A Stepwise Approach for the Isolation and the Identification of Chemically Reactive Exofacial Protein Thiols

The plasma membrane controls the selective internalization of (macro)molecules through different mechanisms, often relaying on specialized outward-facing carriers such as exofacial proteins thiols (EPTs). Although the interchange of critical thiols and disulphides between EPTs and exogenous cargoes is the first critical event, the identification of specific cell interactors remains to be thoroughly explored. Besides, it is likewise evident that only the relatively little suite of EPTs truly reactive can be considered theranostic targets.

We were aimed at developing a stepwise procedure for the isolation and identification of a subset of EPTs, that we named chemically reactive EPS, which are potential theranostic targets.

In the present study, EPTs that displayed permissive sulfhydryls on the surface of live cells in vitro underwent i) chemo-selective capture by means of thiolated superparamagnetic microbeads (isolation step), followed by ii) their prompt release via disulphide breakage through the addition of DTT reducing agent (elution step) and iii) analysis by means of SDS-PAGE and LCMS/ MS (identification step).

In total cell lysates, most of the proteins recovered were intracellular. Conversely, this methodology allowed a 2.6-fold enrichment in chemically reactive EPTs recovered and identified, corresponding up to 37% of the total cellular proteins. The key element of our approach was the reversible chemo-selective capture through disulphide linkages between chemically reactive EPTs and free thiols on microbeads.

We devised an enabling methodology to selectively pick up, recover and characterize chemically reactive EPTs.