Spatial proteomics with 100+ markers with Highly Multiplexed MALDI-IHC

Highly multiplexed, antibody-based protein imaging of tissue has yielded many insights but does not yet allow the routine analysis of more than 50 markers simultaneously. By leveraging the ability of Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging to resolve hundreds of m/z peaks without spectral overlap, MALDI-IHC with photocleavable mass-tag (PCMT)-labelled antibodies offers the possibility to dramatically increase the number of markers that can be imaged. Here, we demonstrate 100+ plex MALDI-IHC imaging and describe our development of gutenTAG, an open-source analysis pipeline for the resulting highly multiplex data. We validated a panel of 133 tumor-ecosystem-focused antibodies, show that it captures expected histopathology in diverse tissues, and demonstrate reproducibility of the method. We further applied a 120-plex subset of this panel to samples from an endometrial cancer cohort, again capturing expected histopathology and identifying novel spatial structures between TCGA molecular subtypes. Our work demonstrates the feasibility of simultaneously detecting hundreds of protein markers in a reproducible and scalable manner.