Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

Abstract

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 is only semi-quantitative and suffers from poor sensitivity. Here, we integrate parallel reaction monitoring mass spectrometry to enable a more quantitative platform for ER import. PRM as opposed to densitometry improves quantification of transthyretin mistargeting while also achieving at least a ten-fold gain in sensitivity. The multiplexing of PRM also enabled us to evaluate a series of normalization approaches, revealing that normalization to auto-labeled APEX2 peroxidase is necessary to account for drug treatment-dependent changes in labeling efficiency. We apply this approach to systematically characterize the relationship between chemical ER stressors and ER pre-QC induction in HEK293T cells. Using dual-FLAG-tagged transthyretin (FLAGTTR) as a model secretory protein, we find that Brefeldin A treatment as well as ER calcium depletion cause pre-QC, while 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. TOC graph O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/549095v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@bfbce5org.highwire.dtl.DTLVardef@a65f3org.highwire.dtl.DTLVardef@ab5c8corg.highwire.dtl.DTLVardef@48f1d3_HPS_FORMAT_FIGEXP M_FIG C_FIG