基于多巴胺衍生物PA的多巴胺化修饰蛋白研究体系的建立

doi:10.3969/j.issn.1674-8115.2026.10.009

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (10): 1392-1405.doi: 10.3969/j.issn.1674-8115.2026.10.009

• 论著 · 技术与方法 • 上一篇

基于多巴胺衍生物PA的多巴胺化修饰蛋白研究体系的建立

洪源鑫, 阿孜古丽·吐拉麦提, 石文韬, 杨田, 朱蕾, 张雪莉, 张志刚(), 李冬雪()

上海交通大学医学院附属仁济医院上海市肿瘤研究所，肿瘤系统医学全国重点实验室，上海 200240

收稿日期:2026-03-26 接受日期:2026-04-21 出版日期:2026-10-20 发布日期:2026-10-20
通讯作者: 李冬雪，副研究员，博士；电子信箱：dxli@shsci.org。
张志刚，研究员，博士；电子信箱：zzhang@shsci.org。
作者简介:第一联系人：为共同第一作者（co-first authors）。
基金资助:
国家自然科学基金(92168111);国家自然科学基金(82230087);上海交通大学医学院“双百人”项目(20181708);上海市高水平地方高校创新团队(SHSMU-ZDCX20210802)

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

Hong Yuanxin, Tulamati Aziguli, Shi Wentao, Yang Tian, Zhu Lei, Zhang Xueli, Zhang Zhigang(), Li Dongxue()

State Key Laboratory of Systems Medicine for Cancer; Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China

Received:2026-03-26 Accepted:2026-04-21 Online:2026-10-20 Published:2026-10-20
Contact: Li Dongxue, E-mail: dxli@shsci.org.
Zhang Zhigang, E-mail: zzhang@shsci.org.
Supported by:
National Natural Science Foundation of China(92168111);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20181708);Innovative Research Team of High-level Local Universities in Shanghai(SHSMU-ZDCX20210802)

摘要/Abstract

摘要：

目的·构建一种适用于多巴胺化修饰蛋白筛选与鉴定的研究体系，并初步分析其在不同细胞和组织中的分布特征。方法·结合癌症基因组图谱（The Cancer Genome Atlas，TCGA）和基因型-组织表达（Genotype-Tissue Expression，GTEx）数据库分析多巴胺转运蛋白、多巴脱羧酶（DOPA decarboxylase，DDC）及转谷氨酰胺酶2（transglutaminase 2，TGM2）在人体不同组织中的表达情况，评估多巴胺化修饰发生的分子基础。以炔基化多巴胺衍生物PA为化学探针，将其加至细胞培养液中，结合点击化学反应、生物素标记富集及质谱分析，对不同细胞类型中的候选多巴胺化修饰蛋白进行筛选，并通过功能富集分析和蛋白相互作用网络分析解析其功能特征。同时利用抗多巴胺化修饰特异性抗体，通过免疫组织化学方法在小鼠及人胰腺相关组织中对多巴胺化修饰进行初步验证。结果·多巴胺转运蛋白、DDC及TGM2在人体不同组织普遍存在，为蛋白质多巴胺化修饰在不同组织中的发生提供了分子基础条件。PA可作为有效化学探针用于候选多巴胺化修饰蛋白的标记、富集与鉴定。分别在小鼠海马HT22细胞、新生小鼠原代小胶质细胞、胰岛α细胞、胰岛β细胞和胰腺癌细胞AsPC-1中筛选到56、327、463、56和86个候选蛋白，同时不同细胞类型中筛选获得的候选多巴胺化修饰蛋白谱存在明显差异，并呈现出细胞类型依赖的功能特征。功能富集分析显示，这些蛋白主要参与代谢调控、蛋白质合成及细胞结构构成相关过程。免疫组织化学结果提示，在小鼠及人胰腺相关组织中均可检测到多巴胺化修饰。结论·成功建立了一种基于炔基化多巴胺衍生物PA的多巴胺化修饰研究体系。该方法兼具可追踪性、富集能力和蛋白质组学拓展性，可用于系统筛选候选多巴胺化修饰蛋白并分析其功能特征，为进一步阐明多巴胺化修饰的分子机制及其在生理和病理过程中的作用提供了方法学参考。

关键词: 多巴胺, 多巴胺化修饰, 化学探针, 蛋白质组

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

中图分类号:

R730.2

洪源鑫, 阿孜古丽·吐拉麦提, 石文韬, 杨田, 朱蕾, 张雪莉, 张志刚, 李冬雪. 基于多巴胺衍生物PA的多巴胺化修饰蛋白研究体系的建立[J]. 上海交通大学学报（医学版）, 2026, 46(10): 1392-1405.

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.

图/表 7

图1 SLC6A3 和 DDC 在各正常组织及肿瘤组织中的表达Note： A/C. SLC6A3 (A) and DDC (C) mRNA expression in various normal tissues based on the GTEx database. B/D. SLC6A3 (B) and DDC (D) mRNA expression in pan-cancer tissues and adjacent normal tissues from the TCGA database.

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

图2 DA衍生物PA的合成路线及结构表征Note： A. Synthetic route of the alkyne-modified dopamine derivative PA. B. 1H NMR spectrum of PA recorded in DMSO-d₆. C. LC-MS spectrum of PA, showing a molecular ion peak corresponding to the expected molecular weight (~205.1 Da).

Fig 2 Synthetic route and structural characterization of dopamine derivative PA

表1 炔基化多巴胺衍生物PA的 1H NMR化学位移归属

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

图3 基于PA探针的多巴胺化修饰蛋白富集策略及候选蛋白谱Note： A. Schematic illustration of the PA-based strategy for the enrichment and identification of dopaminylated proteins in cells. B. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between PA and biotin azide, forming a stable triazole conjugate. C‒G. Heatmaps showing the top 25 candidate dopaminylated proteins identified in different cell types, including hippocampal HT22 cells (C), primary microglia (D), pancreatic α cells αTC1-9 (E), pancreatic β cells SJβ/79-7 (F), and pancreatic ductal adenocarcinoma (PDAC) AsPC-1 cells (G).

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

图4 不同细胞类型中多巴胺化修饰候选蛋白的KEGG富集及交集分析Note： A‒E. KEGG pathway enrichment analysis of candidate dopaminylated proteins identified in hippocampal HT22 cells (A), primary microglia (B), pancreatic α cells (C), pancreatic β cells (D), and PDAC AsPC-1 cells (E). F. Venn diagram showing the overlap of candidate dopaminylated proteins among the five cell types.

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

图5 不同细胞类型中多巴胺化修饰候选蛋白的蛋白相互作用网络分析Note： A‒E. Protein-protein interaction (PPI) networks of candidate dopaminylated proteins identified in hippocampal HT22 cells (A), primary microglia (B), pancreatic α cells αTC1-9 (C), pancreatic β cells SJβ/79-7 (D), and PDAC AsPC-1 cells (E). Hub proteins are highlighted according to network connectivity.

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

图6 小鼠及人胰腺组织中多巴胺化修饰的免疫组织化学检测Note： A. Immunohistochemical detection of pan-dopaminylation (pan-DA) in WT mouse pancreatic tissue, showing positive staining compared with the negative control. B. Immunohistochemical detection of pan-DA in pancreatic tumor tissue from KPC mice. C. Detection of pan-DA in mouse pancreatic tissues at different pathological stages, including normal pancreas, low-grade PanIN, high-grade PanIN, and PDAC lesions. D. Immunohistochemical detection of pan-DA in human pancreatic tissues, including normal pancreas, low-grade PanIN, high-grade PanIN, and PDAC.

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

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基于多巴胺衍生物PA的多巴胺化修饰蛋白研究体系的建立

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

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