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Synthetic Peptide Libraries

A large number of different library strategies using synthetic peptides have been described. These approaches include one-bead/one-peptide libraries (Wu et al. 1994), solution mixtures of peptides (Songyang et al. 1994), one-well/one-peptide strategies (Lee and Lawrence 1999), peptides on chips (Houseman et al. 2002), and even proteins on chips (Zhu et al. 2000). A detailed description of the vast array of peptide library strategies now available is well beyond the scope of this review. However, all of these methods offer a rapid means to quickly identify preferred amino acid sequences in what is typically a single experiment. Peptide-based libraries also permit the use of amino acid derivatives beyond the standard genetically encoded residues (e.g., post-translationally modified residues such as phosphoTyr, hydrox-yPro, etc.). In addition, many of the methods not only identify a preferred consensus sequence, but also often furnish an assessment of the range of residues permitted at a given position on the peptide ligand.

Each of the peptide library strategies enjoys certain advantages while enduring specific disadvantages:

1. One-bead/one-peptide libraries are extremely easy to prepare via split-and-pool synthesis (Lam et al. 2003). However, these libraries are commonly composed of a mixture of millions of beads, with each bead possessing a unique peptide sequence. Consequently, a screening method must be devised so that the bead containing the tightest binding ligand can be readily identified. Possibilities include the use of a target protein that contains an appended fluorophore or is conjugated to an enzymatic reporter. Beads can also be identified via the introduction of radioactivity (i.e., the use of [g-32P]ATP). Once leads have been identified, the beads are isolated and the bound peptides identified by microsequencing. Given the heavy reliance upon the latter, the use of uncommon hypermodified residues is severely restricted.

2. Soluble peptide library mixtures have also been utilized to identify consensus sequences (Songyang and Cantley 1998). These libraries are prepared by treating the growing peptide chain with a mixture of the standard amino acid derivatives. The actual ratio of the amino acids introduced during the coupling reaction is based upon the relative coupling efficiencies of the individual residues. Consequently, a particular residue that couples sluggishly (e.g., Arg) is present at a greater relative ratio than one that couples readily (e.g., Gly). Following completion of the synthesis, the peptide mixture is cleaved from the resin and subsequently employed for consensus sequence identification. The latter is achieved by selective enrichment of the binding sequence, often using an affinity column. For example, protein kinase-cat-alyzed phosphorylation of the mixture is allowed to proceed until a small fraction (<1%) of the total peptide is phosphorylated. The phosphopeptide mixture is subsequently isolated and sequenced as a mixture. Each position on the peptide is not identified as a single residue, but rather as the relative abundance of all the amino acid residues at a particular site. The residue present in the largest amount at a given position is taken as the one most favored at that site. However, since a peptide mixture, as opposed to a single peptide, is sequenced, this strategy does not yield sequences of unique peptides but merely determines the preferences for particular residues at specific positions. An inherent assumption of this method is that selection at each position is independent of the adjacent amino acids. Consequently, this technique ignores the possibility that two or more residues can act in a syn-ergistic fashion to promote target protein affinity.

3. The one-well/one-peptide approach ("parallel synthesis") (Granier 2002) employs pure peptides that are spatially segregated from one another (Lee and Lawrence 1999). This technique has the advantage that the sequence of each peptide in each well is verified in advance. Furthermore, a wide assortment of hypermodified amino acid residues can be employed, since the synthesis history of each peptide in each well in known. An obvious disadvantage is that the size of these libraries, by necessity, is much smaller than those described in points 1 and 2 above. Variations that employ spatially segregated mixtures ("positional scanning") have been reported that address this concern (Houghten et al. 1996).

4. Peptide chips represent the solid phase version of the method described in 3 (Houseman et al. 2002). The added advantage of this system is the higher spatial density, and therefore smaller chip size [membranes have been employed in this technique as well (Frank 2002)]. However, the increased spatial density of the individual peptide "colonies" can come at a cost. Although methods that employ fluorescence detection of target protein binding will work well in this system, other common methods, such as those that utilize radioactivity, cannot be applied to ultra high-density chips.

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