for elucidating the metabolic adaptations of <i>Opuntia</i> using gel-free proteomic analysis.
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