Proteomic data of samples from lipid droplets of ASFV-infected cells

Peptide separations were carried out on an Easy-nLC 1000 nano system (Thermo Scientific). For the analysis, triplicates were loaded into a precolumn Acclaim PepMap 100 (Thermo Scientific) and eluted in a RSLC PepMap C18, 50 cm long, 75 µm inner diameter and 2 µm particle size (Thermo Scientific). The mobile phase flow rate was 300 nL/min using 0.1% formic acid in water (solvent A) and 0.1% formic acid and 100% acetonitrile (solvent B). The gradient profile was set as follows: 5–35% solvent B for 100 min, 35%-45% solvent B 20 min, 45%-100% solvent B 5 min, and 100% solvent B 15 min. Four microliters (about 1 µg) of each sample were injected. MS analysis was performed using a Q-Exactive mass spectrometer (Thermo Scientific). For ionization, 1900 V of liquid junction voltage and 300°C capillary temperature was used. The full scan method employed a m/z 300–1800 mass selection, an Orbitrap resolution of 70,000 (at m/z 200), a target automatic gain control (AGC) value of 3e6, and maximum injection times of 100 ms. After the survey scan, the 15 most intense precursor ions were selected for MS/MS fragmentation. Fragmentation was performed with a normalized collision energy of 27 eV and MS/MS scans were acquired with a starting mass of m/z 200, AGC target was 2e5, resolution of 17500 (at m/z 200), intensity threshold of 8e4, isolation window of 2.0 m/z units and maximum IT was 100 ms. Charge state screening was enabled to reject unassigned, singly charged, and equal or more than seven protonated ions. A dynamic exclusion time of 30s was used to discriminate against previously selected ions.肽段分离实验在赛默飞世尔科技（Thermo Scientific）Easy-nLC 1000纳升液相系统（Easy-nLC 1000 nano system）上完成。将三份平行样品上样至Acclaim PepMap 100预柱（Acclaim PepMap 100），随后在规格为50 cm长、75 μm内径、2 μm粒径的RSLC PepMap C18分析柱（RSLC PepMap C18）上完成洗脱。流动相流速设为300 nL/min，流动相A为含0.1%甲酸的水溶液，流动相B为含0.1%甲酸的纯乙腈溶液。梯度洗脱程序设置如下：100 min内流动相B浓度从5%升至35%，20 min内升至45%，再用5 min升至100%，最后维持100%流动相B运行15 min。每份样品进样量为4 μL（约1 μg）。质谱分析采用Q-Exactive质谱仪（Q-Exactive mass spectrometer，赛默飞世尔科技（Thermo Scientific））。电离参数设置为：液接电压1900 V，毛细管温度300℃。全扫描模式参数包括：质荷比（m/z）扫描范围300~1800，轨道阱（Orbitrap）分辨率为70000（m/z 200处），自动增益控制（automatic gain control, AGC）目标值为3×10^6，最大注入时间为100 ms。一级扫描完成后，选取强度最高的15个前体离子进行串联质谱（MS/MS）碎裂。碎裂采用归一化碰撞能量27 eV，MS/MS扫描起始质量为m/z 200，AGC目标值为2×10^5，分辨率为17500（m/z 200处），强度阈值为8×10^4，隔离窗口为2.0 m/z，最大离子注入时间为100 ms。启用电荷态筛选，排除未指定电荷、单电荷以及质子化电荷数≥7的离子。设置30 s动态排除时长，以避免重复选取已分析的前体离子。