Tissue-ABPP enables high-resolution confocal fluorescence imaging of serine hydrolase activity in cryosections – Application to glioma brain unveils activity hotspots originating from tumor-associated neutrophils

Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. The ABPP approach utilizes cell/tissue proteomes and features the FP warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Here, we advance the ABPP methodology to glioma brain cryosections, enabling high-resolution confocal fluorescence imaging of SH activity in different cell types of the tumor microenvironment, identified by using extensive immunohistochemistry on activity probe labeled sections. We name this technique tissue-ABPP to distinguish it from conventional gel-based ABPP. We show heightened SH activity in glioma vs. normal brain and unveil activity hotspots originating from tumor-associated neutrophils. Thorough optimization and validation is provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Tissue-ABPP enables a wide range of applications for confocal imaging of SH activity in any type of tissue or animal species.