LC-MS/MS screen of day 21 cortical neurons differentiated from <i>UBE3A</i> knockout and <i>UBE3A </i>wild-type isogenic control human induced pluripotent stem cells

We used tandem mass tag (TMT) labeling to profile whole-cell lysates from <i>UBE3A </i>knockout and isogenic control neurons differentiated for 21 days in neuronal culture conditions. Three independent replicates of <i>UBE3A </i>knockout and control cultures were lysed in urea lysis buffer, digested in trypsin, each lysate was labeled with a unique isobaric tandem mass tag (TMT), and the samples combined for analysis by analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). LC-MS/MS analysis identified a total of 5,621 proteins of which 5,590 were detected in all 6 samples. MaxQuant software (version 1.6.14.0.) was used to normalize protein signals per sample and generate relative protein abundances (expressed as fold-change in the KO from the control) and student’s t-tests used to determine significant differences.本研究采用串联质量标签（tandem mass tag, TMT）标记技术，对在神经元培养条件下分化21天的UBE3A基因敲除及同基因对照神经元的全细胞裂解液开展蛋白质组定量分析。本研究设置3次独立生物学重复：将UBE3A基因敲除组与对照组的细胞培养物分别以尿素裂解缓冲液裂解，经胰蛋白酶消化后，每份裂解液使用唯一的同重串联质量标签（TMT）进行标记，随后将所有标记后的样品混合，通过液相色谱-串联质谱（liquid chromatography with tandem mass spectrometry, LC-MS/MS）完成分析。LC-MS/MS分析共鉴定到5621种蛋白质，其中5590种可在全部6份样品中被稳定检测到。本研究使用MaxQuant软件（版本1.6.14.0）对各样品的蛋白质信号进行归一化处理，计算得到相对蛋白质丰度（以敲除组相对于对照组的倍数变化表示），并通过学生t检验判定各组间蛋白质丰度的显著性差异。