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

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.