Abstract
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.
Footnotes
Is the mechanical unfolding of proteins just a technological feat applicable only to synthetic preparations or is it applicable to real biological samples? Here, we describe a method providing all the necessary steps to deal with native membranes, from the isolation of the plasma membrane of single cells, to the characterization and identification of the embedded membrane proteins. We combined single-molecule force spectroscopy with an automatic pattern classification of the obtained Force-distance curves, and we provide a Bayesian identification of the unfolded proteins. The Bayesian identification is based on the cross-matching of Mass Spectrometry datasets with 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.