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Mirror-image trypsin digestion and sequencing of D-proteins

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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Escherichia Coli

SUBMITTER: Guanwei Zhang

LAB HEAD: Ting Zhu

PROVIDER: PXD046228 | Pride | 2024-01-19

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	D-Dpo4-5m_by_synthetic_D-trypsin.raw	Raw
	D-Dpo4-Y12S_by_synthetic_D-trypsin.raw	Raw
	D-L25_by_synthetic_D-trypsin.raw	Raw
	D-protein_sequencing_no-enzyme.pdResult	Other
	D-protein_sequencing_trypsin.pdResult	Other

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