TY - JOUR TI - Unfolding and identification of membrane proteins in situ AU - Galvanetto, Nicola AU - Ye, Zhongjie AU - Marchesi, Arin AU - Mortal, Simone AU - Maity, Sourav AU - Laio, Alessandro AU - Torre, Vincent A2 - Bassereau, Patricia A2 - Aldrich, Richard W A2 - Blanchard, Aaron T A2 - Tapia-Rojo, Rafael VL - 11 PY - 2022 DA - 2022/09/12 SP - e77427 C1 - eLife 2022;11:e77427 DO - 10.7554/eLife.77427 UR - https://doi.org/10.7554/eLife.77427 AB - Single-molecule force spectroscopy (SMFS) uses the cantilever tip of an atomic force microscope (AFM) to apply a force able to unfold a single protein. The obtained force-distance curve encodes the unfolding pathway, and from its analysis it is possible to characterize the folded domains. SMFS has been mostly used to study the unfolding of purified proteins, in solution or reconstituted in a lipid bilayer. Here, we describe a pipeline for analyzing membrane proteins based on SMFS, which involves the isolation of the plasma membrane of single cells and the harvesting of force-distance curves directly from it. We characterized and identified the embedded membrane proteins combining, within a Bayesian framework, the information of the shape of the obtained curves, with the information from mass spectrometry and proteomic databases. The pipeline was tested with purified/reconstituted proteins and applied to five cell types where we classified the unfolding of their most abundant membrane proteins. We validated our pipeline by overexpressing four constructs, and this allowed us to gather structural insights of the identified proteins, revealing variable elements in the loop regions. Our results set the basis for the investigation of the unfolding of membrane proteins in situ, and for performing proteomics from a membrane fragment. KW - atomic force microscopy KW - protein identification KW - single-molecule force spectroscopy JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -