Integrated profiling of proteins, glycoforms and mRNA in single cells reveals dynamic glycan remodeling during T cell differentiation

Protein glycosylation is a critical post-translational modification that plays vital roles in cellular function and disease, including infection, autoimmunity and cancer. Techniques that measure specific glycosylated proteins face challenges in sensitivity and throughput, preventing applications in single cells. We introduce Glycan Proximity Sequencing (GPS) for the integrated profiling of proteins, glycosylation and protein-specific glycoforms, as well as mRNA in single cells. GPS achieves affinity-based detection of proteins and their modifications via proximity-based single-cell RNA sequencing. Experiments using mixtures of T and B cells, together with a GnTI-knockout mutant, demonstrated sensitive detection of both global and protein-specific glycosylation differences. Applied to human peripheral blood mononuclear cells, GPS revealed dynamic glycan remodeling patterns during T cell differentiation and potential galectin-binding glycoproteins for modulating immune responses. Notably, we identified a new CD45:LELhigh subcluster of terminally differentiated effector memory CD8+ T cells that are enriched for poly-LacNAc and 𝛼2,3-sialylation modifications and associated with exhaustion and susceptibility to galectin-1–induced apoptosis. GPS also captured glycosylation changes in response to perturbations like sialidase treatment and T cell activation. GPS is a scalable and sensitive platform for single-cell glycoproteomics and protein-specific biomarker discovery, and it can reveal mechanistic insights on the role of glycosylation in T cell differentiation. Single-cell GPS assays were performed on three human peripheral blood mononuclear cell (PBMC) samples using the 10x Genomics Chromium pipeline. Two samples, both derived from the same donor (donor 1), were processed together in a single run; one of these was stimulated with Concanavalin A (ConA) for 24 hours prior to the GPS workflow. The third sample, obtained from a different donor (donor 2), was processed in a separate run. All human PBMCs were purchased from STEMCELL Technologies. Both mRNA expression and proximity ligation assay (PLA) features were captured in all assays.