A proximity proteomics pipeline with improved reproducibility and throughput.
Xiaofang ZhongQiongyu LiBenjamin J PolaccoTrupti PatilAaron MarleyHelene FoussardPrachi KhareRasika VartakJiewei XuJeffrey F DiBertoBryan L RothManon EckhardtMark Von ZastrowNevan J KroganRuth HüttenhainPublished in: Molecular systems biology (2024)
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 5HT 2A 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 5HT 2A 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.
Keyphrases
- mass spectrometry
- high resolution
- liquid chromatography
- high performance liquid chromatography
- capillary electrophoresis
- genome wide
- multiple sclerosis
- induced apoptosis
- electronic health record
- crispr cas
- big data
- machine learning
- gene expression
- copy number
- oxidative stress
- dna methylation
- small molecule
- transcription factor
- amino acid
- tandem mass spectrometry
- wild type