Development of a laser desorption ionization – ion mobility – mass spectrometry method to accelerate the cyclic peptides stereochemistry determination workflow

Cyclic peptides are a class of compounds with significant therapeutic potential. The increase in number of stereocenters resulting in exponential surge of stereoisomers poses an immense challenge on the stereochemical analysis of these molecules. Current stereochemistry control strategies for synthetic peptides requires chiral LC method development using synthetic markers. However, the synthesis of all possible stereoisomers as well as chiral LC method development to separate all of them is extremely costly and time consuming. Alternatively, cyclic peptides can be hydrolyzed into single amino acids (AA), further derivatized by chiral reagents for LC-MS analysis, and compared to AA standards subjected to identical derivatization protocols. This LC-MS based methodologies could determine chirality for all possible stereoisomers within short time (less than 1hr) avoiding the need to synthesize large number of cyclic peptide stereoisomers. While examining literature reported LC-MS methodologies, we sought opportunities to further reduce the analysis time required for comprehensive determinations of the stereochemistry of cyclic peptides via advanced MS platforms. Here-in we report a new laser desorption/ionization-ion mobility-mass spectrometry (LDI-IM-MS) method for the rapid determination of amino acid stereochemistry in cyclic peptides. Chiral derivatization reagents, l-FDLA (Nα-(2,4-dinitro-5-fluorophenyl)-l-leucinamide) and d-FDLA were used to derivatize the amino acids into diastereomer pairs. The diastereomers were differentiated using LDI-IM-MS on a Bruker timsTOF flex platform, with key ion mobility parameters optimized and experimental CCS (collision cross section) values calculated. Enhanced sample preparation workflow including solid phase extraction (SPE) and CuCl₂ doping exhibited better ion mobility differentiation for selected samples. This approach was successfully applied to the analysis of polymyxin B, a natural product cyclic peptide, providing comprehensive stereochemical determination of all constituent amino acids within 1 min. The new workflow not only accelerates the stereochemical analysis of cyclic peptides but also holds promise for broader applications in pharmaceuticals including chiral quality control and monitoring of peptide stability.