Single-Cell Proteomic Profiling of Human Primary CD8⁺ T Cells

Single-cell proteomics (SCP) has emerged as a transformative technique that overcomes the averaging effects inherent in bulk-cell analyses, enabling high-resolution profiling of protein expression and post-translational modification (PTM) with single-cell resolution. Human primary CD8⁺ T cells serve as central effector cells in the human immune response, playing crucial roles in combating pathogenic infections and mediating immune surveillance. Understanding the proteomic architecture of human immune cells is crucial for elucidating their diverse functional states, yet human primary CD8⁺ T cells remain largely unexplored by single-cell proteomics (SCP) due to their characteristically small cellular size and extremely limited protein content, which collectively pose significant technical obstacles for high-sensitivity protein identification and PTM detection at the single-cell level. This study successfully established a robust SCP analytical workflow to characterize the proteomic landscape of individual human primary CD8⁺ T cells. The workflow first utilized the CellenONE automated single-cell sorter for gentle and precise cell isolation, and was followed by the analysis of protein composition and PTM identification using the high-sensitivity timsTOF Ultra 2 mass spectrometer. This SCP approach consistently identified over 2 500 proteins and more than 16 000 peptides from individual human primary CD8⁺ T cells. Protein abundance across single cells demonstrate strong reproducibility, with pairwise correlations exceeding 0.77 and a median coefficient of variation (CV) of 28.61%. Subcellular localization analysis revealed that the identified proteins are primarily distributed in the cytoplasm (1 186 proteins), nucleus (891 proteins), mitochondria (261 proteins), and cell membrane (181 proteins), reflecting the diverse functional compartments within human primary CD8⁺ T cells. Furthermore, PTM profiling identifies 32 phosphorylated, 64 methylated, and 356 acetylated proteins per cell, with acetylation being the most prevalent modification. These PTM proteins are also mainly localized to the cytoplasm and nucleus. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that the functional categories of proteins in human primary CD8⁺ T cells are predominantly enriched in ribosomal and immune-related pathways, emphasizing the central role of protein synthesis and immune regulatory networks in human primary CD8⁺ T cell function. In conclusion, the SCP workflow developed in this study presents the first high-resolution proteome and PTM atlas of human primary CD8⁺ T cells, providing a valuable foundation for elucidating complex cellular processes and guiding future research into disease mechanisms and therapeutic targeting.