Leveraging High-Resolution\nIon Mobility-Mass Spectrometry\nfor Cyclic Peptide Soft Spot Identification

Abstract

Cyclic peptides are an important class of molecules that\ngained\nsignificant attention in the field of drug discovery due to their\nunique pharmacological characteristics and enhanced proteolytic stability.\nYet, gastrointestinal degradation remains a major hurdle in the discovery\nof orally bioavailable cyclic peptides. Soft spot identification (SSID)\nof the regions in the cyclic peptide sequence susceptible to amide\nhydrolysis by proteases is used in the discovery stage to guide medicinal\nchemistry design. SSID can be an arduous task, traditionally performed\nusing liquid chromatography–tandem mass spectrometry (LC-MS/MS),\noften resulting in complex and time-consuming manual analysis, particularly\nwhen isomeric linear peptide metabolites chromatographically coelute.\nHere, we present an alternative orthogonal approach that entails a\nhigh-resolution ion mobility (HRIM) system based on Structures for\nLossless Ion Manipulation (SLIM) technology interfaced with quadrupole\ntime-of-flight (QTOF) mass spectrometry to address some of the challenges\nassociated with SSID. Two strategies were used to resolve linear isomeric\npeptide metabolites: labeled and label-free, both utilizing the HRIM\nplatform. The label-free strategy leverages negative polarity to ionize\nthe isomers which achieves better separation of the gas phase ions\nin the ion mobility (IM) dimension as compared to positive polarity,\nwhich is a more conventional approach when studying proteins and peptides.\nThe second approach uses an isotope-labeled dimethyl tag on the terminal\namine group, acting as a “shift reagent” to influence\nthe mobility of isomers in the positive mode. This method resulted\nin baseline separation for the isomers of interest and produced unique\nproduct ions in the fragmentation spectra for unambiguous soft spot\nidentification. Both label-free and labeled strategies demonstrated\nthe ability to solve the challenges associated with SSID for cyclic\npeptides.