On-bead chemical synthesis and display of phosphopeptides for affinity pull-down proteomics.
We describe a new method for phosphopeptide proteomics based on the solid-phase synthesis of phosphopeptides on beads suitable for affinity pull-down experiments. Peptide sequences containing the Bad Ser112 and Ser136 phosphorylation motifs were used as bait in affinity pull-down experiments to determine their ability to bind 14-3-3 proteins. Support-bound peptides were assembled directly on the solid support (PEGA) by standard solid-phase synthesis through a BAL-type handle. The peptides were varied in length and sequence. This synthetic strategy also allowed introduction of a soft electrophile (aldehyde) at the C terminus for potential activity-based proteomics. The synthetic support-bound Bad phosphopeptides were able to pull down 14-3-3zeta. Furthermore, Bad phosphopeptides bound endogenous 14-3-3 proteins, and all seven members of the 14-3-3 family were identified by mass spectrometry. In control experiments, none of the unphosphorylated Bad peptides bound transfected 14-3-3zeta or endogenous 14-3-3. We conclude that the combined synthesis and display of phosphopeptides on-bead is a fast and efficient method for affinity pull-down proteomics.
Brandt
Malene
M
Department of Natural Sciences, Section for Bioorganic Chemistry, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Madsen
Jens C
JC
Bunkenborg
Jakob
J
Jensen
Ole N
ON
Gammeltoft
Steen
S
Jensen
Knud J
KJ
D015394:Molecular Structure; D010748:Phosphopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D040901:Proteomics
D010748:Phosphopeptides