Benchmarking and Automating the Biotinylation Proteomics Workflow.
Res Sq
; 2024 Jul 03.
Article
en En
| MEDLINE
| ID: mdl-39011118
ABSTRACT
Protein biotinylation has been widely used in biotechnology with various labeling and enrichment strategies. However, different enrichment strategies have not been systematically evaluated due to the lack of a benchmarking model for fair comparison. Most biotinylation proteomics workflows suffer from lengthy experimental steps, non-specific bindings, limited throughput, and experimental variability. To address these challenges, we designed a two-proteome model, where biotinylated yeast proteins were spiked in unlabeled human proteins, allowing us to distinguish true enrichment from non-specific bindings. Using this benchmarking model, we compared common biotinylation proteomics methods and provided practical selection guidelines. We significantly optimized and shortened sample preparation from 3 days to 9 hours, enabling fully-automated 96-well plate sample processing. Next, we applied this optimized and automated workflow for proximity labeling to investigate the intricate interplay between mitochondria and lysosomes in living cells under both healthy state and mitochondrial damage. Our results suggested a time-dependent proteome remodeling and dynamic translocation within mitochondria and between mitochondria and lysosomes upon mitochondrial damage. This newly established benchmarking model and the fully-automated 9-hour workflow can be readily applied to the broad fields of protein biotinylation to study protein interaction and organelle dynamics.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
Res Sq
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos