A data-independent acquisition-based global phosphoproteomics system enables deep profiling.
A data-independent acquisition-based global phosphoproteomics system enables deep profiling.
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DOI：
10.1038/s41467-021-22759-z
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发表时间：
2021-05-05
影响因子：
16.6
通讯作者：
Chen YJ
中科院分区：
文献类型：
--
作者：
Kitata RB;Choong WK;Tsai CF;Lin PY;Chen BS;Chang YC;Nesvizhskii AI;Sung TY;Chen YJ
磷酸蛋白质组学可以提供对细胞信号动力学的洞察。为了实现对微量样品的深度和稳健的定量磷酸蛋白质组学分析，我们在此开发了一种基于数据独立采集(DIA)质谱学和从数据依赖采集(DDA)和DIA数据衍生的混合谱库的全球磷蛋白质组学策略。结果表明，该方法灵敏度高(~lt；0.1 ng)，定位准确，重现性好(中位变异系数约5%)。作为概念验证，我们使用肺癌细胞系和患者来源的组织构建了一个涵盖159,524个磷酸肽(88,107个亚磷酸盐)的混合磷酸蛋白质组谱文库。基于这个文库，我们的单次简化的DIA工作流程在两小时内对细胞系样本中的36,350个亚磷酸盐(19,755个1类)进行了定量。应用于耐药细胞和患者来源的肺癌组织，描绘了与耐药和肿瘤进展相关的位点特异性磷酸化事件，表明我们的工作流程能够以深度覆盖、高灵敏度和低运行间缺失值的方式表征磷酸化信号。磷酸蛋白质组学可以为疾病相关的细胞信号变化提供系统的见解。在这里，作者提出了一个敏感的工作流程，集成了基于文库和直接独立于数据的获取方法，并提供了一个用于深入磷蛋白组学分析的混合谱库资源。
Phosphoproteomics can provide insights into cellular signaling dynamics. To achieve deep and robust quantitative phosphoproteomics profiling for minute amounts of sample, we here develop a global phosphoproteomics strategy based on data-independent acquisition (DIA) mass spectrometry and hybrid spectral libraries derived from data-dependent acquisition (DDA) and DIA data. Benchmarking the method using 166 synthetic phosphopeptides shows high sensitivity (<0.1 ng), accurate site localization and reproducible quantification (~5% median coefficient of variation). As a proof-of-concept, we use lung cancer cell lines and patient-derived tissue to construct a hybrid phosphoproteome spectral library covering 159,524 phosphopeptides (88,107 phosphosites). Based on this library, our single-shot streamlined DIA workflow quantifies 36,350 phosphosites (19,755 class 1) in cell line samples within two hours. Application to drug-resistant cells and patient-derived lung cancer tissues delineates site-specific phosphorylation events associated with resistance and tumor progression, showing that our workflow enables the characterization of phosphorylation signaling with deep coverage, high sensitivity and low between-run missing values. Phosphoproteomics can provide systematic insights into disease-associated cell signaling changes. Here, the authors present a sensitive workflow integrating library-based and direct data-independent acquisition approaches, and a hybrid spectral library resource for in-depth phosphoproteomic profiling.
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影响因子：
14.9
作者：
Okuda S;Watanabe Y;Moriya Y;Kawano S;Yamamoto T;Matsumoto M;Takami T;Kobayashi D;Araki N;Yoshizawa AC;Tabata T;Sugiyama N;Goto S;Ishihama Y
通讯作者：
Ishihama Y
影响因子：
14.9
作者：
Hornbeck PV;Kornhauser JM;Tkachev S;Zhang B;Skrzypek E;Murray B;Latham V;Sullivan M
通讯作者：
Sullivan M
影响因子：
11.5
作者：
Carlos Montero, Juan;Seoane, Samuel;Pandiella, Atanasio
通讯作者：
Pandiella, Atanasio
影响因子：
2.9
作者：
Muntel, Jan;Gandhi, Tejas;Reiter, Lukas
通讯作者：
Reiter, Lukas
影响因子：
48
作者：
Lawrence RT;Searle BC;Llovet A;Villén J
通讯作者：
Villén J