目的：以磷酸化蛋白质组学技术分析抗菌肽merecidin处理人肺腺癌A549细胞后细胞内磷酸化蛋白质表达的差异，探究merecidin对肺腺癌A549细胞蛋白质活性、功能的影响以及涉及的信号通路。方法：采用9 μmol/L merecidin处理肺腺癌A549细胞6 h，收集并提取总蛋白，SDS-PAGE检验全蛋白提取效果，加入胰酶来对蛋白质进行酶解。酶解所获肽段用TMT标记、采用 HPLC 分级分离、经 IMAC 磷酸化修饰富集以及液相色谱-质谱联用（HPLC-MS/MS）分离肽段。使用 localization probability>0.75 的 标 准 对 鉴 定 数 据 进 行 过 滤 ，利 用 GO（Gene Ontology）数 据 库 、KEGG（KyotoEncyclopedia of Genes and Genomes）数据库和STRING数据库对磷酸化蛋白组学数据进行分析。结果：SDS-PAGE 结果显示，经9 μmol/L merecidin处理后的A549细胞全蛋白分离效果清晰、无明显降解，且实验组与对照组条带差异明显；质谱共鉴定出位于3 089个蛋白上的10 320个磷酸化修饰位点，以|Fold change|>或<1.5且P<0.05为阈值从中筛选出差异明显的753个蛋白质及其1 172个磷酸化位点。蛋白质功能富集显示，磷酸化水平显著变化的蛋白质功能主要集中在蛋白质分子结合、代谢活性、分子功能调节、细胞进程、生物功能调节等方面；整合通路生物信息学分析结果显示，差异蛋白与Ras、PI3K/AKT、mTOR、AMPK等多条通路相关联；经过COG数据库筛选，发现差异性磷酸化蛋白主要集中在细胞信号转导、RNA转录、翻译后加工和修饰、核糖体合成蛋白质、细胞骨架蛋白形成及细胞内的物质转运和分泌、囊泡运输等多个方面；蛋白质互相作用层面分析结果显示，merecidin处理后的A549细胞中形成以MAPK1、RPL23A、SRSF3H、NCBP1等为关键蛋白的相互作用网，其中ATG2B、ULK1等蛋白显著上调，MAPK1、AKT1等蛋白显著下调。结论：磷酸化蛋白组学分析结果显示，抗菌肽merecidin可能通过MAPK、RPL23A、SRSF3H和AKT1等关键蛋白质在多方面生物功能和多条信号通路中发挥作用，促进肺腺癌A549细胞凋亡和自噬，从而抑制细胞的增殖。

Objective: The phosphorproteomics technique was used to analyze the differential expression of phosphorylated proteins in the human lung adenocarcinoma A549 cells treated with antimicrobial peptide merecidin, and to explore the effect of merecidin on the protein activity and function of lung adenocarcinoma A549 cells and the signal pathway involved. Methods: A549 cells were treated with 9 μmol/L merecidin for 6 h. The total protein was collected and extracted, and SDS-PAGE experiment was used to test the total protein extraction efficiency. Pancreatin was added to digest the protein. The peptides obtained by enzymatic hydrolysis were labeled with TMT,fractionated by HPLC, enriching phosphorylated modified peptides by IMAC, and separate peptides by HPLC-MS/MS (liquid chromatographymass spectrometry) technology. The identified data were screened using the standard of localization probability>0.75, and the phosphoproteomics data were analyzed using GO (Gene Ontology) database, KEGG (Kyoto Encyclopedia of Genes and Genomes) database and STRING database. Results: SDS-PAGE results showed that the total protein separation of A549 cells treated with 9 μmol/L merecidin was clear and no obvious degradation, and there was significant difference in protein bands between the experimental group and the control group.A total of 10 320 phosphorylation modification sites on 3 089 proteins were identified by mass spectrometry, of which 753 proteins and 1 172 phosphorylation sites were screened out with |Fold Change| >1.5 and P<0.05 as the threshold. Protein function enrichment showed that functions of proteins with significant changes in phosphorylation level mainly focused on molecular binding, metabolic activity, molecular function regulation, cell process, biological function and other aspects. Bioinformatics results of integrated pathway showed that differentially expressed proteins were associated with Ras, PI3K/AKT, mTor, AMPK etc. COG database screening showed that the differentially expressed phosphorylated proteins were concentrated in cell signal transduction, processing and modification of RNA transcription and translation,protein synthesis of ribosome, formation of cytoskeleton proteins, intracellular substance transportation, secretion and vesicle transportation etc. At the protein interaction level, after merecidin treatment, an interaction network with MAPK1, RPL23A, SRSF3H, NCBP1, etc. as key proteins was formed in A549 cells; proteins such as ATG2B and ULK1 etc. were significantly up-regulated, while proteins such as MAPK1 and AKT1 etc. were significantly down-regulated. Conclusion: Phosphoproteomic analysis shows that the antimicrobial peptide merecidin may play a role in multiple biological functions and multiple signaling pathways through key proteins such as MAPK1, RPL23A, SRSF3H and AKT1, and promote the apoptosis and autophagy of lung adenocarcinoma A549 cells, thereby inhibiting cell proliferation.

国家自然科学基金资助项目（No.81760661；No. 81560573）