The stereoselective separation of serine containing peptides by zwitterionic ion exchanger type chiral stationary phases and the study of serine racemization mechanisms by isotope exchange and tandem mass spectrometry.
The occurrence of d-amino acids (D-AAs) in higher-developed organisms in their free form, and within peptides and proteins, has been investigated with an increasing number of studies. Often the inversion of the stereochemical configuration of an individual amino acid drastically changes its biological activity. Alongside Asn and Asp, Ser is most prone to racemization within peptides. Specific enzymes catalyzing D-Ser generation and breakdown have been described. Hence, the applicability of enantioselective ZWIX(+)(®) and ZWIX(-)(®) chiral stationary phases (CSPs) to peptide separations was assessed and a set of 14 pairs of diastereomeric and enantiomeric Ser and Thr containing di-, tri- and tetra-peptides was chromatographically separated without prior hydrolysis to the individual amino acids. To a certain extent, RP chromatography also enabled the separation of peptide diastereomers. The ZWIX CSPs delivered chromatographic selectivities between 1.04 and 7.23, allowing a change of elution order by switching between the ZWIX(+) and the ZWIX(-) CSP. Coupling these highly selective chromatographic columns with an LTQ-Orbitrap XL™ mass spectrometer and performing high resolution MS(2) measurements enabled us to investigate mechanistic aspects of chemically induced racemization of Ser embedded in short peptides. As reaction medium an alkaline aqueous solution (pH 12.3) was selected. Proton/deuterium exchange experiments provided evidence of a fast Cα proton exchange with simultaneous racemization. Additionally, (18)O/(16)O exchange allowed the identification of an alternative, and somewhat retarded racemization via a reversible β-elimination and reintroduction of water at the hydroxymethyl side chain of Ser. This involved the intermediate generation of the prochiral didehydro alanine unit.
University of Salzburg, Department of Molecular Biology, Division of Chemistry & Bioanalytics, Hellbrunnerstrasse 34, 5020 Salzburg, Austria. Electronic address: roland.reischl@sbg.ac.at.
Roland Johann
RJ
Reischl
University of Vienna, Faculty of Chemistry, Department of Analytical Chemistry, Währingerstrasse 38, 1090 Vienna, Austria. Electronic address: wolfgang.lindner@univie.ac.at.
Wolfgang
W
Lindner
D026921:Amino Acid Transport Systems, Neutral / Q000032:analysis* / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D012694:Serine / Q000032:analysis* / Q000737:chemistry; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
D026921:Amino Acid Transport Systems, Neutral; D010446:Peptide Fragments; D012694:Serine
Enantioselective Chromatography; Isotope Exchange; Peptide racemization; d-Amino Acids; d-Amino acid containing peptides (DAACP)