Multiplatform High-Definition Ion Mobility Separations of the Largest Epimeric Peptides

Area of Science:

• Analytical Chemistry
• Biochemistry
• Proteomics

Background:

• Ion mobility spectrometry-mass spectrometry (IMS-MS) is crucial for separating complex mixtures and analyzing molecular structures.
• D-amino acid-containing peptides (DAACPs) play vital roles in neurotransmission and toxicology, but their characterization remains challenging.
• Previous IMS methods could only resolve d/l epimers up to approximately 30 residues.

Purpose of the Study:

• To demonstrate the capability of combining three advanced IMS techniques to resolve large d/l peptide epimers.
• To expand the analysis of isomeric modified peptides using Field Asymmetric Ion Mobility Spectrometry (FAIMS).
• To develop a method for calibrating cyclic IMS for broader applicability.

Main Methods:

• Utilized a combination of trapped IMS, cyclic IMS, and FAIMS for high-resolution separation.
• Analyzed the largest known d/l peptides (CHH from *Homarus americanus*, 72 residues).
• Investigated epimer separation across multiple charge states and conformers.

Main Results:

• Achieved baseline resolution of 72-residue d/l peptides with a dynamic range up to 100.
• Observed similar spectra for d and l epimers, differing only by a uniform shift in the separation parameter.
• Demonstrated that interepimer resolution correlates with peptide size, suggesting potential for larger DAACP discovery.

Conclusions:

• The combined IMS approach significantly advances the analysis of large d/l peptides, extending capabilities to the size of small proteins.
• The conserved epimer-specific structural elements indicated by spectral shifts offer insights into peptide conformation.
• The developed a priori calibration method for cyclic IMS offers broad utility for future research.