目的·构建5-羟色胺化（serotonylation，5-HT化）修饰蛋白研究体系，为寻找和发现5-HT化修饰蛋白提供方法学基础。方法·基于癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库和基因型-组织表达（Genotype-Tissue Expression，GTEx）数据库分析编码5-HT化修饰的关键酶的基因转谷氨酰胺酶2（transglutaminase 2，TGM2）和编码5-羟色胺转运体（serotonin transporter，SERT）的基因溶质载体家族6（solute carrier family 6，SLC6A4）在正常生理组织和肿瘤组织中的表达情况。利用5-羟色胺盐酸盐分步合成5-羟色胺衍生物5-PT（5-propargyltryptamide），并利用傅里叶变换红外光谱（FT-IR）、核磁共振氢谱¹H-NMR、核磁共振碳谱¹³C-NMR和飞行时间质谱（TOF-MS）等手段进行分析和结构表征。通过流式细胞术检测5-PT在人胰腺癌细胞AsPC-1和小鼠免疫细胞［包括CD4⁺ T细胞、CD8⁺ T细胞以及髓系巨噬细胞（bone marrow derived macrophage，BMDM）］的胞内摄入情况。通过点击化学反应、免疫共沉淀以及质谱分析技术寻找和鉴定发生5-HT化修饰的蛋白，并进行京都基因和基因组数据库（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集分析。结果·生物信息学相关分析显示TGM2和SLC6A4在生物体内正常组织和肿瘤组织广泛存在。流式细胞术结果显示合成的5-PT可通过细胞表面的SERT摄取进入人胰腺癌细胞AsPC-1和小鼠免疫细胞（包括CD4⁺ T细胞、CD8⁺ T细胞以及BMDM）。质谱分析数据显示，在各个细胞蛋白的5-PT处理组都富集到了丰富的5-HT化修饰蛋白。KEGG富集分析显示这些蛋白参与糖酵解和氨基酸合成相关通路。结论·利用合成的5-PT成功构建了5-HT化修饰蛋白的研究体系，在不同的细胞中富集得到多个5-HT化修饰的蛋白，为研究蛋白的5-HT化修饰及其功能提供了相对简单高效的研究手段。

Objective ·To establish a research framework for serotonylation of proteins and to provide a methodological basis for the identification of serotonylated proteins. Methods ·The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases were used to analyze the expression of the transglutaminase 2 (TGM2) gene, which encodes the key enzyme for serotonylation, and the solute carrier family 6 (SLC6A4) gene, which encodes the serotonin transporter (SERT), in normal and pan-cancer tissues. 5-Propargyltryptamide (5-PT), a serotonin derivative, was synthesized stepwise from serotonin hydrochloride, and its structure was characterized by the Fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (¹H-NMR), carbon nuclear magnetic resonance (¹³C-NMR), and time-of-flight mass spectrometry (TOF-MS). The intracellular uptake of 5-PT in the human pancreatic cancer cell line AsPC-1 and mouse immune cells, including CD4⁺ T cells, CD8⁺ T cells, and bone marrow-derived macrophages (BMDMs), was detected by using flow cytometry. Click chemistry, co-immunoprecipitation, and mass spectrometry analysis techniques were employed to identify serotonylated proteins, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed. Results ·Bioinformatics analysis indicated that TGM2 and SLC6A4 were widely expressed in various normal tissues and across pan-cancer tissues. The flow cytometry results showed that the synthesized 5-PT can be taken up into the human pancreatic cancer cell line AsPC-1 and mouse immune cells, including CD4⁺ T cells, CD8⁺ T cells, and BMDMs, via the SERT. Mass spectrometry analysis data showed that a significant amount of serotonylated proteins were enriched in various cells treated with 5-PT. KEGG enrichment analysis revealed that these proteins were involved in important pathways related to glycolysis and amino acid synthesis. Conclusion ·By using the synthesized 5-PT, multiple serotonylated proteins are enriched in various cell types. A research system for identifying serotonylated proteins has been successfully established, providing a relatively simple and efficient method for studying protein serotonylation.