Quantification of proteasome subunits by mass spectroscopy in IP-deficient mice.

20S proteasomes were isolated from murine hearts from control mice and at day 4 and 8 p.i. from β5i/LMP7+/+ and β5i/LMP7-/- mice. The mixture of tryptic 20S proteasome peptides was separated prior to mass spectrometric analysis by reverse phase nano HPLC using a Proxeon System; MS-data were generated on an Orbitrap-MS equipped with a nanoelectrospray ion source as described in material and methods. MS ion intensities are shown for individual proteasome subunits as ratios between β5i/LMP7+/+ and β5i/LMP7-/- mice. Data are mean±SD from two technical replicates that have been performed each with two independent biological replicates. p<0.05 indicates statistical significance. n.d.: ratio was not determined for β5i/LMP7.

本研究从对照组小鼠的心脏组织中分离得到20S蛋白酶体（20S proteasome），并分别从β5i/LMP7+/+与β5i/LMP7-/-小鼠感染后第4天、第8天的心脏组织中分离该蛋白酶体。将胰蛋白酶酶解得到的20S蛋白酶体肽段混合物，通过Proxeon系统搭载的反相纳米高效液相色谱（reverse phase nano HPLC）进行分离，随后开展质谱分析；质谱（MS）数据采集于搭载纳米电喷雾离子源的Orbitrap质谱仪（Orbitrap-MS），具体实验流程详见材料与方法部分。各蛋白酶体亚基的质谱离子强度以β5i/LMP7+/+小鼠与β5i/LMP7-/-小鼠的离子强度比值形式呈现。本次实验数据以平均值±标准差（mean±SD）表示，每组实验包含2次技术重复，且每次技术重复均对应2次独立的生物学重复。p<0.05代表差异具有统计学意义；n.d.表示未测定β5i/LMP7的相对比值。