The challenge of getting the right formula for surface chemistry on a chip has frustrated many would-be array makers in the protein arena. How do you immobilize the ligands without allowing nonspecific binding of proteins to the chip?
Now a group of University of Chicago researchers has detailed, in an article in the March 2002 issue of Nature Biotechnology, a method for producing peptide chips that they claim overcomes these problems. The method involves using a clean gold substrate, which is immersed in alkanethiols to make a monolayer. The glycol groups on this monolayer act to prevent nonspecific protein and radiosotope interactions, and the hydroquinone groups in the monolayer act as a “chemical handle” to selectively immobilize ligands, the researchers wrote.
“Our strategy for preparing peptide chips has two significant advantages over existing formats,” the researchers wrote. The first derives from the ordered structure of the self-assembled monolayer (SAM), which provides a regular, homogeneous environment for immobilized peptide ligands...The second advantage is the low to negligible levels of background signal.”
The researchers tested this surface chemistry on a chip where they selectively immobilized a peptide-cyclopentadiene conjugate of the peptide AclYGEFKKKC-NH2 on the monolayer, then used these chips to characterize the phosphorylation of the peptide by kinase c-Src. First they just used one type of peptide and one concentration of the kinase to test out the basic chemistry, and then they adapted the chip to characterize multiple kinase reactions, using mixtures of the kinases and their inhibitors at a range of concentrations.
For detection, the researchers tried out all of the three major techniques: radioactivity, fluorescence, and surface plasmon resonance spectroscopy, and found that the arrays worked with all three.
The group said it is now working on preparing arrays to identify protein-ligand complexes, to evaluate enzymatic activities such as protease and phosphates activities, and to “determine flanking sequences for enzyme substrates.” They also suggested their chips could be useful for other small molecule arrays. — Marian Moser Jones