Because cyclized peptides have been shown to be more effective than their linear counterparts in many biological systems, the 1995 study focused on methods for producing cyclized peptides. ABRF member laboratories were asked to synthesize the following peptide, ABRF95, by the method most frequently used in their facility:
H-Phe-Cys-Phe-Trp-Lys-Thr-Cys-Thr-NH2
Member laboratories were asked to prepare this octreotide-like peptide with an intact disulfide bond produced by any of four protocols provided by the committee. Coded samples of unpurified peptides were characterized by amino acid analysis, HPLC, electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), Edman sequencing, and spectrophotometric analysis following treatment with Ellman's reagent. These methods permitted quantitation and evaluation of purity, as well as a measure of the amount of oxidized peptide.
A total of 97 samples were received for this study from 41 laboratories, and 37 samples were the linear precursors of peptides cyclized by the post-cleavage procedures. For 33 of the 41 laboratories, more than 50% of the sample had the correct structure, defined as the desired sequence with no remaining protecting groups. Thus, the assembly and cleavage process for the peptide chosen in the study appears to be routinely achieved in the participating core laboratories. Several laboratories submitted samples using protocols not suggested by the committee, including ferricyanide oxidation and use of Ekathiox resin.
The following general conclusions were made as the result of the study. Producing cyclized peptides with the correct structure can be achieved readily by either on-resin or post-cleavage techniques. Post-cleavage techniques are less expensive and provide reasonable yields of the desired product. However, on-resin techniques produce greater yields of the final product. The mercuric acetate method did not yield the correct peptide product but produced a mercury-peptide adduct that was stable by HPLC and mass spectrometry. This adduct had been noted previously during the synthesis of octreotide.
It is best to use a combination of analytical techniques that complement each other in their ability to detect the correct product and unwanted by-products. The use of Ellman's reagent can record the loss of free sulfhydryl groups but does not discriminate between sulfhydryls that become oxidized to disulfide bonds in monomers or dimers, mercury adducts, or peptides with incompletely deprotected cysteine residues. HPLC analysis readily separates peptides that are linear, cyclized, dimeric, mercury adducts, incompletely deprotected, or deletion by-products. However, unless the peaks are identified by mass spectrometry, one cannot assume that even a single peak is the desired product. Mass spectrometry is very useful in discriminating the peptide products, and it is essential to use unit resolution techniques to resolve 1-2 amu differences such as those seen between acids and amides.
Overall, the quality of the peptides produced in this study was excellent. The results of the cyclization tests are encouraging, particularly because many of the participating laboratories may have been performing these protocols for the first time. However, that 5 out of 41 laboratories submitted samples in which the desired peptide had not been made, for reasons that can be attributed only to human error, is a recurring theme in the annual studies performed by this committee.
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