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oai:figshare.com:article/33977805 2026-09-23T20:13:54Z category_1 category_4 category_8 category_12 category_13 category_14 category_15 category_21 category_873 category_46 category_61 category_64 category_128 category_132 portal_63 item_type_3 month_year_09_2026

Rapid Single-Cell N‑glycosylation Profiling of Circulating Human Immune Cells by Mass Spectrometry Imaging Lyndsay E. A. Young (6329558) Lauren E. Hill (25099767) James. W. Dressman (25099770) Caroline G. Kittrell (17770641) Kaitlyn Bejar (25099773) Blake Sells (25099776) Anand S. Mehta (203501) Robin J. Leach (9989758) David G. DeNardo (8873405) Richard R. Drake (625159) Biophysics Biochemistry Microbiology Cell Biology Genetics Molecular Biology Neuroscience Biotechnology Chemical Sciences not elsewhere classified Immunology Developmental Biology Cancer Hematology Infectious Diseases sup >+</ sup recently developed high pdms stamps coated linked sialic acids common glycomic landscape specific glycomic clusters mass spectrometry imaging dendritic cell markers immune cell subtypes specific sialylation shifts associated immune n captured single cells immune cells specific differences brightfield imaging cell resolution cell populations cell n cell level using antibodies surface proteins resolve subtype pronounced enrichment profile n pbmcs ). pancreatic cancer myeloid lineages msi analysis metabolic fitness matrix application level n key determinant healthy donors glycome remodeling findings demonstrate enabled detection effector responses distinct subtype 6 sialylation 000 cells 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. 2026-09-23T00:00:00Z Dataset Dataset 10.1021/jasms.6c00321.s001 https://figshare.com/articles/dataset/Rapid_Single-Cell_N_glycosylation_Profiling_of_Circulating_Human_Immune_Cells_by_Mass_Spectrometry_Imaging/33977805 CC BY-NC 4.0