Analysis of aspartyl peptide degradation products by high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry.
A reversed-phase HPLC method for the analysis of degradation products of the model aspartyl tripeptides Phe-Asp-GlyNH2 and Gly-Asp-PheNH2 after incubation at pH 2 and 10 was developed. Most of the compounds could be separated with a gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. Resolution of the isomeric pairs L-Phe-alpha-L-Asp-GlyNH2/L-Phe-beta-L-Asp-GlyNH2 and L-Phe-alpha-D-Asp-GlyOH/L-Phe-beta-D-Asp-GlyOH was achieved with a gradient of acetonitrile in phosphate buffer, pH 5.0. Under acidic conditions the major degradation pathway was cleavage of the peptide backbone amide bonds yielding dipeptides and amino acids, C-terminal deamidation as well as formation of succidinimyl peptides. At alkaline pH both deamidation of the C-terminal amide as well as isomerization and concomitant enantiomerization of Asp were observed. The peaks were identified both by reference substances and by online electrospray mass spectrometry. The results were compared to a previous developed capillary electrophoresis method. Diastereomeric pairs ofpeptides that could not be separated by capillary electrophoresis were resolved by HPLC while the separation of corresponding pairs of alpha- and beta-Asp peptides was not always achieved by HPLC in contrast to capillary electrophoresis illustrating that both techniques can be complimentary in peptide analysis.
De Boni
Silvia
S
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Jena, Philosophenweg 14, D-07743 Jena, Germany.
Oberthür
Christine
C
Hamburger
Matthias
M
Scriba
Gerhard K E
GK
D001224:Aspartic Acid / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D001224:Aspartic Acid