Mass spectrometric evaluation of synthetic peptides for deletions and insertions

A new technique to evaluate methods for the synthesis of peptides was developed. It is based on the identification and quantitation of peptide by-products by mass spectrometry. Model oligopeptides containing 10 or 20 alanine residues were synthesized by automated solid phase methods using a variety of protocols, and the levels of deletion and insertion peptides were measured by the 252Cf fission fragment ionization time-of-flight spectrometric technique in which the total, unfractionated, synthetic product was deposited on a film of nitrocellulose and analyzed. The introduction of D-alanine at every third residue of the model eliminated peptide conformation problems that led to incomplete reactions in the all L model. Couplings with preformed symmetrical anhydrides in dimethylformamide gave rise to significant levels of both deletion peptides and insertion peptides. The best of the protocols examined was a double coupling of tert-butyloxycarbonyl-alanine by in situ activation with dicyclohexylcarbodiimide in dichloromethane. [D-Ala3,6,9,12,15,18]Ala20-Val was synthesized with an average deletion of only 0.036% per step and an average insertion of only 0.029% per step, which is equivalent to a stepwise yield of 99.93% for the target peptide.