Fig. 1: Schematic representation of a ‘mix-and-split’ peptide synthesis strategy. The orange, magenta and green squares represent single amino acids added to the peptide chain. Analyzing the many different peptides produced from such advanced synthetic techniques had become a procedural bottleneck.
High-throughput screening—a widely used technique in pharmaceutical research—is designed to rapidly test the affinity of all possible compounds to a specified target. Libraries of short peptides are often used in these screens, but the analysis of these libraries to identify the compound that results in a ‘hit’ has become a bottleneck.
Now, Su Seong Lee and co-workers from the Institute of Bioengineering and Nanotechnology of A*STAR, Singapore, in collaboration with researchers from the California Institute of Technology (Caltech), USA, have developed an algorithm that assists in the rapid mass spectrometric analysis of short peptides and reduces this bottleneck.
Peptide synthesis techniques are sufficiently advanced that it is easy to prepare libraries of short peptides (Fig. 1), where multiple copies of single short peptides are attached to small polymer beads known as one-bead–one-compound (OBOC) libraries. Recently, the Caltech researchers reported a method that allows an enzyme to act as a template in selection of the best binding peptide. But the analytical bottleneck remained a problem, which Lee’s team set about resolving.
The current ‘gold standard’ for analyzing these libraries, called Edman degradation, is time consuming. Tandem mass spectrometry methods, which determine both the component amino acids of a peptide and their attachment order, are fast, but less accurate. The inaccuracy occurs, in part, because—in contrast to sequencing natural peptides or proteins—it is not possible to eliminate sequences through a comparison with a database of known proteins for the randomly assembled peptide sequences in an OBOC. Also, using mass spectrometry alone, it is impossible to distinguish between two pairs of the natural amino acids—lysine and glutamine/isoleucine and leucine.
For each of these ‘isobaric pairs’, Lee and his co-workers added 10% of a second amino acid to one of them during the synthesis of the OBOC library. The mass spectrum of these peptides had two sets of peaks, which allowed Lee’s team to distinguish between the isobaric pairs, and pinpoint the exact sequence and position.
Compared with Edman degradation, the success rate of the mass spectrometric screening improved from 44% to 100%. The new procedure also allowed the analysis of several hundred peptides in a few hours compared with several days using Edman degradation. “Our new screening procedure will significantly improve the process for finding affinity agents that can serve as therapeutics and diagnostics for a variety of diseases,” says Lee.
The A*STAR-affiliated researchers mentioned in this highlight are from the Institute of Bioengineering and Nanotechnology.
