Metal-ion free chiral analysis of amino acids as small as proline using high-definition differential ion mobility mass spectrometry

Abstract

The chiral analysis of enantiomers is important because bioactivity can depend strongly on stereochemistry as ligand-protein binding motifs are typically chiral. Ion mobility mass spectrometry-based methods are emerging for the rapid and sensitive chiral analysis of molecules. However, such methods are typically limited by the use of metal-bound trimers, which can be challenging to form owing to ion suppression and the need for extensive pre-screening experiments to identify suitable metal ions. Moreover, the chiral separation of very small molecules, such as cysteine and proline, using ion mobility has remained challenging. Here, using electrospray ionisation high-resolution differential ion mobility mass spectrometry (ESI-DMS-MS), we demonstrate that the enantiomers of benchmark amino acids as small as proline can be rapidly distinguished without the use of metal ions for the first time. ESI-DMS-MS of proton-bound diastereomeric dimer complexes, containing enantiomers of amino acids and a ‘chiral selector’ (N-tert-butoxycarbonyl-O-benzyl-l-serine; BBS) corresponding to [L/D-X(BBS)+H]+ (X = cysteine and proline) resulted in the separation of L and D-enantiomers. By use of DMS-MS and standard solutions of chiral mixtures, these data indicate that the enantiomeric excess of proline can be accurately quantified by differential ion mobility mass spectrometry. Overall, these results provide further evidence that DMS-MS can be used for the rapid and accurate ‘metal-ion free’ chiral analysis of many other biologically important molecules.