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A simple method for developing lysine targeted covalent protein reagents

ABSTRACT: In this project we developed phosphopeptides functionalized with methacrylate ester warheads installed on a cysteine residue, which bind covalently to the protein 14-3-3 sigma. We characterized the binding of these peptides to 14-3-3 proteins in A549 lysates as well as off-targets. We prepared biotinylated forms of the 14-3-3 binding peptides and incubated A549 lysates with these peptides, followed by Streptavidin pull down, trypsin digestion and LCMSMS. All isoforms of 14-3-3 were pull downed as well as multiple proteins with phosphate-containing substrates such as NAD-dependent enzymes.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Ronen Gabizon

LAB HEAD: Nir London

PROVIDER: PXD044294 | Pride | 2024-01-26

REPOSITORIES: pride

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A simple method for developing lysine targeted covalent protein reagents.

Gabizon Ronen R Tivon Barr B Reddi Rambabu N RN van den Oetelaar Maxime C M MCM Amartely Hadar H Cossar Peter J PJ Ottmann Christian C London Nir N

Nature communications 20231201 1

Peptide-based covalent probes can target shallow protein surfaces not typically addressable using small molecules, yet there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters-electrophiles that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on t ...[more]

PMID: 38040731

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