IEC-TMT for pTyr protein complex analysis

This study investigates the downstream signaling pathways of T cell exhaustion by analyzing the changes in phosphotyrosine (pTyr) protein complexes using an integrated ion-exchange chromatography tandem mass tag (IEC-TMT) workflow. Peripheral blood mononuclear cells (PBMCs) from healthy donors were activated and co-cultured with tumor cells to induce T cell exhaustion. A trifunctional probe was developed to enrich pTyr protein complexes from the PBMC lysates. The captured complexes were then fractionated using IEC and analyzed by TMT-based quantitative proteomics. This approach allowed for the identification and quantification of 28 protein complexes. Differential analysis revealed significant changes in the abundance of several complexes, including the upregulation of lysosome-related complexes and downregulation of ER-related complexes, suggesting their potential roles in T cell exhaustion. Further investigation revealed that RTN4, a component of an upregulated lysosome-related complex, contributes to T cell exhaustion. Knockdown of RTN4 reversed T cell exhaustion and enhanced anti-tumor immunity, highlighting RTN4 as a potential therapeutic target.