Sensitive N -Glycopeptide Profiling of Single and Rare Cells Using an Isobaric Labeling Strategy without Enrichment.

Single-cell omics is critical in revealing population heterogeneity, discovering unique features of individual cells, and identifying minority subpopulations of interest. As one of the major post-translational modifications, protein N -glycosylation plays crucial roles in various important biological processes. Elucidation of the variation in N -glycosylation patterns at single-cell resolution may largely facilitate the understanding of their key roles in the tumor microenvironment and immune therapy. However, comprehensive N -glycoproteome profiling for single cells has not been achieved due to the extremely limited sample amount and incompatibility with the available enrichment strategies. Here, we have developed an isobaric labeling-based carrier strategy for highly sensitive intact N -glycopeptide profiling for single cells or a small number of rare cells without enrichment. Isobaric labeling has unique multiplexing properties, by which the "total" signal from all channels triggers MS/MS fragmentation for N -glycopeptide identification, while the reporter ions provide quantitative information. In our strategy, a carrier channel using N -glycopeptides obtained from bulk-cell samples significantly improved the "total" signal of N -glycopeptides and, therefore, promoted the first quantitative analysis of averagely 260 N -glycopeptides from single HeLa cells. We further applied this strategy to study the regional heterogeneity of N -glycosylation of microglia in mouse brain and discovered region-specific N -glycoproteome patterns and cell subtypes. In conclusion, the glycocarrier strategy provides an attractive solution for sensitive and quantitative N -glycopeptide profiling of single/rare cells that cannot be enriched by traditional workflows.