Mirror-image trypsin digestion and sequencing of D-proteins
peer-reviewed · Nature Chemistry · 2024
| Date | 2024-04-01 |
| Type | peer-reviewed |
| Venue | Nature Chemistry |
| Publisher | Springer Science and Business Media LLC |
| Contribution | adjacent |
| DOI | 10.1038/s41557-023-01411-x |
| Citations (OpenAlex) | 21 |
Abstract
The development of mirror-image biology systems and related applications is hindered by the lack of effective methods to sequence mirror-image (D-) proteins. Although natural-chirality (L-) proteins can be sequenced by bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS), the sequencing of long D-peptides and D-proteins with the same strategy requires digestion by a site-specific D-protease before mass analysis. Here we apply solid-phase peptide synthesis and native chemical ligation to chemically synthesize a mirror-image version of trypsin, a widely used protease for site-specific protein digestion. Using mirror-image trypsin digestion and LC-MS/MS, we sequence a mirror-image large subunit ribosomal protein (L25) and a mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), and distinguish between different mutants of D-Dpo4. We also perform writing and reading of digital information in a long D-peptide of 50 amino acids. Thus, mirror-image trypsin digestion in conjunction with LC-MS/MS may facilitate practical applications of D-peptides and D-proteins as potential therapeutic and informational tools.
Methods and tools
- Mirror-image trypsin D-protein sequencing: A chemically synthesized D-trypsin digests mirror-image proteins into peptides that can be sequenced by LC-MS/MS, used to sequence D-proteins and read data stored in a long D-peptide.
Methods it uses
- PEAKS: Commercial DP-based de novo
Data deposited
- Mirror-image trypsin digestion and sequencing of D-proteins — as deposited · PXD046228