To identify proteins enriched in functional membrane microdomains organized by stomatin—hereafter referred to as STOM cargo proteins—we designed mass spectrometry (MS)–based approaches combining co-immunoprecipitation (co-IP) MS and enzymatic proximity labeling MS. For these experiments, we generated a series of stable HEK293T cell lines via lentiviral transduction. For the Co-IP MS analysis, we employed GFP-STOM-WT to capture stomatin and its interacting partners from cellular lysates. Additionally, we generated a cytoplasm-localized GFP-NES control by fusing GFP to a nuclear export signal (NES), enabling subtraction of GFP tag-derived contaminants. Enrichment was performed using a GFP nanobody. GDN was chosen for membrane extraction due to its ability to preserve the membrane region within the stomatin assemblies. This approach enriches cargo proteins interacting with stomatin directly or embedded in the membrane region within the stomatin bowl-like structure. We also applied proximity labeling MS to profile STOM cargo proteins. The labeling enzyme was fused at the N-terminus of stomatin, such that labeled proteins should reside outside the closed stomatin bowl.