AUTHOR=Lyu Ziqi , Genereux Joseph C. 

TITLE=Quantitative measurement of transthyretin mistargeting by proximity labeling and parallel reaction monitoring

JOURNAL=Frontiers in Chemical Biology

VOLUME=2

YEAR=2023

URL=https://www.frontiersin.org/journals/chemical-biology/articles/10.3389/fchbi.2023.1288188

DOI=10.3389/fchbi.2023.1288188

ISSN=2813-530X

ABSTRACT=<p><bold>Introduction:</bold> Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which sometimes suffers from poor sensitivity.</p><p><bold>Methods:</bold> Here, we integrate parallel reaction monitoring (PRM) mass spectrometry to enable a more quantitative platform and assess how chemical ER stressors impact pre-QC of the model secretory protein transthyretin in HEK293T cells.</p><p><bold>Results and Discussion:</bold> We find that some drug treatments affect labeling efficiency, which can be controlled for by normalizing to APEX2 autolabeling. While some chemical ER stress inducers including Brefeldin A and thapsigargin induce pre-QC, tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.</p>