Dehydration/1,6-addition-based Site-specific Bioconjugation Unveils Norepinephrinylation as a Widespread Post-translational Modification in the Cellular Proteome

Lin, Z.; Ma, X.; Cai, Z.; Bai, Y.; Wang, Q.; Li, H.; Symasek, A.; Lovato, A. R.; Lyon, S.; Zhao, Y.; Gao, F.; Mabe, N. W.; Yuan, C.; Zhang, Z.-Y.; Zheng, Q.

doi:10.64898/2026.06.11.731782

bioRxiv · 10.64898/2026.06.11.731782

Dehydration/1,6-addition-based Site-specific Bioconjugation Unveils Norepinephrinylation as a Widespread Post-translational Modification in the Cellular Proteome

Lin, Z.·Ma, X.·Cai, Z.·Bai, Y.·Wang, Q.·Li, H.·Symasek, A.·Lovato, A. R.·Lyon, S.·Zhao, Y.·Gao, F.·Mabe, N. W.·Yuan, C.·Zhang, Z.-Y.·Zheng, Q.

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Abstract

Norepinephrine (NE) is a key neurotransmitter and hormone involved in diverse physiological and pathological processes. Beyond its canonical non-covalent signaling through binding adrenergic receptors, NE also induces protein post-translational modifications (PTMs), representing an emerging regulatory mechanism. Two major forms of NE-derived PTMs have been proposed: non-enzymatic norepinephrinylation (NEylation) of cysteine residues mediated by NE quinone and transglutaminase 2 (TG2)-catalyzed NEylation of glutamine residues. However, the cellular abundance, biochemical basis, and pathophysiological roles of NEylation remain poorly understood due to limited detection tools. Here, we report a novel bioconjugation chemistry for selective labeling and enrichment of the endogenous NEylation proteome in cell lines and tissues, which is based on acid-catalyzed dehydration and 1,6-addition to thiol probes. This strategy enables fluorescence imaging and chemical proteomic profiling, revealing NEylation as a widespread PTM that affects enzymatic activities of modified proteins, including protein tyrosine-protein phosphatase non-receptor type 11 (PTPN11). Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/731782v3_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@406ac5org.highwire.dtl.DTLVardef@121d6b9org.highwire.dtl.DTLVardef@98354corg.highwire.dtl.DTLVardef@d312ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Lin, Z., Ma, X., Cai, Z., Bai, Y., Wang, Q., Li, H., Symasek, A., Lovato, A. R., Lyon, S., Zhao, Y., Gao, F., Mabe, N. W., Yuan, C., Zhang, Z.-Y., Zheng, Q.. 2026-06-15. Dehydration/1,6-addition-based Site-specific Bioconjugation Unveils Norepinephrinylation as a Widespread Post-translational Modification in the Cellular Proteome. https://doi.org/10.64898/2026.06.11.731782

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