Rapid Single-Cell N-glycosylation Profiling of Circulating Human Immune Cells by Mass Spectrometry Imaging

The metabolic fitness of immune cells, reflected in part through glycosylation of surface proteins, is a key determinant of their activation, persistence, and effector responses. A recently developed high-throughput antibody capture cell array platform was further optimized to profile N-glycosylation across bulk cell and single-cell populations of peripheral blood mononuclear cells (PBMCs). Using antibodies against lymphocyte, monocyte, and dendritic cell markers (CD4+, CD8+, CD11c+, CD14,+ CD16+, CD3+, CD19+, CD56+, CD68+), N-glycosylation patterns of these immune cell subtypes were mapped across human PBMC cultures from healthy donors and those with pancreatic cancer. In this platform, PDMS stamps coated with targeted antibodies create patterned capture coordinates. Captured single cells are confirmed by brightfield imaging and selected via SoloCell software. Following PNGase F digestion and matrix application for MALDI-MSI analysis, N-glycans are acquired from a single laser burst per cell at a rate of 7.5 cells per second (18,000 cells in approximately 40 min). Sialic acid isomer stabilization by chemical amidation provided resolution of α2,6- versus α2,3-linked sialic acids and enabled detection of larger mono-, di-, tri- and tetra-sialylated glycans at the single-cell level. Bulk- and single-cell analyses showed concordant glycomic organization across immune cell subtypes. Bulk-level N-glycan profiles clearly distinguished lymphoid from myeloid lineages, and at single-cell resolution, healthy donor PBMCs similarly segregated into distinct subtype-specific glycomic clusters. In contrast, immune cell subtypes in pancreatic cancer PBMC converged into a common glycomic landscape, with marker-defined populations becoming highly intermixed. This convergence was accompanied by linkage-specific sialylation shifts, including a predominance of α2,6 sialylation and pronounced enrichment in CD3+ and CD8+ T cells. Together, these findings demonstrate that single-cell N-glycomic profiling extends bulk analysis to resolve subtype-specific differences in disease-associated immune N-glycome remodeling.