Quantitative Measurement of Transthyretin Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

Area of Science:

• Cell Biology
• Proteomics
• Biochemistry

Background:

• Proximity labeling is a key technique for mapping subcellular proteomes.
• Pre-emptive quality control (pre-QC) manages mistrafficked secretory proteins after endoplasmic reticulum (ER) stress.
• Current methods using immunoblotting for pre-QC lack sensitivity.

Purpose of the Study:

• To develop a more quantitative proximity labeling assay for pre-QC.
• To investigate the impact of chemical ER stressors on pre-QC of transthyretin.
• To establish a robust platform for studying protein mistrafficking.

Main Methods:

• Integration of parallel reaction monitoring (PRM) mass spectrometry with proximity labeling.
• Utilizing HEK293T cells expressing transthyretin as a model secretory protein.
• Quantification of labeling efficiency and normalization to APEX2 auto-labeling.

Main Results:

• PRM mass spectrometry enhances the quantitative accuracy of proximity labeling assays.
• Drug treatments impacting labeling efficiency can be normalized using APEX2 auto-labeling.
• Brefeldin A and thapsigargin induced pre-QC, but tunicamycin and dithiothreitol did not.
• ER stress alone is not sufficient to induce pre-QC.

Conclusions:

• The developed PRM-based proximity labeling platform offers improved sensitivity and quantitation for studying protein mistrafficking.
• Specific chemical ER stressors, not just general ER stress, are required to induce pre-QC.
• Findings challenge the canonical model of pre-QC induction and highlight the complexity of protein quality control.