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.