Multicolumn Two-Dimensional Liquid Chromatography Screening Platform for Stereopeptidomics and Application to Antimicrobial Peptide Polyene and Lipopeptide

Therapeutic peptides are a rapidly growing field in research and drug development. While the majority of natural and synthetic therapeutic peptides have l-amino acids as building blocks, d-amino acid-containing peptides are found frequently in nonribosomal peptides or can be formed during peptide synthesis by epimerization. Thus, analytical methods are needed for the quality control of stereointegrity and the determination of absolute configurations. Enantioselective amino acid analysis following complete hydrolysis is indispensable in the field but leads to the loss of sequence information, i.e., the position of d-amino acids can no longer be unambiguously assigned. Here, we propose a multicolumn two-dimensional liquid chromatography-tandem high-resolution mass spectrometry (2D LC-HRMS) platform with multiple reversed-phase type columns (C18, charged surface hybrid C18, mixed-mode C18 AX) in the first dimension (¹D) and multiple chiral columns in the second dimension (²D) (teicoplanin, teicoplanin aglycone, crown ether, and zwitterionic quinine and quinidine carbamate-based chiral stationary phases). It allows the combination of distinct ¹D columns (for peptide epimer/diastereomer separations) and ²D columns (for peptide enantiomer separations), enabling the full resolution of complex peptide stereoisomer mixtures. The utility of this 2D-LC platform for peptide analyses was demonstrated for a tetrapeptide amide from an antimicrobial peptide polyene natural product and a lipopeptide, digested into dipeptides for middle-down/middle-up stereoselective peptide analysis. Multiple heart-cutting and selective comprehensive 2D-LC, respectively, with active solvent modulation and sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) enabled the full separation of all stereoisomers and the clarification of the configurations of all sample peptides. Such a 2D-LC-HRMS screening platform can be valuable as an efficient and fast generic approach for streamlining method development in the pharmaceutical industry.