Imaging Peptide and Protein Chirality via Amino Acid Analysis by Chiral × Chiral Two-Dimensional Correlation Liquid Chromatography

Abstract

The present contribution illustrates the utilization of a chiral × chiral two-dimensional liquid chromatography (2DLC) setup with tert-butylcarbamoyl quinine chiral stationary phase (CSP) in the first dimension (D) and tert-butylcarbamoyl quinidine CSP in the second dimension (D) to analyze FMOC-derivatized d and l amino acids from peptide hydrolysates. Hereby, in the D andD chiral separation dimensions factors such as selector and immobilization chemistry of the CSPs, mobile phase, temperature, column hardware dimensions, stationary phase supports, particle type and packing were identical. Orthogonality between D andD CSPs was solely based on their stereochemistry, i.e. their opposite configurations in two chiral centers of the selector molecules, which results in inversion of enantiomer elution orders in the two dimensions. Using Coreshell CSPs for fast chromatography allowed D-flow rates which were 60 times faster than theD-flow rates to enable online comprehensive two-dimensional chromatography (LC × LC). Due to very similar chemoselectivity, yet opposite elution orders of corresponding enantiomers in D andD, characteristic 2D-elution patterns for achiral and chiral components can be generated. Peaks of achiral components and impurities are lined up on the diagonal line in the 2D separation space (contour plot) and thereby removed from the chromatographic space of the target enantiomers avoiding overlaps with potential interferences. Corresponding enantiomers provide cross peaks on the 2D chromatogram. Moreover, enantioselectivity of both single CSPs is combined to result in an enhanced overall 2D enantioselectivity. The concept is illustrated for the therapeutic peptides gramicidin and bacitracin. Since all amino acids give a consistent elution order as FMOC-derivatives, all enantiomers of the same configuration are either above or below the diagonal line allowing straightforward imaging of the configuration of the amino acids in peptides by the 2D chromatogram.

Enantioselectivity Effects in Clinical Metabolomics and Lipidomics

“…An innovative approach has been described for providing direct stereochemical information of peptides samples [ 33 ]. To meet this end, a chiral × chiral 2D LC system was designed using two CSP of the same type but of opposite stereochemistry (quinine and quinidine carbamates).…”

Molecular basis for chirality-regulated Aβ self-assembly and receptor recognition revealed by ion mobility-mass spectrometry

“…D/L peptide epimer separation is also vital for peptide functional interrogation and peptide-based drug discovery targeting several neurodegenerative diseases. Typically, epimers can be separated with a chiral column by HPLC or capillary electrophoresis 8,32 . In this study, we provide a general, effective multi-dimensional strategy as a faster alternative to HPLC separation.…”

Recent Developments in Two-Dimensional Liquid Chromatography: Fundamental Improvements for Practical Applications

“…Woiwode et al describe what they refer to as “two-dimensional correlation liquid chromatography” for characterizing the chirality of peptides and proteins by chiral × chiral separations of their constituent amino acids. 118 In this work they deployed stationary phases for the 1 D and 2 D separations that were very similar except for the configuration of the chiral selector bonded to the stationary phase. In this way they refer to “R columns and S columns”.…”