Leveraging High-Resolution Ion Mobility-Mass Spectrometry for Cyclic Peptide Soft Spot Identification.

Area of Science:

• Analytical Chemistry
• Biochemistry
• Drug Discovery

Background:

• Cyclic peptides offer unique pharmacological properties and stability, making them attractive drug candidates.
• Oral bioavailability of cyclic peptides is limited by gastrointestinal degradation.
• Soft spot identification (SSID) is crucial for designing orally bioavailable cyclic peptides but is often complex and time-consuming.

Purpose of the Study:

• To present an orthogonal approach using high-resolution ion mobility (HRIM) coupled with Structures for Lossless Ion Manipulation (SLIM) technology for soft spot identification (SSID) of cyclic peptides.
• To overcome challenges in SSID, particularly the coelution of isomeric linear peptide metabolites.
• To develop efficient and unambiguous methods for SSID to guide medicinal chemistry efforts.

Main Methods:

• Utilized a high-resolution ion mobility (HRIM) system based on Structures for Lossless Ion Manipulation (SLIM) technology interfaced with quadrupole time-of-flight (QTOF) mass spectrometry.
• Employed two strategies for resolving linear isomeric peptide metabolites: a label-free approach using negative polarity ionization and an isotope-labeled dimethyl tag approach.
• Investigated the separation of gas-phase ions in the ion mobility (IM) dimension and analyzed unique product ions in fragmentation spectra.

Main Results:

• The HRIM-SLIM platform effectively addressed challenges in SSID.
• The label-free strategy using negative polarity provided better separation of isomeric peptide metabolites in the IM dimension.
• The isotope-labeled dimethyl tag strategy achieved baseline separation of isomers and generated unique product ions for unambiguous SSID.

Conclusions:

• Both label-free and labeled HRIM-SLIM strategies offer effective solutions for soft spot identification in cyclic peptides.
• These methods enhance the analysis of cyclic peptides, overcoming limitations of traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• The developed approach facilitates medicinal chemistry design for orally bioavailable cyclic peptide therapeutics.