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Subcellular proteomics include, in its experimental workflow, steps aimed at purifying organelles. The purity of the subcellular fraction should be assessed before mass spectrometry analysis, in order to confidently conclude the presence of associated specific proteoforms, deepening the knowledge of its biological function. In this chapter, a protocol for isolating endoplasmic reticulum (ER) and purity assessment is reported, and it precedes the proteomic analysis through a gel-free/label-free proteomic approach. Dysfunction