iProXProteomicsYang ChenHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0017683000chenyang1816185048@bjmu.edu.cnThe Center for Precision Medicine Multi-omics Research (CPMMR)iProXfalseER-IP enables rapid isolation and proteomic characterization of the endoplasmic reticulumIsolation of the endoplasmic reticulum (ER) is essential for characterizing organelle-specific molecular processes, yet existing ER purification methods often require lengthy workflows and involve trade-offs between speed, specificity, and compatibility with downstream analyses. Here, we developed ER-IP, a rapid immunoaffinity-based method for ER isolation. ER-IP utilizes a Sec61β-3×HA-EGFP fusion protein to label ER membranes and enables affinity capture of ER fractions using anti-HA magnetic beads. We validated ER-IP through fluorescence microscopy, transmission electron microscopy, and biochemical analyses. The isolated fractions contained intact ER membrane structures and were enriched for canonical ER proteins. Compared with an ER-IP workflow incorporating a pre-clearing centrifugation step and a commercially available ER isolation kit, ER-IP substantially reduced processing time, enabling ER enrichment within approximately 15minutes while maintaining comparable recovery of ER-associated proteins. To further evaluate the molecular composition of ER-IP isolates, we performed quantitative proteomic analysis and identified 8,540 proteins. ER-IP fractions showed enrichment of proteins involved in protein translocation, protein folding, ER-associated degradation, lipid metabolism, and calcium homeostasis. In addition, the resulting proteomic dataset enabled identification of BAG2 as a previously unannotated ER-associated protein, illustrating the utility of ER-IP for discovery-oriented applications. Together, ER-IP provides a rapid and experimentally accessible approach for ER enrichment and downstream molecular characterization, expanding the toolkit available for investigating ER biology.Wed Jun 10 00:00:00 GMT+01:00 2026PXD0795439606