A proximity proteomics pipeline with improved reproducibility and throughput.
Mol Syst Biol
; 20(8): 952-971, 2024 Aug.
Article
in En
| MEDLINE
| ID: mdl-38951684
ABSTRACT
Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biotinylation
/
Proteome
/
Proteomics
Limits:
Humans
Language:
En
Journal:
Mol Syst Biol
Journal subject:
BIOLOGIA MOLECULAR
/
BIOTECNOLOGIA
Year:
2024
Document type:
Article
Affiliation country:
Country of publication: