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Elife. 2018 Oct 22;7. pii: e40982. doi: 10.7554/eLife.40982.

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions.

Author information

1
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
2
Department of Ophthalmology, West China Hospital, Sichuan University, Chengdu, China.
3
Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
4
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
5
Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States.
#
Contributed equally

Abstract

Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein 'bait'. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs was analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.

KEYWORDS:

RNAseq; biochemistry; chemical biology; extracellular interactome; mouse; protocadherin; secretome

PMID:
30345971
PMCID:
PMC6197854
DOI:
10.7554/eLife.40982
[Indexed for MEDLINE]
Free PMC Article

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