Endoplasmic reticulum (ER) fractions were purified from HEK293T cells expressing ER-tag or control-tag constructs and analyzed by quantitative proteomics to assess enrichment of ER proteins and define ER proteome composition. Proteomic analysis demonstrated selective enrichment of annotated ER proteins relative to other cellular compartments. A curated reference set of ER-associated proteins was used to benchmark coverage. Approximately 800 proteins were significantly enriched over control immunoprecipitations, of which 508 matched curated ER-associated proteins.