Application of in-cell fast photochemical oxidation of proteins (IC-FPOP) for the study of organoids.

Abstract

Fast photochemical oxidation of proteins (FPOP) is a powerful, mass spectrometry (MS)-based, biophysical method used to probe protein structure, interactions, and conformations. FPOP was recently extended into cells (IC-FPOP) and can modify thousands of proteins in a single experiment, enabling proteome-wide structural biology. Although IC-FPOP can reveal critical structural information in 2D cell culture, the conditions do not emulate an in-vivo environment. Organoids are multicellular three-dimensional model systems that resemble the corresponding organ. They are the ideal 3D model system for evaluating drug penetration, drug efficacy, and chemoresistance since they mimic in vivo conditions. But the complexity of organoids makes them difficult for structural studies. Therefore, we have extended IC-FPOP into 3D bioprinted Huh-7 liver organoids. To obtain spatial resolution within the model, we integrated cryosectioning into the IC-FPOP workflow. This novel application of IC-FPOP revealed modifications in each section of the organoid, the top middle, and bottom. Pathway analysis revealed the interrogation of over 5 native pathways stemming from each region. Peptide level analysis revealed differences in the extent of modification for peptides identified in each region of the organoid, which confirms the acquisition of structural information. By coupling the organoid model with IC-FPOP we aim further validate its implementation for complex proteome-wide structural studies.