Identification of degradation products of aspartyl tripeptides by capillary electrophoresis-tandem mass spectrometry.
Capillary electrophoresis-electrospray tandem mass spectrometry (CE-MS/MS) has been used to identify degradation products of the aspartyl tripeptides Phe-Asp-GlyNH(2) and Gly-Asp-PheNH(2) following incubation of the peptides in acidic and alkaline solution. At pH 2, the dominant decomposition products resulted from cleavage of the peptide backbone amide bonds to yield the respective dipeptides and amino acids. In addition, the cyclic aspartyl succinimide intermediate was identified by its [M+H](+) at m/z = 319 and the MS/MS spectrum exhibiting a simple fragmentation pattern with the [C(8)H(10)N](+)-ion as the principal daughter ion (a(1) of Phe-Asp-GlyNH(2)). Deamidation of the C-terminal amide as well as isomerization and enantiomerization of the Asp residue occurred upon incubation at pH 10. alpha-Asp and the isomeric beta-Asp and most of the diastereomeric forms (corresponding to D/L-Asp) could be separated by CE. All isomers could be identified based on their MS/MS spectra. Peptides with the amino acid sequence Phe-Asp-Gly containing the regular alpha-Asp bond displayed a highly intense b(2) fragment ion and a low abundant y(2) ion. In contrast, the y(2) and a(1) fragment were high abundant daughter ions in the mass spectra of beta-Asp peptides while the b(2) ion exhibited a lower abundance. Differences in the MS/MS spectra of the isomers of the peptides with the sequence Gly-Asp-Phe were obvious but less pronounced. In conclusion, CE-MS/MS proved to be a useful tool to study the decomposition and enantiomerization of peptides including the isomerization of Asp residues, due to the combination of efficient separation of isomers by CE and their identification by MS/MS.
De Boni
Silvia
S
University of Jena, School of Pharmacy, Department of Pharmaceutical Chemistry, Jena, Germany.
Neusüss
Christian
C
Pelzing
Matthias
M
Scriba
Gerhard K E
GK
D001224:Aspartic Acid / Q000032:analysis* / Q000737:chemistry; D002021:Buffers; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization; D013237:Stereoisomerism
D002021:Buffers; D009842:Oligopeptides; D010446:Peptide Fragments; D001224:Aspartic Acid