An automated proximity proteomics pipeline for subcellular proteome and protein interaction mapping.
bioRxiv
; 2023 Apr 12.
Article
en En
| MEDLINE
| ID: mdl-37090610
ABSTRACT
Proximity labeling (PL) coupled with mass spectrometry has emerged as a powerful technique to map proximal protein interactions in living cells. Large-scale sample processing for proximity proteomics necessitates a high-throughput workflow to reduce hands-on time and increase quantitative reproducibility. To address this issue, we developed a scalable and automated PL pipeline, including generation and characterization of monoclonal cell lines, automated enrichment of biotinylated proteins in a 96-well format, and optimization of the quantitative mass spectrometry (MS) acquisition method. Combined with data-independent acquisition (DIA) MS, our pipeline outperforms manual enrichment and data-dependent acquisition (DDA) MS regarding reproducibility of protein identification and quantification. We apply the pipeline to map subcellular proteomes for endosomes, late endosomes/lysosomes, the Golgi apparatus, and the plasma membrane. Moreover, using serotonin receptor (5HT2A) as a model, we investigated agonist-induced dynamics in protein-protein interactions. Importantly, the approach presented here is universally applicable for PL proteomics using all biotinylation-based PL enzymes, increasing both throughput and reproducibility of standard protocols.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Idioma:
En
Revista:
BioRxiv
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos