磷酸蛋白质组学
蛋白质组学
磷酸肽
计算生物学
定量蛋白质组学
串联质谱法
生物信息学
细胞培养中氨基酸的稳定同位素标记
串联质量标签
计算机科学
生物
化学
磷酸化
质谱法
生物信息学
细胞生物学
蛋白质磷酸化
生物化学
蛋白激酶A
色谱法
基因
作者
Ana Martínez-Val,Kyle L. Fort,Claire Koenig,Leander Van der Hoeven,Giulia Franciosa,Thomas J. Moehring,Yasushi Ishihama,Yu‐Ju Chen,Alexander Makarov,Yue Xuan,Jesper V. Olsen
标识
DOI:10.1038/s41467-023-39347-y
摘要
Abstract Achieving sufficient coverage of regulatory phosphorylation sites by mass spectrometry (MS)-based phosphoproteomics for signaling pathway reconstitution is challenging, especially when analyzing tiny sample amounts. To address this, we present a hybrid data-independent acquisition (DIA) strategy (hybrid-DIA) that combines targeted and discovery proteomics through an Application Programming Interface (API) to dynamically intercalate DIA scans with accurate triggering of multiplexed tandem mass spectrometry (MSx) scans of predefined (phospho)peptide targets. By spiking-in heavy stable isotope labeled phosphopeptide standards covering seven major signaling pathways, we benchmark hybrid-DIA against state-of-the-art targeted MS methods (i.e., SureQuant) using EGF-stimulated HeLa cells and find the quantitative accuracy and sensitivity to be comparable while hybrid-DIA also profiles the global phosphoproteome. To demonstrate the robustness, sensitivity, and biomedical potential of hybrid-DIA, we profile chemotherapeutic agents in single colon carcinoma multicellular spheroids and evaluate the phospho-signaling difference of cancer cells in 2D vs 3D culture.
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