UC Riverside Genereux Lab - Mistargeting Assay Data

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring
Data License: CC BY 4.0 | ProteomeXchange: PXD044008 | doi: https://doi.org/10.6069/ckq1-da18
  • Organism: Homo sapiens
  • Instrument: LTQ Orbitrap Velos Pro,LTQ Orbitrap Velos
  • SpikeIn: No
  • Keywords: Proximity Labeling, Parallel Reaction Monitoring, ER Pre-Emptive Quality Control
  • Lab head: Joseph Genereux Submitter: Joseph Genereux
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 (PRM) mass spectrometry to enable a quantitative platform for ER import. PRM as opposed to densitometry improves quantification of tranthyretin mistargeting while 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 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.
Experiment Description
Plasmids of cytosolic proximity labeling enzyme FLAG-APEX2-NES (cyt_APEX), and the model secretory protein FLAG_TTR, are transiently transfected into HEK293T cells, followed by reseeding into smaller dishes for later 16-h drug treatment versus vehicle control. 30 min before proximity labeling reaction, biotin-phenol was introduced into conditioned media. The 1-min labeling was initiated with hydrogen peroxide. After quenching, cells were harvested and lysed. Whole cell lysates were brought to the same concentration. The same amount of lysates were loaded onto avidin beads for affinity purification. Avidin-enriched samples (eluates) were processed for PRM experiments and eluate digests were injected in random order.
Sample Description
Proteins from eluate samples were diluted by water and precipitated in Methanol/chloroform. Pelleted proteins were washed with methanol for four times and air dried. Cleaned-up protein pellets were then resuspended in 9 M urea, reduced by 5 mM Tris(2-carboxyethyl)phosphine at ambient temperature (~20 °C), carbamidomethylated by 10 mM iodoacetamide in dark at ambient temperature, digested by trypsin overnight, and acidified by formic acid.
Created on 7/23/23, 11:06 AM
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This is a demonstration of how we use Prosit to predict peptide CID spectra of non-human origins. The original CSV files cannot be found (APEX2-NES, 8/17/22 and avidin, 8/19/22).

 

Two CSV files were created separately for FLAG-APEX2-NES and chicken avidin, based on the instruction below:

 

Tryptic peptides of interest were pasted to CSV files. For methionine-containing peptides, both intact and oxidized (ox) versions were included. Collision energy was set to 35. Charge states of interest were 2 and 3. Fragmentation method was set to CID for LTQ Orbitrap Velos Pro.

 

The intensity prediction model and iRT predict