Ion-spray tandem mass spectrometry in peptide synthesis: Structural characterization of minor by-products in the synthesis of ACP(65–74)

Abstract

Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65–74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Gly-OCH 2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF p- cresol for 1 h at 0°C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65–74). The significant minor component was apparently from incomplete deprotection of Asp 70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp 70, succinimide formation at Asn 73, acylation of the Tyr 71 side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods.