GoDig, a platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standards, is a powerful, flexible, and easy-to-use method that uses tandem mass tags to increase sample throughput up to 18-fold relative to label-free methods. Though the protein-level success rates of GoDig are high, the peptide-level success rates are more limited, hampering assays of harder-to-quantify proteins and site-specific phenomena. To guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of “priming runs,” an acquisition mode with significantly higher success rates. In this mode, two or more chromatographic priming runs are automatically performed to improve the accuracy and precision of target elution orders, followed by analytical runs which quantify targets. Using priming runs, success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy expression profiles across four human cell lines.

中文翻译：

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插入分析前色谱引发运行可通过样品复用显着改善靶向途径蛋白质组学

GoDig 是一个用于靶向途径蛋白质组学的平台，无需手动测定安排或合成标准品，是一种强大、灵活且易于使用的方法，它使用串联质量标签将样品通量相对于标签提高高达 18 倍。免费方法。尽管 GoDig 的蛋白质水平成功率很高，但肽水平的成功率更为有限，阻碍了难以量化的蛋白质和位点特异性现象的分析。为了指导 GoDig 检测的优化以及 GoDig 平台的改进，我们创建了 GoDigViewer，这是一款新的独立软件，可提供 GoDig 运行的详细可视化。 GoDigViewer 指导实施“启动运行”，这是一种成功率显着提高的采集模式。在此模式下，会自动执行两次或多次色谱启动运行，以提高目标洗脱顺序的准确度和精密度，然后进行量化目标的分析运行。使用启动运行，400 个肽靶点列表的成功率超过 97%，200 个通常无法使用非靶向质谱定量的靶点列表的成功率超过 95%。我们使用启动运行对 125 种巨自噬蛋白进行了定量测定，成功率 >95%，并揭示了四种人类细胞系中巨自噬表达谱的差异。