A Cross-Linking-Aided Immunoprecipitation/Mass Spectrometry Workflow Reveals Extensive Intracellular Trafficking in Time-Resolved, Signal-Dependent Epidermal Growth Factor Receptor Proteome.
A Cross-Linking-Aided Immunoprecipitation/Mass Spectrometry Workflow Reveals Extensive Intracellular Trafficking in Time-Resolved, Signal-Dependent Epidermal Growth Factor Receptor Proteome.
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交联辅助免疫沉淀/质谱工作流程揭示了时间分辨、信号依赖性表皮生长因子受体蛋白质组中广泛的细胞内运输
DOI：
10.1021/acs.jproteome.9b00427
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发表时间：
2019-10-04
影响因子：
4.4
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Chen Y;Leng M;Gao Y;Zhan D;Choi JM;Song L;Li K;Xia X;Zhang C;Liu M;Ji S;Jain A;Saltzman AB;Malovannaya A;Qin J;Jung SY;Wang Y
配体与细胞表面受体的结合启动信号传导级联，其通常通过蛋白质-蛋白质相互作用（PPI）网络转导以激活过多的反应途径。然而，缺乏捕获膜PPI网络的工具。在这里，我们描述了一个交联辅助质谱工作流程的信号依赖性表皮生长因子受体（EGFR）蛋白质组的分离和鉴定。我们在EGF治疗后的不同时间点在细胞培养物中进行蛋白质交联，然后通过内源性EGFR的免疫沉淀和通过定量质谱分析相关蛋白质。我们确定了140个蛋白质在2小时的时间过程中的数据依赖性采集，并进一步验证了平行反应监测的结果具有高的信心。EGFR蛋白质组中的大部分蛋白质在内吞作用和细胞内蛋白质转运中起作用。EGFR蛋白质组是高度动态的，具有不同的时间行为;在所有时间点出现的10种蛋白质构成核心蛋白质组。功能表征显示，含FYVE结构域的蛋白质的丢失改变了EGFR的细胞内分布，但对EGFR蛋白质组或信号传导有微小影响。因此，我们的研究结果表明，EGFR蛋白质组包括影响EGFR信号传导的功能调节剂和作为内吞囊泡组分被捕获的旁观者。这些分子的高分辨率时空信息有助于描绘许多途径，这些途径可以确定信号传导的强度和持续时间，以及受体的位置和目的地。
Ligand binding to the cell surface receptors initiates signaling cascades that are commonly transduced through a protein–protein interaction (PPI) network to activate a plethora of response pathways. However, tools to capture the membrane PPI network are lacking. Here, we describe a cross-linking-aided mass spectrometry workflow for isolation and identification of signal-dependent epidermal growth factor receptor (EGFR) proteome. We performed protein cross-linking in cell culture at various time points following EGF treatment, followed by immunoprecipitation of endogenous EGFR and analysis of the associated proteins by quantitative mass spectrometry. We identified 140 proteins with high confidence during a 2 h time course by data-dependent acquisition and further validated the results by parallel reaction monitoring. A large proportion of proteins in the EGFR proteome function in endocytosis and intracellular protein transport. The EGFR proteome was highly dynamic with distinct temporal behavior; 10 proteins that appeared in all time points constitute the core proteome. Functional characterization showed that loss of the FYVE domain-containing proteins altered the EGFR intracellular distribution but had a minor effect on EGFR proteome or signaling. Thus, our results suggest that the EGFR proteome include functional regulators that influence EGFR signaling and bystanders that are captured as the components of endocytic vesicles. The high-resolution spatiotemporal information of these molecules facilitates the delineation of many pathways that could determine the strength and duration of the signaling, as well as the location and destination of the receptor.
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影响因子：
4.8
作者：
de Araujo, Mariana E. G.;Stasyk, Taras;Huber, Lukas A.
通讯作者：
Huber, Lukas A.
影响因子：
--
作者：
Akimov, Vyacheslav;Rigbolt, Kristoffer T. G.;Blagoev, Blagoy
通讯作者：
Blagoev, Blagoy
影响因子：
4.4
作者：
Engholm-Keller, Kasper;Hansen, Thomas Aarup;Larsen, Martin R.
通讯作者：
Larsen, Martin R.
影响因子：
1.2
作者：
D'Santos C;Taylor C;Carroll JS;Mohammed H
通讯作者：
Mohammed H
影响因子：
4
作者：
Balderhaar, Henning J. Kleine;Ungermann, Christian
通讯作者：
Ungermann, Christian