Transformative R01 Program
MASSIVELY PARALLEL IDENTIFICATION OF PROTEIN LIGANDS
The figure depicts a schematic that represents the general strategy of the T-RO1 project. At the left, the general chemical structure of a peptoid is shown. This is a class of compounds where synthetic methods exist to create “libraries” of millions of different compounds, each differing by the chemical nature of the side chain projecting from the nitrogen atoms on the main chain. The library is made on small beads in such a way that each bead displays billions of copied of a single peptoid. The crux of this proposal is to incubate millions of such beads with a protein mixture (such as a tissue homogenate) that contains dozens of fluorescently-labeled proteins that have been tagged by virtue of their enzymatic properties. The figure shows a cartoon of a protein with a red asterisk representing the fluorescent label. Again, there will be dozens of such labeled proteins in the mixture. This will then be incubated with the bead-displayed peptoids and those that retain the fluorescently labeled proteins will be identified by the characteristic fluorescent halo at the surface of the bead. A micrograph depicting such a bead is shown at the right of the figure. This looks like a blue ball (the bead) surrounded by a corona of red (the labeled protein bound by peptoids on the surface of the bead). These “hits” will be picked and a technique called mass spectrometry will be employed to identify the chemical structure of the peptoid and the identity of the bound protein. Since this protocol allows dozens of proteins to be targeted in a single screen, it should greatly accelerate the isolation of highly specific protein binding compounds, which are useful tools for biological research and may also serve as drug leads.