采用组合前体同位素标记和同量异位素标记（cPILOT）进行自动样品多路复用。
Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT).
机构信息
Department of Chemistry, Vanderbilt University.
Department of Chemistry, Vanderbilt University; Vanderbilt Memory & Alzheimer's Center, Vanderbilt University Medical Center; Vanderbilt Brain Institute, Vanderbilt University Medical Center; Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center;
出版信息
J Vis Exp. 2020 Dec 18(166). doi: 10.3791/61342.
We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.
我们引入了一种高通量定量蛋白质组学工作流程，结合了前体同位素标记和等重标记（cPILOT）技术，能够在单个实验中分别对多达 22 或 24 个样本进行串联质量标签或等重 N,N-二甲基亮氨酸等重标签的多重标记。这种增强的样品多路复用技术大大减少了质谱采集时间，并提高了昂贵的商业等重试剂的利用率。然而，该策略中样品处理和移液步骤的手动过程可能既费力又耗时，并且会导致样品损失和定量误差。这些限制可以通过自动化来克服。在这里，我们将手动的 cPILOT 方案转移到自动化液体处理设备上，该设备可以并行处理大量的样品（例如，96 个样品）。总的来说，自动化提高了 cPILOT 的可行性和可重复性，并允许具有类似自动化设备的其他研究人员广泛使用。
Zotero 插件