A Universal Modular High-Plex MALDI Mass Spectrometry Imaging Platform

Abstract High-plex spatial molecular profiling is needed to resolve tissue heterogeneity, but conventional immunohistochemistry and in situ hybridization remain limited in multiplexing capacity, probe flexibility, and analytical scalability. Here we introduce a universal, modular TagMass platform for mass spectrometry imaging that uses bioorthogonal click chemistry to couple photocleavable arginine-based reporters to antibodies, aptamers, oligonucleotides, and lectins. This design enables rapid generation of customizable probes and sensitive multiplexed imaging of proteins, RNAs, and glycans in both fresh-frozen and formalin-fixed paraffin-embedded tissues. We demonstrate single-cycle 10-plex MALDI imaging, extend the workflow to aptamer-based and RNA-targeted imaging, and achieve 44-plex immune profiling of triple-negative breast cancer through iterative acquisition. TagMass resolves spatially organized proliferative, immune, and immunosuppressive programs within the tumor microenvironment and supports computational interpretation of high-dimensional imaging data. The platform is further compatible with ambient ionization workflows, including nano-DESI. TagMass therefore provides a broadly adaptable framework for high-content spatial omics and translational tissue phenotyping.