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

Abstract

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.