Locus-Specific Chromatin Proteome Revealed by Mass Spectrometry-Based CasID

Ugur, Enes; Bartoschek, Michael D. und Leonhardt, Heinrich (2020): Locus-Specific Chromatin Proteome Revealed by Mass Spectrometry-Based CasID. In: Hancock, R. (Hrsg.): The Nucleus. Methods in molecular biology, Bd. 2175. Humana, NY: Springer. S. 109-121

Abstract

Biotin proximity labeling has largely extended the toolbox of mass spectrometry-based interactomics. To date, BirA, engineered BirA variants, or other biotinylating enzymes have been widely applied to characterize protein interactions. By implementing chromatin purification-based methods the genome-wide interactome of proteins can be defined. However, acquiring a high-resolution interactome of a single genomic locus preferably by multiplexed measurements of several distinct genomic loci in parallel remains challenging. We recently developed CasID, a novel approach where the catalytically inactive Cas9 (dCas9) is coupled to the promiscuous biotin ligase BirA (BirA∗). With CasID, first the local proteome at repetitive telomeric, major satellite, and minor satellite regions was determined. With more efficient biotin ligases and sensitive mass spectrometry, others have successfully identified the chromatin composition at even smaller genomic, non-repetitive regions of a few hundred base pairs in length. Here, we summarize the most recent developments towards interactomics at a single genomic locus and provide a step-by-step protocol based on the CasID approach.

• Dokumententyp: Buchbeitrag
• Fakultät: Biologie > Department Biologie II
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
• ISBN: 978-1-0716-0762-6 (print) ; 978-1-0716-0763-3 (online)
• Ort: Humana, NY
• Sprache: Englisch