metabolites relative contents of all samples

erwent independent analysis with three biological replicates. The data acquisition system primarily comprised UPLC (SHIMADZU Nexera X2) coupled with Tandem mass spectrometry (Applied Biosystems 4500 QTRAP). The liquid phase conditions predominantly featured Agilent SB-C18 (1.8 μm, 2.1 mm*100 mm), with the solvent system comprising ultrapure water (0.1% formic acid, referred to as solvent A) and acetonitrile (0.1% formic acid, referred to as solvent B). The elution gradient commenced at 95:5 for A/B at 0 minutes, transitioned to 5:95 for A/B at 9 minutes, reverted to 95:5 for A/B at 10-11 minutes, and maintained 95:5 for A/B at 14 minutes. The flow rate was set at 0.35 mL/min, the column temperature maintained at 40°C, and the injection volume set at 4 μL.The mass spectrometry conditions primarily encompassed LIT and triple quadrupole (QQQ) scans conducted on a state-of-art triple quadrupole linear ion trap mass spectrometer (Q TRAP), specifically the AB4500 Q TRAP UPLC-MS/MS system equipped with an ESI Turbo ion spray interface. This system is adeptly controlled by the Analyst 1.6.3 software (AB Sciex), allowing operation in positive and negative ionization modes. The ESI source was configured with a source temperature of 550°C, an ion spray voltage of 5500 V (in positive ion mode) and -4500 V (in negative ion mode), while the ion source gas I, gas II, and curtain gas were set to 50, 60, and 25 psi, respectively. The QQQ scans were executed in MRM mode, with DP and CE parameters meticulously calibrated for each MRM ion pair. Tailored to the eluted metabolites in each period, a distinct set of MRM ion pairs was diligently monitored.本研究开展了含三次生物学重复的独立分析实验。数据采集系统主要由超高效液相色谱（UPLC，SHIMADZU Nexera X2）与串联质谱（Tandem mass spectrometry，Applied Biosystems 4500 QTRAP）联用构成。液相色谱分离采用安捷伦SB-C18色谱柱（1.8 μm，2.1 mm×100 mm），流动相体系为含0.1%甲酸的超纯水（记为流动相A）与含0.1%甲酸的乙腈（记为流动相B）。洗脱梯度设置如下：0 min时A与B的体积比为95:5，9 min时切换为5:95，10~11 min时恢复为95:5，14 min时维持95:5。流速设定为0.35 mL/min，柱温保持40℃，进样体积为4 μL。质谱检测采用搭载ESI Turbo离子喷雾接口的AB4500 Q TRAP UPLC-MS/MS系统，即先进的三重四极杆线性离子阱质谱仪（Q TRAP），主要开展线性离子阱（LIT）与三重四极杆（QQQ）扫描。该系统由Analyst 1.6.3软件（AB Sciex）控制，支持正、负两种电离模式运行。ESI源参数配置如下：源温度550℃，正离子模式下离子喷雾电压为5500 V，负离子模式下为-4500 V；离子源气I、气II与帘气（curtain gas）分别设置为50 psi、60 psi与25 psi。三重四极杆扫描采用多反应监测（MRM）模式，针对每个MRM离子对的去簇电压（DP）与碰撞能量（CE）参数进行了精准校准。针对每个时间段的洗脱代谢物，均针对性监测专属的MRM离子对集合。