Interaction of Plasmodium yoelii tryptophan-rich antigen 7 with CD71 on macrophage membrane regulates host inflammatory response

To screen for candidate interacting proteins of PyTRAg7 protein and macrophage membrane proteins, differential bands were identified by immunoprecipitation combined with silver staining and mass spectrometry. Excised protein bands were destained, reduced, allylated, and digested with trypsin for 20h at 37°C. Eluted peptides were analyzed using the Q-Exactive mass spectrometer (Thermo Scientific) for 60 min. The mass spectrometer was operated in positive ion mode. Full scan MS spectra (m/z 300–1800) were acquired with a resolution of 70000 at 100 m/z. Automatic gain control (AGC) target was set to 1e6; Maximum inject time was 50 ms, and dynamic exclusion was 30.0s. Up to 20 most intense ions were selected for higher-energy collisional dissociation fragmentation depending on signal intensity. Isolation window was 1.5 m/z; MS/MS fragment spectra were acquired with a resolution of 17500 at 100 m/z, AGC target was 1e5; Maximum inject time was 50 ms; Normalized collision energy was 27eV, and underfill was 0.1%. MS raw data was analyzed with Proteome Discoverer Daemon 2.5.为筛选PyTRAg7蛋白与巨噬细胞膜蛋白的候选互作蛋白，本研究通过免疫沉淀结合银染与质谱技术鉴定差异条带。切取的蛋白条带经脱色、还原、烯丙基化修饰后，于37℃下用胰蛋白酶消化20小时。洗脱的肽段采用赛默飞世尔科技（Thermo Scientific）Q-Exactive质谱仪进行60分钟的分析。质谱采用正离子模式运行：以100 m/z处分辨率70000采集m/z范围300–1800的全扫描质谱光谱；自动增益控制（AGC）目标值设为1×10⁶，最大注入时间为50 ms，动态排除时长为30.0 s。根据信号强度选取前20个强度最高的离子进行高能碰撞解离碎裂，隔离窗口为1.5 m/z；以100 m/z处分辨率17500采集MS/MS碎片光谱，自动增益控制目标值设为1×10⁵，最大注入时间为50 ms，归一化碰撞能量为27 eV，填充不足阈值为0.1%。原始质谱数据采用Proteome Discoverer Daemon 2.5进行分析。