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Combined effort reveals beads |
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| Separating the wheat from the chaff in combinatorial peptide synthesis is still a major problem, especially when using solid-state resin beads in which high-throughput screening generates many hits. According to Dehua Pei of Ohio State University (Columbus), this has led to a demand for a fast and inexpensive way of sequencing library-derived peptides. Direct sequencing using Edman degradation is one option, but it is expensive and time-consuming. Sequencing pools of selected peptides is quicker, but information about individual peptides is lost.
Pei and colleagues have developed a method in which a peptide library is synthesized on a solid support without tagging or partial chain termination. This means that most beads in the library carry an equal quantity of full-length peptides and no encoding molecules that can potentially interfere with library screening. The team screens the library against a target protein or other biomolecule to identify the beads that carry sequences with the desired properties. The peptides on the positive beads are subjected to partial Edman degradation, resulting in a peptide ladder that is analyzed using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS; J. Comb. Chem. 2001, 3, 251–254). “The MALDI-MS method is ideally suited for such applications for its speed, ease in sample preparation, and the formation of predominantly singly charged ions,” explains Pei. To distinguish between amino acids of the same mass, a small amount of chain-termination products is generated along with the coupling reactions during library synthesis. The Ohio State team tested the validity of its approach using a synthetic peptide library screened against E. coli peptide deformylase (PDF). The screening technique generated turquoise beads for those that carried the most reactive substrates of PDF, and the most intensely colored beads were picked out under the microscope for sequencing. “We were able to sequence 72 beads in 2–3 hours at a 90% success rate and at a cost of $2–$5 per peptide,” says Pei. |
