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. 2023 Jul 3;15(10):1422–1430. doi: 10.1038/s41557-023-01252-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, Overall tyramine excision (TyrEx) strategy for labelling of His₆-PlpA3-M5G: PlpXY expressed in the cell installs aminopyruvate site-specifically in the target biomolecule at a GYG-containing motif. Splicing results in a mass loss of −135.0757 Da (−C₈H₉NO). The post-translationally modified product reacts tetrazine derivatives to form the pyridazyl protein conjugates a–c. Structures are based on models calculated with AlphaFold^47,48. b, Conjugation of His₆-PlpA3-M5G with a fluorescent tetrazine probe analysed by SDS–PAGE. GYG: unmodified His₆-PlpA3-M5G molecular weight (MW) 13.3 kDa. +PlpXY: His₆-PlpA3-M5G was co-produced with PlpXY to yield the aminopyruvate-containing product (MW 13.2 kDa). The CF®488A fluorophore (6) was imaged by gel fluorescence using a 488 nm excitation and 532/528 nm emission filter. The experiment was performed once. c, Deconvoluted electrospray ionization–mass spectra (ESI–MS) showing ions for the substrate (His₆-PlpA3-M5G, m/z calculated 13,326.2112, found 13,326.2278) and the cycloaddition product using tetrazine 2 (m/z calculated 13,381.2071, found 13,381.2350). d, Observed y- and b-ions for the ESI–MS/MS fragmentation of a trypsinized reaction product localizing the cycloaddition site (blue).

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