新型在线三维分离技术扩展了细胞磷酸化蛋白质组的可检测功能景观。
Novel Online Three-Dimensional Separation Expands the Detectable Functional Landscape of Cellular Phosphoproteome.
机构信息
Department of Chemistry, Center for Proteogenome Research, Korea University, Seoul 02841, Republic of Korea.
School of Biological Sciences, Seoul National University, Seoul 08826, Republic of Korea.
出版信息
Anal Chem. 2022 Sep 6;94(35):12185-12195. doi: 10.1021/acs.analchem.2c02641. Epub 2022 Aug 22.
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.
蛋白质磷酸化是一种普遍的翻译后修饰，它调节细胞过程的几乎所有方面。目前，带有广泛离线样品分级和磷酸肽富集方法的液相色谱-串联质谱（LC-MS/MS）是深度磷酸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。我们提出了一种用于超深度磷酸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附加的气相分离。这种方法在 1.5 天的数据采集过程中，在 HeLa 细胞中鉴定出超过 100,000 个磷酸肽（>60,000 个磷酸化位点），最大的 HeLa 细胞磷酸蛋白质组显著扩展了细胞磷酸蛋白质组的可检测功能图谱。
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