Exploring the Landscape of Microproteins in T Cells: Insights Gained from Nascent Protein Profiling and Global Proteomics Approaches

Microproteins, as hidden players in the proteomic club, are encoded by small open reading frames (sORFs) and have garnered increasing attention in recent years. T cells play a critical role in the immune system's response to cancer, however, the exploration of novel microproteins in T cells remains an unexplored area. In this study, we employed an integrated approach combined nascent protein profiling and quantitative global proteomics to identify novel microproteins in human T cells. Overall design: In brief, we isolated CD3+ T cells from human peripheral blood mononuclear cells (PBMCs). T cell activation was performed either with phorbol PMA 25 ng/mL and Iono 1 µg/mL or with anti-CD3/CD28-coated microbeads (ThermoFisher Scientific, #11161D) at a bead to cell ratio of 1: 1, at 37 °C with 5% CO2 for 24 hours. Then RNA-Seq data were acquired for inactivated T cells, activated T cells with PMA/Iono treatment, and activated T cells with anti-CD3/CD28 stimulate.