A sensitive S-trap-based approach to the analysis of T-cell raft proteome

The development of sensitive technologies for T cell-raft proteomics is highly challenging. We explored an innovative strategy, based on suspension trapping (S-trap) sample preparation. Mouse splenocytes were subjected to negative immunoselection for T cell preparation. Rafts were isolated by a detergent-free method and Optiprep gradient ultracentrifugation. Flotillin-1-, LAT- and cholesterol-rich microdomains were subjected to proteomic analysis through an optimized protocol based on S-Trap and high pH fractionation, followed by nano-LC-MS/MS. We identified 2680 proteins in the raft-rich fraction and established a database of 894 bona fide T cell-raft proteins. We performed a differential analysis on the raft-rich fraction from non-stimulated vs. anti-CD3/CD28 TCR-stimulated T cells. Our results revealed 42 proteins present in one condition and absent in the other, such as Akt2 and Nck1. For the first time a proteomic analysis is performed on rafts from ex-vivo T cells obtained from individual mice, before and after TCR activation. Our results show an increased specificity and sensitivity of the proposed method.