RT Journal Article
SR Electronic
T1 Unfolding and identification of membrane proteins from native cell membranes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 732933
DO 10.1101/732933
A1 Galvanetto, Nicola
A1 Maity, Sourav
A1 Ilieva, Nina
A1 Ye, Zhongjie
A1 Laio, Alessandro
A1 Torre, Vincent
YR 2019
UL http://biorxiv.org/content/early/2019/09/30/732933.abstract
AB Is the mechanical unfolding of proteins just a technological feat applicable only to synthetic preparations or can it provide useful information even for real biological samples? Here, we describe a pipeline for analyzing native membranes based on high throughput single-molecule force spectroscopy. The protocol includes a technique for the isolation of the plasma membrane of single cells. Afterwards, one harvests hundreds of thousands SMSF traces from the sample. Finally, one characterizes and identifies the embedded membrane proteins. This latter step is the cornerstone of our approach and involves combining, within a Bayesian framework, the information of the shape of the SMFS Force-distance which are observed more frequently, with the information from Mass Spectrometry and from proteomic databases (Uniprot, PDB). We applied this method to four cell types where we classified the unfolding of 5-10% of their total content of membrane proteins. The ability to mechanically probe membrane proteins directly in their native membrane enables the phenotyping of different cell types with almost single-cell level of resolution.