Two-Level Spatially Localized Proximity Labeling for Cross-Biological-Hierarchy Measurement and Manipulation

Proximity labeling (PL) has emerged as a powerful technique for the in situ elucidation of biomolecular interaction networks. However, PL methods generally rely on single-biological-hierarchy control of spatial localization at the labeling site, which limits their application in multi-tiered biological systems. Here, we introduced another enzymatic reaction upstream of an enzyme-based PL reaction and targeted the two enzymes to markers indicating different biological hierarchies, establishing a two-level spatially localized proximity labeling (P²L) platform for in situ molecular measurement and manipulation. Using the cellular- and glycan-level as the hierarchical models, we demonstrated the ability of P²L to efficiently execute a two-step logic operation and to discriminate target cells with different levels of glycosylation within mixed cell populations. By mounting clickable handles via P²L, we reprogrammed the robust covalent assembly of cells at designated sites. The combination of P²L with proteomics led to the profiling of the protein microenvironment of specific glycans on target cells, revealing changes in tumor-cell-surface interactions under immune pressure from a glycan perspective. P²L provides not only a solution for revealing the heterogeneity of biological systems, but also new insights in the fields of intelligent logic computation, enzyme engineering, tissue engineering, etc.