Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
Thermo Fisher Scientific, Rockford, IL, USA.
Nat Methods. 2020 Apr;17(4):399-404. doi: 10.1038/s41592-020-0781-4. Epub 2020 Mar 16.
Isobaric labeling empowers proteome-wide expression measurements simultaneously across multiple samples. Here an expanded set of 16 isobaric reagents based on an isobutyl-proline immonium ion reporter structure (TMTpro) is presented. These reagents have similar characteristics to existing tandem mass tag reagents but with increased fragmentation efficiency and signal. In a proteome-scale example dataset, we compared eight common cell lines with and without Torin1 treatment with three replicates, quantifying more than 8,800 proteins (mean of 7.5 peptides per protein) per replicate with an analysis time of only 1.1 h per proteome. Finally, we modified the thermal stability assay to examine proteome-wide melting shifts after treatment with DMSO, 1 or 20 µM staurosporine with five replicates. This assay identified and dose-stratified staurosporine binding to 228 cellular kinases in just one, 18-h experiment. TMTpro reagents allow complex experimental designs-all with essentially no missing values across the 16 samples and no loss in quantitative integrity.
同重标记技术能够在多个样本中同时进行全蛋白质组表达测量。这里提出了一套扩展的基于异丁基脯氨酸亚铵离子报告基团结构（TMTpro）的 16 种同重标记试剂。这些试剂与现有的串联质量标签试剂具有相似的特性，但具有更高的片段化效率和信号。在一个蛋白质组规模的示例数据集上，我们比较了未经 Torin1 处理和经 Torin1 处理的八种常见细胞系，每个重复定量了超过 8800 种蛋白质（每种蛋白质平均有 7.5 个肽段），每个蛋白质组的分析时间仅为 1.1 小时。最后，我们修改了热稳定性测定法，以在 5 个重复中检测 DMSO、1µM 或 20µM 星形孢菌素处理后的全蛋白质组融解位移。这项测定法仅用一个 18 小时的实验就鉴定并按剂量分层了星形孢菌素与 228 种细胞激酶的结合。TMTpro 试剂可以实现复杂的实验设计——16 个样本几乎没有缺失值，定量完整性也没有损失。
