A mass spectrometric technique for detecting and identifying by-products in the synthesis of peptides

Abstract

The utility of a new mass spectrometric technique for detecting and identifying peptide by-products produced in the synthesis of peptides is demonstrated. The technique involves three sequential steps: (1) practically nondestructive 252Cf plasma desorption mass spectrometric analysis of monolayer amounts of the peptide(s) of interest bound to a thin layer of nitrocellulose; (2) enzyme-catalyzed microscale chemical reaction of the surface-bound peptide(s) to produce structurally informative hydrolysis products; (3) plasma desorption mass spectrometric analysis of these hydrolysis products. The first step determines the presence and the molecular weights of unwanted by-products resulting from errors or incomplete reactions during synthesis. The subsequent two steps provide information on the precise location in the peptides where errors have occurred. In the present paper, the technique is applied to an investigation of unwanted peptide by-products associated with the use of tryptophan during stepwise solid-phase peptide synthesis. Synthetic preparations of melittin and [Bpa-8]dynorphin A (1–17) were each found to contain a major impurity with molecular weight 28 Da higher than that of the desired product. The impurity in the melittin preparation, in which the final deprotection step involved the high-low HF procedure, was shown to result from incomplete removal of the formyl group from Trp-19. On the other hand, the impurity in the [Bpa-8]dynorphin A (1–17) preparation, where the removal of the formyl group from Trp-14 was carried out using piperidine, was shown to result from migration of the formyl group to Lys-11 or Lys-13. Findings concerning the relative rates of hydrolysis at Ni-formylated tryptophan and Trp by chymotrypsin and Nε-formylated lysine and Lys by carboxypeptidase B are also presented.