PT  - JOURNAL ARTICLE
AU  - Galvanetto, Nicola
AU  - Maity, Sourav
AU  - Ilieva, Nina
AU  - Ye, Zhongjie
AU  - Laio, Alessandro
AU  - Torre, Vincent
TI  - Unfolding and identification of membrane proteins from native cell membranes
AID  - 10.1101/732933
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 732933
4099  - http://biorxiv.org/content/early/2019/09/30/732933.short
4100  - http://biorxiv.org/content/early/2019/09/30/732933.full
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.