Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.
J Proteome Res. 2022 Aug 5;21(8):1789-1799. doi: 10.1021/acs.jproteome.2c00172. Epub 2022 Jul 25.
Mass spectrometry-based profiling of the phosphoproteome is a powerful method of identifying phosphorylation events at a systems level. Most phosphoproteomics studies have used data-dependent acquisition (DDA) mass spectrometry as their method of choice. In this Perspective, we review some recent studies benchmarking DDA and DIA methods for phosphoproteomics and discuss data analysis options for DIA phosphoproteomics. In order to evaluate the impact of data-dependent and data-independent acquisition (DIA) on identification and quantification, we analyze a previously published phosphopeptide-enriched data set consisting of 10 replicates acquired by DDA and DIA each. We find that though more unique identifications are made in DDA data, phosphopeptides are identified more consistently across replicates in DIA. We further discuss the challenges of identifying chromatographically coeluting phosphopeptide isomers and investigate the impact on reproducibility of identifying high-confidence site-localized phosphopeptides in replicates.
基于质谱的磷酸化蛋白质组学分析是一种在系统水平上鉴定磷酸化事件的强大方法。大多数磷酸蛋白质组学研究都使用基于数据依赖性采集（DDA）的质谱作为首选方法。在本观点中，我们回顾了一些最近的基准测试 DDA 和 DIA 方法用于磷酸蛋白质组学的研究，并讨论了 DIA 磷酸蛋白质组学的数据分析选项。为了评估数据依赖和数据独立采集（DIA）对鉴定和定量的影响，我们分析了先前发表的由 DDA 和 DIA 分别获得的 10 个重复的磷酸肽富集数据集。我们发现，尽管 DDA 数据中可以鉴定出更多的独特鉴定物，但在 DIA 中，磷酸肽在重复中更一致地被鉴定出来。我们进一步讨论了鉴定色谱共洗脱磷酸肽异构体的挑战，并研究了在重复中鉴定高可信度定位磷酸肽的重现性的影响。
