Protein myristoylation modification omics mass spectrometry of iWAT

Sixteen 8-week-old male C57BL/6J mice were injected with adenovirus expressing Eepd1-Flag (1x10^8/mouse) into multiple sites in their iWAT. Three days later, each mouse was administered 50 mg/kg of myristic acid solution (prepared in 0.1% BSA) via oral gavage. Half of the mice were then exposed to 8°C for 16 hours in a cold chamber, while the other half remained at room temperature (8 mice per group). Subsequently, iWAT was collected from the mice, and proteins were lysed using IP lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.5% sodium deoxycholate). Eepd1 protein was enriched using flag beads (Smart-Lifescience). The enriched beads were processed with non-contact ultrasound, and protein concentration was determined. Proteins were reduced, alkylated, and precipitated overnight with acetone : acetic acid (100 : 0.1). After centrifugation and washing, trypsin digestion was performed overnight at 37°C. The supernatant was concentrated, desalted, and dissolved in 0.1% formic acid for detection by nano-LC-IMS-MS in diaPASEF mode. MS scanning ranged from 100-1700 m/z, with 32 DIA windows of 26 Da width. Spectronaut 18 software was used for qualitative analysis, and MaxLFQ for protein group quantification. Differences between groups were analyzed by t-test with BH-corrected Q-values.

在本项研究中，对十六只8周龄的雄性C57BL/6J小鼠进行了Eepd1-Flag表达腺病毒的注射，注射剂量为每只小鼠1x10^8个病毒颗粒，注射部位为其内脏脂肪（iWAT）。注射三天后，每只小鼠通过口服灌胃给予50 mg/kg的月桂酸溶液（该溶液由0.1%的牛血清白蛋白配制）。随后，将其中一半小鼠置于8°C的低温箱中暴露16小时，另一半小鼠则保持室温（每组8只小鼠）。收集小鼠的iWAT后，使用IP裂解缓冲液（50 mM Tris-HCl，pH 8.0，150 mM NaCl，1 mM EDTA，1% Triton X-100，和0.5%的胆酸钠）进行蛋白裂解。利用flag磁珠（Smart-Lifescience）富集Eepd1蛋白。经非接触式超声波处理后，测定蛋白浓度。蛋白经过还原、烷基化，并在乙腈：乙酸（100：0.1）溶液中过夜沉淀。离心洗涤后，于37°C下过夜进行胰蛋白酶消化。消化液经过浓缩、脱盐，并以0.1%甲酸溶解，用于diaPASEF模式下的纳米液相色谱-飞行时间质谱-质谱联用（nano-LC-IMS-MS）检测。质谱扫描范围为100-1700 m/z，具有32个26 Da宽度的DIA窗口。采用Spectronaut 18软件进行定性分析，MaxLFQ软件进行蛋白质组定量分析。通过t检验及BH校正的Q值分析，对各组之间的差异进行了比较。