Abstract

One of the biggest challenges to studying membrane proteins is their quantitative characterization under native-like conditions that preserve their structural and functional integrity. Here, we introduce an integrated preparative and analytical method that enables researchers to simultaneously measure both the concentration of an endogenous membrane-protein target and its binding affinity to a specific ligand in a native lipid-bilayer environment directly extracted from crude cellular membranes. To demonstrate our approach, we first used the nanodisc-forming polymer Glyco-DIBMA to create a native membrane-protein library of a breast cancer cell line. Using microfluidic diffusional sizing (MDS) on the Fluidity One-M, we then determined both the concentration of the endogenous oncoprotein HER2 and its affinity to trastuzumab, a therapeutic HER2-specific antibody. The combination of native membrane-protein libraries with MDS provides quantitative information on membrane-proteins without the need for laborious and costly protein-purification campaigns. In addition, the capability of determining absolute cellular concentrations of endogenous membrane proteins provides a much-needed opportunity for calibrating relative protein-copy numbers obtained from proteomics, microscopy, or flow cytometry. The method is straightforward to adapt in standard research laboratories, takes only a few hours to complete, and has the potential to be expanded from cell lines to tissues and tumor biopsies.