Establishment of a research platform for protein dopaminylation based on the dopamine-derived probe PA

doi:10.3969/j.issn.1674-8115.2026.10.009

Abstract

Abstract:

Objective ·To establish a research platform for the screening and identification of dopaminylated proteins, and to preliminarily characterize their distribution across different cells and tissues. Methods ·The Cancer Genome Atlas and Genotype-Tissue Expression databases were analyzed to examine the expression of dopamine transporter, DOPA decarboxylase (DDC), and transglutaminase 2 (TGM2) across different tissues, in order to evaluate the molecular basis for protein dopaminylation. An alkynylated dopamine derivative, PA, was used as a chemical probe and added to the cell culture medium. Click chemistry, biotin-based enrichment, and mass spectrometry were then used to screen candidate dopaminylated proteins in different cell types. Functional enrichment analysis and protein-protein interaction network analysis were further performed to characterize their functional features. In addition, an antibody specific for dopaminylation was used for preliminary validation of dopaminylation in mouse and human pancreatic tissues by immunohistochemistry. Results ·The dopamine transporter, DDC, and TGM2 were widely expressed across diverse tissues, providing a molecular basis for protein dopaminylation in multiple tissues. PA served as an effective chemical probe for the labeling, enrichment, and identification of candidate dopaminylated proteins. A total of 56, 327, 463, 56, and 86 candidate proteins were screened in mouse hippocampal HT22 cells, neonatal mouse primary microglia, pancreatic α-cells, pancreatic β-cells, and AsPC-1 cells, respectively. The candidate dopaminylated proteomes identified in different cell types showed marked differences and displayed cell type-dependent functional characteristics. Functional enrichment analysis indicated that these proteins were mainly involved in metabolic regulation, protein synthesis, and cellular structure organization-related processes. Immunohistochemical results further suggested that dopaminylation could be detected in both mouse and human pancreatic tissues. Conclusion ·A research platform for protein dopaminylation based on the alkynylated dopamine-derived probe PA was established. This method integrates traceability, enrichment capability, and proteomics scalability, enabling the systematic screening of candidate dopaminylated proteins and the analysis of their functional characteristics. It provides a methodological foundation for further elucidating the molecular mechanisms of dopaminylation and its roles in physiological and pathological processes.

Key words: dopamine, dopaminylation, chemical probe, proteomics

CLC Number:

R730.2

Hong Yuanxin, Tulamati Aziguli, Shi Wentao, Yang Tian, Zhu Lei, Zhang Xueli, Zhang Zhigang, Li Dongxue. Establishment of a research platform for protein dopaminylation based on the dopamine-derived probe PA[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(10): 1392-1405.

Figures/Tables 7

Fig 1 Expression of SLC6A3 and DDC in various normal tissues and pan-cancer tissues

Fig 2 Synthetic route and structural characterization of dopamine derivative PA

Tab 1 1H NMR chemical shift assignments of the alkynylated dopamine derivative PA

Chemical shift (δ)/ppm	Assigned proton	Integration	Description
6.93‒6.90	Aromatic H-2	1H	1, 2, 4-trisubstituted benzene ring
6.82	Aromatic H-5	1H	Aromatic region
6.70‒6.68	Aromatic H-6	1H	Aromatic region
4.71‒4.70	O‒CH₂‒C≡CH	2H	OCH₂ group of the propargyloxy moiety
3.75	OCH₃	3H	Methoxy group on the aromatic ring
2.76‒2.73	CH₂‒NH₂	2H	Methylene group adjacent to the amino group
2.62‒2.55	Ar‒CH₂	2H	Benzylic CH₂ group
ca. 2.50‒3.10	C≡CH	1H	Terminal alkyne proton, possibly overlapped
Broad peak, variable	NH₂	2H	Exchangeable protons, may be broad or not clearly observed

Fig 3 PA-based strategy for the enrichment and profiling of candidate dopaminylated proteins

Fig 4 KEGG enrichment and overlap analysis of candidate dopaminylated proteins in different cell types

Fig 5 Protein-protein interaction networks of candidate dopaminylated proteins in different cell types

Fig 6 Immunohistochemical detection of dopaminylated in mouse and human pancreatic tissues

TrendMD

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