Ion-spray tandem mass spectrometry in peptide synthesis: structural characterization of minor by-products in the synthesis of ACP(65-74).
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 Bly-OCH2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF/p-cresol for 1 h at 0 degrees 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 Asp70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp70, succinimide formation at Asn73, acylation of the Tyr71 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.
Schnölzer
M
M
Scripps Research Institute, La Jolla, California 92037.
Jones
A
A
Alewood
P F
PF
Kent
S B
SB
D000213:Acyl Carrier Protein / Q000737:chemistry*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D007477:Ions; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry
D000213:Acyl Carrier Protein; D007477:Ions; D009842:Oligopeptides; D013388:Succinimides