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Mirror-image trypsin digestion and sequencing of D-proteins

Nature Chemistry volume 16, pages 592–598 (2024)Cite this article

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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.

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Fig. 1: Synthetic natural-chirality (l-) and synthetic mirror-image (d-) trypsins.
Fig. 2: Digestion and sequencing of ribosomal protein d-L25.
Fig. 3: Digestion and sequencing of d-Dpo4.
Fig. 4: Writing and reading information in a long d-peptide.

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Data availability

All data are available in the main text or the Supplementary Information. The E. coli proteome database (Taxonomy 83333) was downloaded from UniProt (https://www.uniprot.org). The LC–MS/MS data were deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD046228. Source data are provided with this paper.

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Acknowledgements

We thank J. Chen, Q. Deng, C. Fan, H. Liu, G. Wang, Y. Xu, J. Zhang and R. Zhao for assistance with the experiments, and H. Deng and X. Tian at the Tsinghua Technology Center for Protein Sciences, C. C. Wong at the Peking Union Medical College Hospital State Key Laboratory of Complex Severe and Rare Diseases, and T. Guo and Y. Zhu at the Westlake iMarker Lab for assistance with the tandem mass analysis. The work was supported by the National Natural Science Foundation of China (grant nos. 21925702 and 32050178), the Research Center for Industries of the Future (RCIF) at Westlake University, the Westlake Education Foundation, the New Cornerstone Science Foundation, the Tsinghua-Peking Center for Life Sciences (CLS) and the Beijing Frontier Research Center for Biological Structure. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the paper.

Author information

Authors and Affiliations

  1. School of Life Sciences, Tsinghua-Peking Center for Life Sciences, Beijing Frontier Research Center for Biological Structure, Tsinghua University, Beijing, China

    Guanwei Zhang

  2. School of Life Sciences, New Cornerstone Science Laboratory, Research Center for Industries of the Future, Westlake University, Hangzhou, China

    Guanwei Zhang & Ting F. Zhu

  3. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Guanwei Zhang & Ting F. Zhu

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  1. Guanwei Zhang
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Contributions

G.Z. performed the experiments. Both authors analysed the data and wrote the paper. T.F.Z. designed and supervised the study.

Corresponding author

Correspondence to Ting F. Zhu.

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A provisional US patent application (no. 63/546,881) has been filed by Westlake University with T.F.Z. and G.Z. listed as inventors. The authors declare no other competing interests.

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Nature Chemistry thanks Michael Kay, Stephen Kent and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Design of the synthetic l-/d-trypsinogen.

a, Amino acid sequence of the synthetic l-/d-trypsinogen (UniProt P00761). The amino acid colours correspond to the peptide segment colours used in b and Fig. 1a. b, Synthetic route for the total chemical synthesis of l-/d-trypsinogen.

Extended Data Fig. 2 Chiral specificity of trypsin digestion of substrate peptides.

a,b, Analytical RP–HPLC chromatograms of the chemically synthesized l-LYAARLYAVR (a) and d-LYAARLYAVR (b) before digestion. c,d, Analytical RP–HPLC chromatograms of l-LYAARLYAVR (c) and d-LYAARLYAVR (d) digested by the recombinant l-trypsin. e,f, Analytical RP–HPLC chromatograms of l-LYAARLYAVR (e) and d-LYAARLYAVR (f) digested by the synthetic l-trypsin. g,h, Analytical RP–HPLC chromatograms of l-LYAARLYAVR (g) and d-LYAARLYAVR (h) digested by the synthetic d-trypsin. The experiments were performed three times with similar results.

Source data

Extended Data Fig. 3 Chiral specificity of trypsin digestion of ribosomal protein L25 and Dpo4.

a–c, Analytical RP–HPLC chromatograms of the synthetic d-L25 before (a) and after digestion by the recombinant l- (b) and synthetic l-trypsin (c). d,e, Analytical RP–HPLC chromatograms of the synthetic l-L25 before (d) and after digestion by the synthetic d-trypsin (e). f–h, Analytical RP–HPLC chromatograms of the synthetic d-Dpo4-5m before (f) and after digestion by the recombinant l- (g) and synthetic l-trypsin (h). i,j, Analytical RP–HPLC chromatograms of the recombinant l-Dpo4-5m before (i) and after digestion by the synthetic d-trypsin (j). The experiments were performed twice with similar results.

Source data

Extended Data Fig. 4 Cleavage site specificity of trypsin digestion of ribosomal protein L25 and Dpo4.

a, Observed cleavage frequency at the P1 site of l-L25 digested by the recombinant l- and synthetic l-trypsin, and of d-L25 by the synthetic d-trypsin. b, Observed cleavage frequency at the P1 site of l-Dpo4-5m digested by the recombinant l- and synthetic l-trypsin, and of d-Dpo4-5m by the synthetic d-trypsin. c, Observed cleavage frequency of l-L25 digested by the recombinant l- and synthetic l-trypsin, and of d-L25 by the synthetic d-trypsin, at trypsin cleavage sites with lysine at the P1 site and with or without proline at the P1′ site, displayed on a log scale. d, Observed cleavage frequency of l-Dpo4-5m digested by the recombinant l- and synthetic l-trypsin, and of d-Dpo4-5m by the synthetic d-trypsin, at trypsin cleavage sites with lysine at the P1 site and with or without proline at the P1′ site, displayed on a log scale. The experiments were performed twice with similar results.

Source data

Extended Data Fig. 5 Sorting of de novo sequencing results.

a–e, Sorting of de novo sequencing results by the sums of the ALC of the potential 10-aa d-peptide sequences indexed by alanine (a, also shown in Fig. 4e), phenylalanine (b), glycine (c), histidine (d), and leucine (e). The experiment was performed twice with similar results.

Source data

Extended Data Fig. 6 LC–MS/MS analysis of the undigested information-storing 50-aa d-peptide.

a, Design of an information-storing 50-aa d-peptide, also shown in Fig. 5a. b,c, ESI–MS spectrum of the undigested information-storing 50-aa d-peptide (b), with an example of the tandem mass spectra of the undigested d-peptide shown (c). No 50-aa sequence was present in the de novo sequencing results. The experiment was performed twice with similar results.

Supplementary information

Supplementary Information

Supplementary Figs. 1–22 and Tables 1–3.

Reporting Summary

Supplementary Data

PSMs of trypsin-digested ribosomal protein L25 and Dpo4.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 1

Uncropped gel from Fig. 1b.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 2

Statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

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Zhang, G., Zhu, T.F. Mirror-image trypsin digestion and sequencing of D-proteins. Nat. Chem. 16, 592–598 (2024). https://doi.org/10.1038/s41557-023-01411-x

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