Encoding Human Serine Phosphopeptides in Bacteria for Proteome-wide Identification of Phosphorylation-dependent Interactions

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2018 Jun 12

PMID 29889213

Citations 15

Authors

Karl W Barber

Paul Muir

Richard V Szeligowski

Svetlana Rogulina

Mark Gerstein

Jeffrey R Sampson

Farren J Isaacs

Jesse Rinehart

Affiliations

Soon will be listed here.

Abstract

Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems. We developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. We genetically encoded phosphoserine in recoded E. coli and generated a peptide-based heterologous representation of the human serine phosphoproteome. We designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. We then integrated our phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, we found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.

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