A simple method for developing lysine targeted covalent protein reagents

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.