Secretory proteins are canonically targeted to the endoplasmic reticulum (ER). During ER stress, preemptive quality control (preQC) protects the ER by preventing nascent protein import into the ER, presenting a threat to cytosolic proteostasis. This process has been difficult to observe in living cells, where mistargeted protein populations can be small compared to the amount of properly targeted protein. We have demonstrated that peroxidase proximity labeling is an effective approach for quantifying mistargeted secretory proteins in the cytosol. In combination with mass spectrometry, our approach is simple, quantitative, and inherently multiplexed. We have used this approach to identify factors that contribute to protein mistargeting, including the poorly characterized ER Hsp70 Hspa13. We have also identified a quality control process for cytosolically mistargeted secretory protein that triages mistargeted proteins on the basis of their thermodynamic stability.
