Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome
Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome
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DOI：
10.1021/acs.analchem.2c02641
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发表时间：
2022-08-22
影响因子：
7.4
通讯作者：
Lee，Sang-Won
中科院分区：
文献类型：
--
作者：
Kang，Chaewon;Huh，Sunghyun;Lee，Sang-Won
蛋白质磷酸化是一种普遍的翻译后修饰，基本上调节细胞过程的各个方面。目前，液相色谱-串联质谱（LC-MS/MS）与广泛的离线样品分离和磷酸肽富集方法是深度磷蛋白质组分析的最佳实践，但平衡通量和分析深度仍然是一个实际的挑战。我们提出了一种超深磷蛋白质组分析的在线三维分离方法，该方法结合了在线二维液相色谱分离和气相分离。该方法在1.5天的数据采集过程中，在HeLa细胞中鉴定出超过10万个磷酸化肽（bbb60000个磷酸化位点），最大的HeLa细胞磷酸化蛋白质组显著扩展了细胞磷酸化蛋白质组的可检测功能景观。
Protein phosphorylation is a prevalent post-translational modification that regulates essentially every aspect of cellular processes. Currently, liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an extensive offline sample fractionation and a phosphopeptide enrichment method is a best practice for deep phosphoproteome profiling, but balancing throughput and profiling depth remains a practical challenge. We present an online three-dimensional separation method for ultradeep phosphoproteome profiling that combines an online two-dimensional liquid chromatography separation and an additional gas-phase separation. This method identified over 100,000 phosphopeptides (>60,000 phosphosites) in HeLa cells during 1.5 days of data acquisition, and the largest HeLa cell phosphoproteome significantly expanded the detectable functional landscape of cellular phosphoproteome.