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Region Hovedstaden - en del af Københavns Universitetshospital
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On-bead chemical synthesis and display of phosphopeptides for affinity pull-down proteomics

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  • Malene Brandt
  • Jens C Madsen
  • Jakob Bunkenborg
  • Ole N Jensen
  • Steen Gammeltoft
  • Knud Jørgen Jensen
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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.
OriginalsprogEngelsk
TidsskriftChemBioChem
Vol/bind7
Udgave nummer4
Sider (fra-til)623-30
Antal sider8
ISSN1439-4227
DOI
StatusUdgivet - 2006

ID: 42360977