Uncovering the glutamate carboxypeptidase II microenvironment using a multi-labeling proteomic approach.

Glutamate carboxypeptidase II (GCPII) is a membrane-bound metallopeptidase predominantly expressed in neural and prostatic tissues, with significantly elevated levels in prostate carcinoma that increase with tumor grade. Despite its significance as a target for imaging and therapy in prostate cancer, its physiological function in this tissue remains poorly understood. To help fill this knowledge gap, we developed an integrated approach combining proximity labeling technologies for proteomic profiling (horseradish peroxidase, µMap, and riboflavin tetraacetate labeling) with our previously established iBody platform, which targets GCPII with a small-molecule specific inhibitor with proven efficacy as a chemical probe. Proximity labeling proteomic experiments on U251 MG-GCPII cells were followed by mass spectrometry and statistical analysis of protein abundances obtained by label-free quantification. Additionally, selected identified proteins were further validated through Western blot analyses and GCPII pulldown assays using cell lysates. This work identifies a network of GCPII-associated proteins that are potentially involved in cancer metabolism, migration, invasiveness, progression, and immune evasion. Our novel proximity proteomics labeling strategy provides a low-background framework, efficient biotinylation, and enhanced target binding via the avidity effect. Among the approaches tested, riboflavin tetraacetate-based iBody labeling exhibited the highest precision, underscoring its potential for membrane protein interactome mapping.