Peptides can be purchased from several companies specializing in contract synthesis and if only a few are required, this is the most straightforward way to obtain the desired reagents. Custom synthesis of peptides can be expensive, with specific modifications costing even more. However, in-house synthesis is labor-intensive, requires significant knowledge of peptide chemistry and, if performed using an automated machine, involves large capital expenditure. In general, acquisition of an automated peptide synthesizer is probably best suited to laboratories or institutes with substantial and ongoing requirements for synthetic peptides, and preferably with their own dedicated personnel. An in-house peptide synthesis facility is a particularly attractive alternative to custom synthesis because it allows much greater flexibility in the design and production of peptides. This can be important if specially derivatized pep-tides are needed, or if, for example, chemically defined immunogens such as multiple antigen peptides are to be synthesized (9,10).
Solid-phase peptide synthesis is based on the sequential addition of protected amino acids onto an insoluble support. Addition proceeds from carboxy terminus to amino terminus. The first amino acid is attached to a solid support by a linker and, if necessary, side-chain amino acid function is protected throughout chain assembly. The carboxy group of the in-coming, acylating amino acid is activated for coupling while its amino group is protected temporarily for each coupling step and then deprotected for the next cycle. The cycles of deprotection and coupling are continued until the amino acid chain is complete. The peptide is then cleaved from the solid support and the amino acid side-chains are deprotected to give the final peptide product. In general, once a peptide is made the sequence cannot be altered. Modifications such as acetylation, phos-phorylation or the introduction of additional residues (e.g., for use in conjugation) should be planned before synthesis. The modifications can then be incorporated into the synthesis procedure. For example, phosphopeptides are synthesized using amino acid residues with specially derivatized side-chains. The final product is usually evaluated by reverse-phase high-performance liquid chromatogra-phy (C8 or C18 columns with water/acetonitrile gradients) and mass spectrometry. For antibody production, a suitable amount of pep-tide is about 50 mg. This allows for affinity purification of antibodies and antibody blocking experiments, to demonstrate antibody specificity, as well as immunization. The synthesis of most peptide sequences in the region of 20 residues in length is currently considered to be quite routine. There are, however, always exceptions. Certain sequences can be extremely difficult to synthesize and may require alternative synthetic strategies (11,12).
Was this article helpful?