Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation

Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guid...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2024-05, Vol.96 (19), p.7756-7762
Main Authors: Feng, Yu, Qirjollari, Athanasia, Fawaz, Maria V., Cancilla, Mark T., Gonzalez, Raymond J., Pearson, Kara
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino acids
Fragmentation
Hydrolysis
Isotope Labeling
Linearization
Mass spectrometry
Mass spectroscopy
Methylation
Oral administration
Oxidation-Reduction
Peptides
Peptides, Cyclic - chemistry
Proteolysis
Scientific imaging
Tandem Mass Spectrometry - methods
Workflow
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Summary:Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m/z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.
ISSN:0003-2700
1520-6882
1520-6882
DOI:10.1021/acs.analchem.4c01372