De Novo Sequencing of Synthetic Bis -cysteine Peptide Macrocycles Enabled by “Chemical Linearization” of Compound Mixtures
peer-reviewed · Analytical Chemistry · 2023
| Date | 2023-10-10 |
| Type | peer-reviewed |
| Venue | Analytical Chemistry |
| Publisher | American Chemical Society (ACS) |
| Contribution | adjacent |
| DOI | 10.1021/acs.analchem.3c01742 |
| Citations (OpenAlex) | 5 |
| Venue 2-year citedness | 7.10 |
Abstract
A “chemical linearization” approach was applied to synthetic peptide macrocycles to enable their de novo sequencing from mixtures using nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS). This approach─previously applied to individual macrocycles but not to mixtures─involves cleavage of the peptide backbone at a defined position to give a product capable of generating sequence-determining fragment ions. Here, we first established the compatibility of “chemical linearization” by Edman degradation with a prominent macrocycle scaffold based on bis -Cys peptides cross-linked with the m -xylene linker, which are of major significance in therapeutics discovery. Then, using macrocycle libraries of known sequence composition, the ability to recover accurate de novo assignments to linearized products was critically tested using performance metrics unique to mixtures. Significantly, we show that linearized macrocycles can be sequenced with lower recall compared to linear peptides but with similar accuracy, which establishes the potential of using “chemical linearization” with synthetic libraries and selection procedures that yield compound mixtures. Sodiated precursor ions were identified as a significant source of high-scoring but inaccurate assignments, with potential implications for improving automated de novo sequencing more generally.
Methods and tools
- Bis-cysteine macrocycle chemical linearization: Cleaves synthetic bis-Cys macrocyclic peptides at a defined backbone position by Edman chemistry so that mixtures from macrocycle libraries can be sequenced de novo by nLC-MS/MS.