Proteomic analysis of injured and uninjured nerves

Murine injured sciatic nerves were harvested from C57BL/6J mice at 24h post-transection. Uninjured nerves were set as control group. Nerve samples were frozen and then ground into fine and uniform powder in liquid nitrogen for protein extraction. Then the samples were digested and fractionated. The data independent acquisition (DIA) mass spectrometry analysis was acquired by TimsTOF Pro2 mass spectrometer (Bruker) and nanoElute (Bruker). For data independent acquisition (DIA), 56 DIA windows were acquired and the collision energy was ramped linearly as a function of the mobility from 59 eV at 1/K0 = 1.6 Vs/cm2 to 20 eV at 1/K0 = 0.6 Vs/cm2. The MS/MS spectra were recorded from 100 to 1700 m/z. The default factory settings were used for the Spectronaut Pulsar™ 15.3.210906.50606 (Biognosys, Swiss) search and library generation.

本实验从横断损伤24小时后的C57BL/6J小鼠体内采集损伤坐骨神经样本，以未损伤的坐骨神经作为对照组。将神经样本速冻后置于液氮中研磨为细腻均匀的粉末，用于蛋白质提取。随后对样本进行酶解与分级分离。采用布鲁克（Bruker）timsTOF Pro2质谱仪与nanoElute纳升液相系统完成数据非依赖采集（Data Independent Acquisition, DIA）质谱分析。本次DIA分析共设置56个采集窗口，碰撞能量随离子迁移率呈线性变化：当1/K0为1.6 Vs/cm²时碰撞能量为59 eV，当1/K0为0.6 Vs/cm²时碰撞能量为20 eV。质谱二级谱（MS/MS）的采集范围为100~1700 m/z。使用瑞士Biognosys公司的Spectronaut Pulsar™ 15.3.210906.50606软件进行谱库构建与数据库检索时，采用其默认出厂参数设置。