Abstract: The imbalance of the protein homeostasis network leads to the formation of insoluble aggregates by misfolded proteins, which is a core pathological feature of neurodegenerative diseases such as Alzheimer's disease. However, there is still a technical blind spot in the proteomic analysis of insoluble aggregates. This study employed the previously developed aggregate microenvironment-targeted photocatalytic probe D6, which has many advantages such as white light excitation that can avoid the damage of traditional ultraviolet light to cells, higher ROS generation efficiency, and aggregate protein targeting. First, the D6 probe binds to aggregate proteins, then the photocatalytic reaction generates ROS to oxidize neighboring aggregate proteins into active intermediates, followed by nucleophilic addition of aniline-biotin to attach a biotin tag. Next, streptavidin agarose microspheres are used to capture proteins labeled with the biotin tag. Finally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is used to perform qualitative and quantitative analysis on the enriched components. Using human hippocampal neuronal cells as a model, we systematically studied the effects of four inhibitors targeting the ubiquitin-proteasome system (bortezomib), HSP90 molecular chaperone (17-AAG), autophagy-lysosomal pathway (chloroquine), and translation process (cycloheximide) on the aggregate proteome. Proteomic results showed that more than 200 significantly changed aggregate proteins were identified under the four treatment conditions. Among them, bortezomib treatment induced the most significant aggregation effect, with a total of 1β341 significantly enriched aggregate proteins identified, indicating that the acute inhibition of proteasome function had the most severe impact on protein homeostasis, leading to a large number of proteins that could not be normally degraded and thus aggregated. Although the number of enriched proteins in the 17-AAG, chloroquine, and cycloheximide treatments was less than that in the bortezomib group, they also revealed their unique homeostatic perturbation patterns. These data collectively demonstrate that the aggregate proteomics analysis method based on photocatalytic proximity labeling adopted in this study can sensitively and specifically analyze the dynamic changes and molecular characteristics of cell aggregate proteomes under different perturbations of the protein homeostasis network. Further Gene Ontology analysis of the proteomic results revealed that, although the perturbed nodes were different, various protein homeostatic stresses led to the aggregation of a large number of core factors involved in RNA metabolism and protein synthesis (such as ribosomal proteins and splicing factors), suggesting that the gene expression machinery is a universal vulnerable link in the protein homeostasis network. At the same time, different perturbations induced distinctly characteristic aggregation spectra: proteasome inhibition had the most extensive effect; molecular chaperone inhibition was more selective; autophagy inhibition had a diversified impact, involving energy metabolism and signal transduction; and translation inhibition highly specifically targeted mRNA processing machinery. This study successfully integrated the conformational stability dimension of proteins into proteomic analysis, not only providing a high-resolution molecular fingerprint for parsing the mechanisms of protein homeostasis imbalance but also offering new insights for discovering potential biomarkers and intervention targets for related diseases.

Key words: photocatalysis, proximity labeling, aggregated proteome, proteostasis, neurodegenerative diseases