Mammalian MemPrep establishes the lipid composition of ER membranes in HEK293T cells.

The endoplasmic reticulum (ER) forms a dynamic network of sheets and tubules, whose lipid composition remains incompletely defined. Using an optimized MemPrep workflow, we establish a high‑confidence lipidome of the mammalian ER and selectively enrich membrane vesicles originating from ER tubules as a major ER subdomain. Quantitative lipidomics show ER membranes are dominated by phosphatidylcholine and monounsaturated glycerophospholipids, consistent with a highly compressible bilayer. Although proteomics suggests a functional specialization of ER tubules and an enrichment of tubule-associated proteins, the lipidome of an ER tubule-enriched isolate is indistinguishable from the general ER, indicating that principal ER architectures share a common lipid composition. Integration of lipidomic data with bioinformatic analyses of transmembrane helices demonstrates that the physicochemical features of ER lipids mirror those of ER-resident membrane proteins, including reduced hydrophobicity and increased polarity compared to plasma membrane proteins. These findings support a coordinated evolution of ER proteins and lipids based on shared biophysical constraints. Together, this work provides a definitive characterization of the mammalian ER lipidome and suggests that membrane properties are maintained across the ER.