Identification of Lasso Peptide Topologies Using Native Nanoelectrospray Ionization-Trapped Ion Mobility Spectrometry–Mass Spectrometry

Lasso peptides are a fascinating class of bioactive ribosomal natural products characterized by a mechanically interlocked topology. In contrast to their branched-cyclic forms, lasso peptides have higher stability and have become a scaffold for drug development. However, the identification and separation of lasso peptides from their unthreaded topoisomers (branched-cyclic peptides) is analytically challenging since the higher stability is based solely on differences in their tertiary structures. In the present work, a fast and effective workflow is proposed for the separation and identification of lasso from branched cyclic peptides based on differences in their mobility space under native nanoelectrospray ionization–trapped ion mobility spectrometry–mass spectrometry (nESI-TIMS-MS). The high mobility resolving power (R) of TIMS resulted in the separation of lasso and branched-cyclic topoisomers (R up to 250, 150 needed on average). The advantages of alkali metalation reagents (e.g., Na, K, and Cs salts) ...